CN117503690A - Long-acting co-carried hydrogel preparation for pulmonary arterial hypertension treatment, preparation method and application - Google Patents

Long-acting co-carried hydrogel preparation for pulmonary arterial hypertension treatment, preparation method and application Download PDF

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CN117503690A
CN117503690A CN202311494988.7A CN202311494988A CN117503690A CN 117503690 A CN117503690 A CN 117503690A CN 202311494988 A CN202311494988 A CN 202311494988A CN 117503690 A CN117503690 A CN 117503690A
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何伟
林辰诗
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China Pharmaceutical University
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Abstract

The invention belongs to the field of pharmaceutical preparations, and in particular relates to a long-acting co-carried hydrogel preparation for pulmonary arterial hypertension treatment, and a preparation method and application thereof. A long-acting co-carried hydrogel is characterized in that a biological macromolecular drug phospholipid compound and a micromolecular chemical drug are co-carried in an in-situ temperature-sensitive hydrogel, and an in-situ hydrogel skeleton is poloxamer. The invention improves the stability of biological macromolecules by preparing the phospholipid complex, and then loads the biological macromolecule phospholipid complex and the micromolecular compound into the in-situ temperature-sensitive hydrogel. The long-acting co-carried hydrogel can improve the stability of biological macromolecular drugs, and can be released in vivo for a long time after administration. The prepared long-acting co-carried hydrogel can simultaneously deliver biological macromolecular drugs and small molecular compounds and cooperatively treat pulmonary hypertension. The long-acting treatment of pulmonary hypertension is realized through single intramuscular injection administration, the defect of implanted pump administration is overcome, and the compliance and the living convenience of patients are expected to be improved.

Description

Long-acting co-carried hydrogel preparation for pulmonary arterial hypertension treatment, preparation method and application
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to a long-acting co-carried hydrogel preparation for pulmonary arterial hypertension treatment, and a preparation method and application thereof.
Background
Pulmonary hypertension (Pulmonary arterial hypertension, PAH) is an inflammatory disease with highly lethal cardiovascular and pulmonary vascular abnormalities, and persistent inflammation induces excessive proliferation of pulmonary vascular smooth muscle cells (Pulmonary artery smooth muscle cells, PASMCs) to trigger pulmonary vascular remodeling, progressive increases in vascular resistance, and severe cases leading to heart failure and even death. Currently approved clinical drug therapies for PAH mainly involve three signaling pathways: the prostacyclin pathway, the endothelin 1 pathway, and the nitric oxide pathway. The clinical first-line medicine treprostinil sodium (Treprostinil Sodium, TRE) is a tricyclic prostacyclin analogue, and can promote vasodilation of pulmonary and systemic arterial blood vessels, inhibit platelet aggregation and alleviate symptoms of PAH patients. PAH is clinically treated by adopting a single drug, and can only achieve the effects of relieving symptoms or reducing pulmonary arterial pressure, the treatment effect is unsatisfactory, the average survival rate of 5 years is only 34 percent, and the disease progress can only be delayed for advanced patients. The leading-edge strategy in the field of PAH therapy is currently combination therapy. The targeted drug combination therapy has certain curative effects on improving the hemodynamics and exercise tolerance of PAH patients, however, the targeted drug at the present stage is mainly vasodilating drugs, the pathological processes of focal inflammation and pulmonary artery reconstruction cannot be changed, and the diseases cannot be radically cured.
Studies have shown that the occurrence and progression of PAH disease is closely related to inflammation, persistent vascular inflammation and immune dysregulation can produce excessive vasoconstriction in PASMCs, forming a pro-proliferative, anti-apoptotic phenotype, leading to pulmonary vascular remodeling. The invention firstly proposes to treat PAH by using biological macromolecular medicine Interleukin 2 (Interleukin-2, IL-2). IL-2 induces proliferation of endogenous regulatory T lymphocytes (Tregs) on the one hand, and the resulting immunomodulatory effects inhibit local inflammation, and on the other hand, down-regulates cyclin CDK4/cyclin D1 via the SMAD3 signaling pathway, prevents proliferation of PASMCs, inhibits or even reverses pulmonary artery remodeling. Based on the above, the invention provides the combined delivery of the vasodilator TRE and the immunomodulator IL-2, and the combined treatment of pulmonary arterial hypertension is realized by simultaneously treating both symptoms and root causes under the two ways of dilating pulmonary artery blood vessels and inhibiting PASMCs proliferation.
Because PAH is a chronic heart and lung disease, the long-acting sustained-release preparation can prolong the effective blood concentration of the medicine, reduce the administration times and the dosage of patients, greatly improve the compliance of the patients and the treatment effect of the medicine, and is the first choice of PAH treatment. Many attempts have been made in the industry to address the problems of poor stability of IL-2 biomacromolecule drugs and short TRE half-life. There are a number of formulations for TREs currently approved by the FDA:(oral solid preparation), ->(inhalable solution) and->(subcutaneous infusion). Clinical reports show that the oral solid preparation is taken three times a day, and long-term high-frequency taking of the medicine causes symptoms such as gastrointestinal bleeding and the like of patients; the long-term use of the aerosol inhalant brings throat irritation and pain to exacerbate lung inflammation; in addition, in the case of the optical fiber,the patient is required to carry the hypodermic pump with him, which brings great inconvenience to the patient's life. There are studies on the preparation of IL-2 into bionic nano-loaded particle vaccine, fusion protein dimer liquid preparation and the like, and long-acting slow release of IL-2 cannot be realized. At present, no co-carrier preparation can meet the long-acting stable delivery of IL-2 and TRE.
Therefore, the invention constructs a long-acting co-carried hydrogel, adopts phospholipid complex to encapsulate IL-2 to improve the stability in vivo and prolong the half life of the drug; the IL-2 phospholipid complex and TRE are co-carried in the in-situ temperature sensitive hydrogel, and a reservoir is formed by intramuscular injection, so that the PAH is treated by slow release of the drug. The hydrogel co-carrier system is beneficial to realizing the release rate requirement of the double medicaments, and the small-molecule TRE is released in advance through a gel water channel and enters into a blood vessel to quickly relax the blood vessel, so that the symptoms are relieved; the macromolecular IL-2 slowly releases, and long-acting inhibits PASMCs proliferation and focal inflammation through lymphatic transport, thus fundamentally reversing pulmonary vascular remodeling.
Disclosure of Invention
One of the purposes of the invention is to construct a long-acting IL-2/TRE co-loaded hydrogel which is easy to prepare. The phospholipid complex is prepared by using phospholipid-entrapped biomacromolecules with high safety and good biocompatibility, and the phospholipid complex and the small-molecule drugs are co-loaded in the temperature-sensitive in-situ hydrogel to prepare the long-acting co-loaded hydrogel, so that the effective co-delivery of the small-molecule drugs and the biomacromolecule drugs is realized.
The second purpose of the invention is to provide the application of the long-acting co-carried hydrogel in-vitro release and in-vivo pharmacokinetic slow release.
The third purpose of the invention is to verify the long-acting application of the long-acting co-carried hydrogel in the rat model induced by the monocrotaline to the pulmonary arterial hypertension.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the invention provides a long-acting co-carried hydrogel for treating pulmonary arterial hypertension, which is characterized in that: the biological macromolecular drug phospholipid complex and the micromolecular drug are co-carried in the in-situ temperature-sensitive hydrogel.
The long-acting co-carried hydrogel is characterized in that: the small molecule drug is a pulmonary vasodilation drug and mainly comprises treprostinil sodium, treprostinil, epoprostenol, iloprost, beraprost, sildenafil, vardenafil, tadalafil, revamping, bosentan, ambrisentan, macitentan and the like. The biological macromolecule medicine comprises cytokines such as IL-2, IL-3, IFN-alpha, IL-10, IL-11, IL-12, IL-15, IFN-alpha, IFN-beta, TNF-alpha and the like, polypeptide medicines such as enkephalin, thymus peptide, pancreas polypeptide and the like, protein hormone such as auxin, thyrotropin, insulin, relaxin and the like, plasma proteins such as albumin, coagulation factors, antithrombin, plasminogen, globulin and the like. The phospholipid is a generic name of phosphate-containing lipid substances, and mainly comprises lecithin (PC), cephalin, inositol phospholipid, phosphatidic acid and the like.
The preparation method of the long-acting co-carried hydrogel is characterized by comprising the following steps of:
the molar ratio of the small molecular drug TRE to the biological macromolecular drug IL-2 is 8000:1-500:1; the mass ratio of the biological macromolecular drug IL-2 to the phospholipid is 1:200-1:2000; the concentration (w: w) of the in-situ hydrogel poloxamer P407 is 15% -20%;
1) Dissolving IL-2 in PBS with pH of 7.4 to obtain IL-2 solution;
2) Adding the IL-2 solution and the phospholipid into 3mL of organic reagent, and stirring and dissolving at room temperature;
3) Removing residual reaction reagent by adopting a solvent evaporation method and rotary evaporation at 45 ℃ for 1h;
4) Adding 4mL of PBS 7.4 solution to dissolve in the steps, and performing rotary evaporation at room temperature under reduced pressure for 40min;
5) Ultrasonic treatment is carried out on the reaction solution for 10min under the ice bath condition, and IL-2 phospholipid complex IL-2plex can be obtained;
6) In the stirring reaction condition, poloxamer P407 and 2mg of small molecular medicine treprostinil sodium are added into the IL-2plex solution, and the mixture is stirred for 12 hours in an ice bath, so that the IL-2 phospholipid complex/TRE in-situ hydrogel is obtained.
As a preferred mode: the preparation method of the IL-2 phospholipid complex/TRE in-situ hydrogel is characterized by comprising the following steps of:
1) TRE and IL-2 in the molar ratio of 2000:1;
2) The reaction solvent in the step 2) comprises organic reagents such as methanol, chloroform, tetrahydrofuran, diethyl ether and the like, and diethyl ether is most preferred as the reaction solvent;
3) The stirring and dissolving reaction time in the step 2) is 0.25-2h, and most preferably 1h is the stirring reaction time;
4) The ultrasonic power of the probe in the step 5) is 80-200W, and the most preferable 150W is the ultrasonic reaction power.
5) The poloxamer P407 concentration (w: w) in step 6) is 15-20%, most preferably 17% is poloxamer P407 concentration.
Wherein under optimal conditions, the entrapment rate of the IL-2 phospholipid complex is 76.36 +/-2.65%; the particle size of the IL-2 phospholipid complex is 77.8+/-0.3 nm; the potential of the IL-2 phospholipid complex is 8.31+/-4.03 mv; the phase transition temperature of the long-acting co-carried hydrogel is 30 ℃; the phase change time of the long-acting co-carried hydrogel at 37 ℃ is 90s.
The specific technical scheme of the invention is as follows: the preparation method of the long-acting co-carried hydrogel for intramuscular injection comprises the following specific preparation steps (taking papain (Pap) phospholipid complex/TRE in-situ hydrogel and IL-2 phospholipid complex/TRE in-situ hydrogel as examples):
preparation of Pap phospholipid Complex/TRE in situ hydrogel (Pap is used as model drug in the prescription process screening process because the amount of IL-2 drug is small and Pap is 23kDa and the molecular weight of IL-2 is close to each other)
1) 1.5mg of Pap and 30mg of PC are added into 3mL of organic reagent, and stirred and dissolved at room temperature;
2) Removing residual reaction reagent by adopting a solvent evaporation method and rotary evaporation at 45 ℃ for 1h;
3) Adding 4mL of Phosphate Buffer Solution (PBS) with pH of 7.4 to dissolve, and performing rotary evaporation at room temperature under reduced pressure for 40min;
4) Ultrasonic treatment is carried out on the reaction solution for 10 minutes under the ice bath condition, so that the Pap phospholipid complex can be obtained;
5) In the stirring reaction condition, poloxamer P407 and 2mg TRE are added into the Pap phospholipid complex solution, and the mixture is stirred for 12 hours in an ice bath, so that the Pap phospholipid complex/TRE in-situ hydrogel is obtained.
The preparation method of the Pap phospholipid complex/TRE in-situ hydrogel is characterized by comprising the following steps of:
1) The reaction solvent in the step 1) comprises organic reagents such as methanol, chloroform, tetrahydrofuran, diethyl ether and the like, and preferably diethyl ether is used as the reaction solvent;
2) The stirring and dissolving reaction time in the step 1) is 0.25-2h, and preferably 1h is the stirring reaction time;
3) The ultrasonic power of the probe in the step 4) is 80-200W, and the preferable 150W is the ultrasonic reaction time.
4) The concentration of poloxamer P407 (w: w) in step 5) is 15-20%, preferably 17% is poloxamer P407.
Application of long-acting co-carried hydrogel in preparation of pulmonary arterial hypertension drugs
As a long-acting co-supported hydrogel, the following problems are faced:
1. the release problem, that is, whether the macromolecular drug and the small molecular drug can be released simultaneously, is that the co-carried hydrogel contains the macromolecular drug and the small molecular drug at the same time in fact, and may face the mutual influence of phospholipid, poloxamer, the macromolecular drug and the small molecular drug, for example, the macromolecular drug is difficult to release due to the influence, the small molecular drug is fast released, and the slow release effect is difficult to realize.
2. Synergistic problem: as a complex system, especially containing macromolecular drugs, the activity of the complex system is influenced by various factors, and whether the complex system can synergistically enhance the activity of the complex system with small-molecule drugs is a technical problem in the field.
3. Long-acting problem: although the liposome can prolong the half life of the medicine, whether the liposome can be released continuously for 15 days and stably maintain a certain concentration level is one of the problems in the field. Is required to coordinate each reasonable formula in the medicament to form the optimal formulation.
The key steps in the invention include: prescription and process screening (types of reaction reagents, screening of reaction time and screening of ultrasonic power) in the preparation process of the IL-2 phospholipid complex, screening of poloxamer P407 concentration in the preparation process of the long-acting co-carried hydrogel, and screening and synergistic effect of TRE: molar ratio of IL-2.
Further, the method comprises the following steps: the key point of the invention is that the formation of the biological macromolecular phospholipid complex is optimized through the screening of a prescription process, the in vivo stability of the biological macromolecular phospholipid complex is improved, and the in vivo half-life period of the biological macromolecular medicament is prolonged. The round two chromatographic results show that the phospholipid complex can maintain the conformational stability of IL-2 to a large extent, and is a material basis for exerting the drug effect. The release results of the long-acting co-carried hydrogel show that the TRE is completely released in the long-acting co-carried hydrogel for 24 hours, and the free TRE only needs to be released4 hours; IL-2 was released completely in the long-acting co-loaded hydrogel over 24 hours, whereas the release of the IL-2 phospholipid complex alone (IL-2 plex) took 8 hours and the release of free IL-2 was complete over 4 hours. The synergy experimental synergy index value of the two drugs in the long-acting co-carried hydrogel is 10.579, which shows that the two drugs are combined to show a synergistic effect, wherein when the molar ratio of TRE to IL-2 is 2000:1 (40 mu M to 20 nM), the long-acting co-carried hydrogel has the highest inhibition rate on PASMCs proliferation. The pharmacokinetic results of the long-acting co-carried hydrogel in the rat body show that TRE in the intramuscular injection long-acting co-carried hydrogel can be released continuously for 15 days, and the half life is obviously improved by 1.68 times compared with that of the intramuscular injection non-preparation form; drug concentration levels in lymph nodes showed that IL-2 in long-acting co-loaded hydrogels reached C on day 4 max Sustained release is achieved for 15 days, maintaining a relatively smooth concentration level in the lymphatic circulation.
The pharmacodynamic study of the Monocrotaline (MCT) induced rat model for resisting pulmonary hypertension shows that the long-acting co-carried hydrogel has remarkable antihypertensive effect compared with a free drug group, can effectively exert the synergistic effect of TRE and IL-2, and reduces pulmonary hypertension. Compared with the free drug group, the long-acting co-carried hydrogel group reduces the expression of inflammatory factor IL-6, plays an anti-inflammatory role, can continuously reduce the expression of actin alpha, effectively inhibits the proliferation of pulmonary artery smooth muscle cells and inhibits the reconstruction of pulmonary vessels.
Advantageous effects
The invention constructs a long-acting co-carried hydrogel system through prescription process screening, can be released in rats continuously for 15 days, prolongs the half life of the medicine, and exerts continuous treatment effect. The long-acting co-carried hydrogel has the advantages of safe materials, high biocompatibility, simple preparation process and the like, can realize slow release of two medicaments, and can meet the requirement of long-acting treatment of chronic diseases such as pulmonary hypertension and the like.
Drawings
FIG. 1 is a graph showing the effect of reagent species on Pap conformation during preparation of phospholipid complexes according to the present invention.
FIG. 2 is a graph showing the effect of reaction time on IL-2plex entrapment rate during preparation of phospholipid complexes according to the present invention.
FIG. 3 is a graph showing the effect of ultrasonic power on IL-2plex particle size during the preparation of phospholipid complexes of the present invention.
FIG. 4 is a graph showing the effect of ultrasonic power on IL-2plex potential during the preparation of phospholipid complexes of the present invention.
FIG. 5 is a graph of FITR results for Pap-plex in accordance with the present invention.
FIG. 6 is a DSC chart of Pap-plex of the present invention.
FIG. 7 is a graph of XRPD results for Pap-plex in accordance with the present invention.
FIG. 8 is a graph showing the particle size distribution of IL-2plex of the present invention.
FIG. 9 is a graph showing the effect of poloxamer P407 concentration on ITG phase transition temperature during the preparation of the long-acting co-loaded hydrogel of the present invention.
FIG. 10 is a graph showing the effect of poloxamer P407 concentration on ITG phase transition time at 37℃during the preparation of the long-acting co-loaded hydrogel of the present invention.
FIG. 11 is a graph showing the viscosity effect of poloxamer P407 concentration on ITG during the preparation of the long-acting co-loaded hydrogel of the present invention.
FIG. 12 is a graph of temperature-modulus results for long-acting co-supported hydrogel rheology in accordance with the present invention.
FIG. 13 is a graph of shear rate-viscosity results for the rheology of a long-acting co-supported hydrogel in accordance with the present invention.
FIG. 14 is a SEM image of the ITG of the present invention.
FIG. 15 is a graph showing the in vitro release results of the long-acting co-carrier hydrogels of the present invention; wherein A is an in vitro release result graph of TRE in the long-acting co-carried hydrogel; b is a graph of the in vitro release result of IL-2 in the long-acting co-carried hydrogel.
FIG. 16 is a graph showing the investigation of the synergistic effect of two drugs in a long-acting co-carried hydrogel, wherein A is the proliferation inhibition rate of drugs TRE/IL-2 on PASMCs in different ratios; b is a visual collaborative map; c is a perspective view.
FIG. 17 is a graph showing the pharmacokinetic profile of TRE in rats in the long-acting co-loaded hydrogels of the present invention.
FIG. 18 is a graph showing the results of concentration of IL-2 in lymph node drug in rat in the long-acting co-carrier hydrogel of the present invention.
FIG. 19 is a graph showing the average pulmonary artery pressure results of the long-acting co-loaded hydrogels of the present invention in animal models.
FIG. 20 is a graph showing the anti-inflammatory results of the long-acting co-loaded hydrogels of the present invention in animal models.
FIG. 21 is a graph showing the results of anti-vascular remodeling in animal models of the long-acting co-loaded hydrogels of the present invention.
Detailed Description
The raw materials, the pharmaceutical excipients or the reagents used in the invention are all sold in the market or can be prepared by the laboratory at self.
The invention adopts a circular dichroscope, a dynamic light scattering nano particle diameter meter, a Fourier transform infrared meter, a differential scanning calorimeter, an XRPD powder diffractometer, a rotary viscometer, a rotary rheometer, a scanning electron microscope and the like to characterize the long-acting co-carried hydrogel.
Healthy male Sprague Dawley rats were used.
The invention is further illustrated below with reference to specific examples.
PC: soybean lecithin
TRE: treprostinil sodium
IL-2: interleukin-2
IL-2plex: IL-2 phospholipid complexes
ITG: IL-2 phospholipid complex/TRE hydrogels
Pap: papain
Pap-plex: papain phospholipid complexes
PTG: papain phospholipid complex/TRE hydrogel
PBS: phosphate buffer (pH 7.4)
MCT: monocrotaline alkali
PAH: pulmonary hypertension
Example 1: screening of organic solvent in preparation process of phospholipid complex
Because of the high price of IL-2, papain (Pap) has similar molecular weight to IL-2, so that the Pap is used for replacing IL-2 to screen, and the obtained result has reference value.
The preparation process comprises the following steps:
1.5mg of Pap is weighed, 3mL of organic solvents such as diethyl ether, methanol, chloroform and tetrahydrofuran are respectively added under stirring, stirring is continued for 4 hours at room temperature, the organic reagent is removed by reduced pressure rotary evaporation, and then 3mL of PBS (pH 7.4) solution is used for dilution to 0.5mg/mL concentration to be measured. The sample solution is measured by a circular dichroscope, far-ultraviolet and near-ultraviolet spectra are recorded, and compared with a Pap control solution which is not treated by an organic solvent, the influence of the organic solvent on the Pap conformation is analyzed.
FIG. 1 is a graph showing the effect of the types of reactants on the conformation of Pap in the preparation of Pap-plex according to the present invention, and it is understood from FIG. 1 that tetrahydrofuran and chloroform may cause irreversible conformational changes or even denaturation of Pap to inactivate Pap, and that diethyl ether has a relatively minimal effect on the conformation of Pap, so diethyl ether is preferred as the reaction solvent.
Example 2: screening of reaction time during preparation of phospholipid Complex
The preparation process comprises the following steps:
40. Mu.g of IL-2 was weighed and dissolved in 40. Mu.L of PBS pH 7.4 to give an IL-2 solution. The IL-2 solution and 30mg of PC were dissolved in 3mL of diethyl ether, and the mixture was stirred at room temperature. The solvent evaporation method is adopted, the residual reaction reagent is removed by rotary evaporation reaction at 45 ℃, and the reaction time is respectively 0.25, 0.5, 1.0, 1.5 and 2.0h. 4mL of PBS 7.4 solution was added for dissolution, and the mixture was distilled under reduced pressure at room temperature for 40min. And (3) carrying out ultrasonic treatment on the reaction solution for 10min under the ice bath condition by using a probe 180W to obtain IL-2plex. The free IL-2 in the solution is removed by ultrafiltration centrifugation, the content of the free IL-2 is determined by an Elisa kit, and the effect of the reaction time on the encapsulation efficiency of the IL-2-plex is analyzed according to the formula (1).
Encapsulation Efficiency (EE)%＝(W Dosage of medicine －W Free IL-2 )/W Dosage of medicine ×100％(1)
Fig. 2 is a graph showing the effect of reaction time on the encapsulation efficiency of the phospholipid complex in the preparation process of the phospholipid complex according to the invention, and as can be seen from fig. 2, the encapsulation efficiency is 80% after 1h, and the reaction time does not continuously increase along with the extension of the reaction time, so that the process with the shortest time is selected on the premise of meeting the sufficiency, and the preferable reaction time is 1h.
Example 3: screening of ultrasonic power in preparation process of phospholipid complex
The preparation process comprises the following steps:
40. Mu.g of IL-2 was weighed and dissolved in 40. Mu.L of PBS pH 7.4 to give an IL-2 solution. The IL-2 solution and 30mg of PC were dissolved in 3mL of diethyl ether, and the mixture was stirred at room temperature. The residual reagent was removed by solvent evaporation at 45℃for 1h. 4mL of PBS 7.4 solution was added for dissolution, and the mixture was distilled under reduced pressure at room temperature for 40min. And (3) carrying out probe ultrasonic treatment on the reaction solution for 10min under the ice bath condition, wherein the ultrasonic power of the probe is respectively 80, 100, 120, 150, 180 and 200W, and the IL-2plex can be obtained. And detecting the prepared IL-2plex by adopting a dynamic light scattering nano particle size meter, and analyzing the influence of ultrasonic power on the particle size and potential of the IL-2plex.
FIG. 3 is a graph showing the effect of ultrasonic power on the particle size of IL-2plex in the preparation process of the phospholipid complex of the invention, and as shown in FIG. 3, the particle size of IL-2plex is in a decreasing trend along with the increase of ultrasonic power, 83.6+/-0.3 nm at 150W, and the particle size is increased along with the increase of ultrasonic power to 180 and 200W, preferably, the ultrasonic power is 150W. FIG. 4 is a graph showing the effect of ultrasonic power on the potential of the IL-2plex according to the invention, and it is clear from FIG. 4 that the effect of ultrasonic power on the particle size is small, and the IL-2plex prepared at 150W is about 8mV.
Example 4: preparation and validation of Pap-plex
The preparation process comprises the following steps:
1.5mg of Pap and 30mg of PC were weighed and dissolved in 3mL of diethyl ether, and the mixture was stirred at room temperature. The residual reagent was removed by solvent evaporation at 45℃for 1h. 4mL of PBS 7.4 solution was added for dissolution, and the mixture was distilled under reduced pressure at room temperature for 40min. And carrying out ultrasonic treatment on the reaction solution for 10min under ice bath conditions by a 150W probe to obtain the Pap-plex.
And detecting the prepared Pap-plex by adopting a Fourier transform infrared instrument, and verifying the generation of the phospholipid complex. By infrared scanning of Pap, PC, physical mixture of the two, pap-plex respectively by tabletting method, FIG. 5 is a FITR result diagram of Pap-plex in the invention, and it is known from the diagram that Pap and PC respectively have obvious characteristic absorption peaks, and the characteristic absorption peak (dotted line circle) of Pap is mainly 1390cm -1 The characteristic absorption peaks (solid circles) of PC are mainly 1734, 1378cm -1 The spectra of Pap-plex and physical mixtures are significantly different, the spectra of physical mixtures are shown as a superposition of PC and Pap, at 1390cm -1 There is still a characteristic absorption peak of Pap, and in the spectrum of the complex, this characteristic absorption peak is covered, and is mainly represented by the characteristic absorption of PC. No new absorption peaks appear in both the Pap-plex and physical mixtures, indicating no new chemical bonds are formed, demonstrating the formation of phospholipid complexes.
And detecting the prepared Pap-plex by using a differential scanning calorimeter, and verifying the generation of the phospholipid complex. Samples were taken and placed in a shallow crucible, temperature programmed, and DSC analysis was performed on Pap, PC, physical mixtures of both, and Pap-plex, respectively. The temperature is 30-200 ℃, and the temperature rising rate is 10 ℃/min. FIG. 6 is a DSC graph of a Pap-plex of the present invention featuring a rapidly decreasing endothermic peak at 30-80℃due to a slow unfolding process by heating the Pap to denature. And has a broad and weak exothermic peak in the range of 100-150 c due to Pap degradation. However, the heat absorption behavior of Pap-plex is closer to that of PC compared with that of Pap+PC physical mixture, the structure of Pap is protected in the heat absorption process, and the degradation peak is also disappeared, so that the structural stability of Pap-plex to Pap is shown. In addition, there was no new peak generated in the Pap-plex compared to the Pap and PC thermograms, suggesting that no new chemical bond was formed between Pap and PC in Pap-plex, demonstrating the formation of phospholipid complexes.
The prepared Pap-plex was detected by XRPD powder diffractometer and the formation of phospholipid complexes was verified. X-ray diffraction analysis was performed on Pap, PC, physical mixtures of both, and Pap-plex under detection conditions of 2 theta of 3 DEG to 40 DEG, 4 DEG/min, respectively. FIG. 7 is a graph showing the XRPD results for Pap-plex of the present invention, showing that Pap has a relatively sharp crystal diffraction peak in the range of 3-15, and PC has a smooth diffraction pattern without a crystal diffraction peak, comparing the diffraction patterns of Pap-plex and physical mixtures, and finding that the physical mixtures still have a crystal diffraction peak, and Pap-plex is similar to PC without a crystal diffraction peak, indicating that Pap exists in a molecular or amorphous state in Pap-plex, demonstrating the formation of phospholipid complexes.
Example 5: preparation of IL-2plex
The preparation process comprises the following steps:
40. Mu.g of IL-2 was weighed and dissolved in 40. Mu.L of PBS pH 7.4 to give an IL-2 solution. The IL-2 solution and 30mg of PC were dissolved in 3mL of diethyl ether, and the mixture was stirred at room temperature. The residual reagent was removed by solvent evaporation at 45℃for 1h. 4mL of PBS 7.4 solution was added for dissolution, and the mixture was distilled under reduced pressure at room temperature for 40min. And (3) carrying out ultrasonic treatment on the reaction solution for 10min under the ice bath condition by a 150W probe to obtain IL-2plex.
And detecting the prepared IL-2 by adopting a dynamic light scattering nano particle size meter. FIG. 8 is a graph showing the particle size distribution of IL-2plex of the present invention, wherein the particle size of IL-2plex obtained in example 5 was 77.8.+ -. 0.3nm, and the potential was 8.31.+ -. 4.03mv.
Example 6: screening of poloxamer P407 concentration in preparation process of long-acting co-carried gel
40. Mu.g of IL-2 was weighed and dissolved in 40. Mu.L of PBS pH 7.4 to give an IL-2 solution. The IL-2 solution and 30mg of PC were dissolved in 3mL of diethyl ether, and the mixture was stirred at room temperature. The residual reagent was removed by solvent evaporation at 45℃for 1h. 4mL of PBS 7.4 solution was added for dissolution, and the mixture was distilled under reduced pressure at room temperature for 40min. And (3) carrying out ultrasonic treatment on the reaction solution for 10min under the ice bath condition by a 150W probe to obtain IL-2plex. In the stirring reaction condition, poloxamer P407 (the concentration of poloxamer P407 is considered to be 15%, 16%, 17%, 18%, 19% and 20%, corresponding to 0.60g, 0.64g, 0.68g, 0.72g, 0.76g and 0.80g respectively) and 2mg TRE are added into IL-2plex solution respectively, and the mixture is stirred for 12 hours in an ice bath to obtain IL-2 phospholipid complex/TRE in situ hydrogel (ITG).
The ITG phase transition temperature was measured by tube inversion, 4mL of gel solution stored at 4℃was placed in a clean dry 10mL sealed EP tube, and a thermometer was inserted into the gel solution. The EP tube was placed in a thermostatic water bath for 10min of equilibration, the liquid level in the EP tube being lower than the water bath level. The initial temperature of the water bath kettle is set to be 20 ℃, the temperature is continuously and slowly increased, and the temperature increasing rate is 1 ℃/3min. When the temperature is increased by 1 ℃, the sealed EP pipe is quickly turned over, and the gel flow condition is observed. When no further gel flow in the EP tube was observed and maintained for more than 30s, the reading from the thermometer at this time was recorded as the gelation temperature. FIG. 9 is a graph showing the effect of poloxamer P407 concentration on the phase transition temperature of the hydrogel during the ITG preparation process of the present invention, wherein the hydrogel having poloxamer P407 concentration of 15% cannot be coagulated, and the gelation temperature of the hydrogel decreases with increasing poloxamer P407 concentration in the range of 16% -20%. In view of the fact that the intramuscular injection in-situ hydrogel needs to meet the condition that the hydrogel is liquid at room temperature of 25 ℃ and is gel at human body temperature of 37 ℃, poloxamer P407 concentration is 16% and 17%.
The gel time of ITG at 37 ℃ is measured by adopting a test tube inversion method, 4mL of gel solution stored at 4 ℃ is taken and placed in a clean and dry 10mL sealed EP tube, the EP tube is placed in a constant temperature water bath at 37 ℃, the liquid level in the EP tube is required to be lower than the liquid level of the water bath, and timing is started. The EP tube was turned over rapidly every 10s and the gel flow was observed. When no further gel flow was observed in the EP tube, the time at this point was noted as the gel time at 37 ℃. FIG. 10 is a graph showing the effect of poloxamer P407 concentration on the phase transition time of the hydrogel at 37℃during the ITG preparation according to the present invention, wherein the gel time of the hydrogel at 37℃is 210s for poloxamer P407 concentration of 16% and 90s for 17% for hydrogel at 37℃and preferably 17% for poloxamer P407 concentration, since the hydrogel is formed in vivo as soon as possible during intramuscular injection.
Measuring apparent viscosity values of ITG under different temperature conditions by adopting a rotary viscometer, taking a proper amount of gel solution into a beaker, respectively placing the beaker into a constant-temperature water bath for balancing for 10min, setting the initial temperature of a water bath kettle to be 27 ℃, and continuously and slowly heating the water bath kettle at a heating rate of 1 ℃/3min. The apparent viscosity of the hydrogels was measured for each 1℃increase in temperature. A No. 5 rotor is selected, the rotating speed is set to be 30rpm, and the liquid level in the beaker is required to submerge scale marks on the rotor. 3 samples were taken for each prescription and the results averaged. FIG. 11 is a graph showing the effect of poloxamer P407 concentration on PTG viscosity during the ITG preparation process according to the present invention, wherein the viscosity gradually increases with increasing temperature, and gel state starts to appear when the viscosity suddenly changes. The hydrogel with poloxamer P407 concentration of 17% has viscosity transition at 30 ℃, the gelation temperature is 30 ℃, the result is consistent with the figure 9, and the solution viscosity is 186.25 +/-3.4 mPa.s at 27 ℃, so that the hydrogel has higher needle penetrating property and is easy to inject; the gel viscosity is up to 11486.3 +/-110.2 mPa.s at 37 ℃, the formability is high, and the gel is not easy to erode.
Example 7: preparation and characterization of long-acting co-carried gel
40. Mu.g of IL-2 was weighed and dissolved in 40. Mu.L of PBS pH 7.4 to give an IL-2 solution. The IL-2 solution and 30mg of PC were dissolved in 3mL of diethyl ether, and the mixture was stirred at room temperature. The residual reagent was removed by solvent evaporation at 45℃for 1h. 4mL of PBS 7.4 solution was added for dissolution, and the mixture was distilled under reduced pressure at room temperature for 40min. And (3) carrying out ultrasonic treatment on the reaction solution for 10min under the ice bath condition by a 150W probe to obtain IL-2plex. Under stirring reaction conditions, 0.68g of poloxamer P407 and 2mg of TRE were added to the IL-2plex solution, respectively, and stirred in an ice bath for 12 hours to obtain ITG.
The ITG rheological properties were determined using a rotational rheometer: a modulus versus temperature profile and a viscosity versus shear rate profile. A flat plate with the diameter of 40mm is used as a rotor, the plate spacing is 0.5mm, the modulus detection condition is adopted, the temperature is 25-45 ℃, the shearing stress is 0.5Pa, the heating rate is 2 ℃/min, and the frequency is 1Hz; the viscosity detection condition is that the temperature is 25 ℃ and the shear rate is 30-3000s -1 . FIG. 12 is a graph of the results of the ITG rheology temperature-modulus in accordance with the present invention. The temperature-modulus data shows hydrogel temperature sensitivity. ITG undergoes a phase transition at 30 ℃, and the elastic modulus (G ') and the viscous modulus (G ") rise rapidly at the phase transition temperature, and G' =g" =30 ℃ is the critical gelation temperature, and the results are consistent with fig. 9 and 11. FIG. 13 is a graph of ITG rheology shear rate versus viscosity results in accordance with the present invention. The shear rate-viscosity data shows the shear thinning behavior of ITG as a non-newtonian fluid. As the shear rate increases, the shear viscosity decreases, mainly due to the velocity gradient between the layers of the non-newtonian fluid as it flows. Both verify the formation of hydrogels.
And (3) evaluating the surface condition of the freeze-dried ITG by adopting a scanning electron microscope, cutting the sheet, fixing the sheet on a conductive copper plate, performing metal spraying treatment on the sheet by adopting an ion sputtering instrument, and observing and collecting images by adopting the scanning electron microscope. FIG. 14 is a SEM image of the ITG of the present invention. As can be seen from the figure, there are spherical particles, which have a particle size of 80nm as measured by particle size, and these particles are round in appearance and have a particle size conforming to the particle size of IL-2plex, thus it is inferred that they are IL-2plex of composite gel. IL-2plex is distributed in the gel solution and forms a hydrogel skeleton together with poloxamer P407, so that protruding particles can be seen on the surface of the hydrogel hole compounded with the IL-2plex.
Example 8: in-vitro slow release behavior investigation of long-acting co-carried hydrogel
In order to verify that the long-acting co-carried hydrogel has a slow release effect, the drug release behavior of the preparation in vitro is examined.
The in vitro release of IL-2 and TRE was determined by the following steps: 1mL of ITG prepared in example 7, 1mL of IL-2plex and free TRE/IL-2 were transferred to a 25kDa dialysis bag, and the two ends were tied with a cotton thread, and the excess cotton thread and the dialysis bag were cut off. The dialysis bags were immersed in PBS buffer, pH values were 6.4, 7.2, 7.4, respectively, and examined for in vitro release in a shaking table at 37℃with a shaking table rotation speed of 50rpm. Taking 1mL of sample solution from the release medium at 1, 2, 4, 8, 12, 24, 48, 72 and 96 hours respectively, supplementing 1mL of PBS solution, measuring the IL-2 content in the release medium by using an ELISA kit, measuring the TRE content in the release medium by using a high performance liquid chromatography, and calculating the cumulative release rate.
FIG. 15 is a graph showing the in vitro release results of the long-acting co-carrier hydrogels of the present invention: wherein (a) is a graph of the in vitro release results of the TRE in the long-acting co-loaded hydrogel, and the results show that the TRE in the long-acting co-loaded hydrogel takes 24 hours to release completely, while the free TRE drug takes only 4 hours. (B) In order to show the in vitro release results of IL-2 in the long-acting co-carried hydrogel, the IL-2 in the long-acting co-carried hydrogel needs to be released completely in 24 hours, while IL-2plex needs to be released completely in 8 hours, and free IL-2 needs to be released completely in only 4 hours.
Example 9: investigation of the synergistic action of TRE and IL-2
Application example 7 Long acting Co-carried gel examined the in vitro synergistic therapeutic effect of TRE and IL-2 in this system.
(1) Tregs cell extraction: the peripheral blood of the rat is taken to obtain mononuclear cells through Ficoll lymphocyte separation liquid. The anti-FITC interlabel magnetic beads and the CD4 rate-FITC streaming antibody are used for separating the magnetic beads to obtain the rat CD4 + T cells of (a); adding a dissociation reagent to dissociate the anti-FITC inter-label magnetic beads, selecting a column, and stopping the reaction by a stopping reagent; CD4 was isolated by anti-APC inter-label beads+CD25rate-APC flow antibody + CD25 + Is noted to keep the environment closed and sterile throughout. Taking a small amount of sample, and detecting CD4 by using a flow cytometer + CD25 + FoxP3 + Proportion of Tregs cells, if CD4 + CD25 + FoxP3 + The next step of activating and amplifying experiment is carried out when the proportion of the Tregs cells is more than 60%.
(2) In vitro activation and expansion of Tregs cells: t cell activation medium configuration method: texMACs serum-freeThe medium was supplemented with final concentration of 10% FBS, 25. Mu.L/mL CD3/CD 28T cell activator. T cell induction medium configuration method: texMACs serum-free medium was supplemented with final concentration of 10% FBS, 200IU/mL IL-2. Enrichment of Tregs cells by centrifugation and 5X 10 cells in T cell activation medium 3 The culture was resuspended and incubated. On day 3 of culture, the harvested cells were cultured with T cell induction medium by centrifugation. On day 5 of culture, cells were collected by centrifugation to replace T cell induction medium. 7 days is an in-vitro activation and expansion period, and after the period is finished, the Tregs cells are expanded by 10 times, and the requirement of a subsequent co-incubation experiment is met only by performing activation and expansion for the second time. Cells were collected after 15 days of culture for Tregs cell purity determination and cell count, if CD4 + CD25 + FoxP3 + The ratio of Tregs cells is greater than 80%, and the number of cells is greater than 1×10 5 The next co-incubation experiment was performed per mL.
(3) PASMCs cells were co-incubated with Tregs cells: rat PAH smooth muscle cells (PASMCs) with good growth state are digested and centrifuged by pancreatin, counted by a cell counter and resuspended by RPMI 1640 culture medium, inoculated into a 96-well plate with a cell density of 5000 cells/well, placed into a cell incubator for culturing for 24 hours, and the serum-free RPMI 1640 culture medium is replaced for culturing for 12 hours when the cell attachment rate reaches 60%. The rat Tregs cells in good growth state were counted by a cell counter and resuspended in RPMI 1640 medium (containing 10% fbs, 2 μg/mL double antibody) and seeded into 96-well plates at a cell density of 3000 cells/well and cultured in a cell incubator for 24h.
(4) MTT assay to determine proliferation inhibition of cells: 200 mu L of long-acting co-carried gel leaching solutions (10 times diluted RPMI 1640 medium) with different TRE/IL-2 concentration ratios are respectively added into each hole, and 3 repeated holes, 6 control holes and 6 positive control holes of each sample to be tested are respectively added. After 48h incubation, the broth and non-adherent Tregs cells were aspirated and washed with PBS. mu.L of MTT (5 mg/mL) and RPMI 1640 medium were added to each well and the culture was continued for 4 hours. The supernatant was aspirated, 150. Mu.L of DMSO was added to each well to dissolve the crystallized particles, absorbance (D) was measured at 570nm using a microplate reader, and the proliferation inhibition rates of PASMCs by the long-acting co-loaded gels at different TRE and IL-2 ratios were calculated.
Based on the cell proliferation inhibition, the potential synergy of the combination drug TRE with IL-2 was assessed by comparing the change in potency using the synergy Finder 3.0 software, zero Interaction Potency (ZIP) model. The efficacy of the combination treatment was assessed using a Synergy Score (SC), which can be interpreted as the average over-response due to drug interactions (i.e., a Synergy score of 10 corresponds to a response that is 10% beyond expectations). When the SC value is < -10, the combination of the two medicaments shows antagonism; when the SC value is less than 10 and less than 10, the combination of the two medicaments shows an additive effect; when the SC value is > 10, the combination of the two medicines shows a synergistic effect.
Fig. 16 is a view of the synergistic effect of two drugs in the long-acting co-carried hydrogel, wherein a graph A shows the proliferation inhibition rate of PASMCs by different drug ratios, and a graph B shows a visual synergistic map. The results show that in the co-incubation experiment of PASMCs and Tregs, the long-acting co-carried hydrogel can successfully deliver two drugs of TRE and IL-2, and the SC value of the two drugs is 10.579 in combination, so that the synergistic effect is shown. Of the ratios studied, the proliferation inhibition rate was highest, up to 89.76%, at a TRE/IL-2 ratio of 2000:1 (60. Mu.M: 20 nM).
Example 10: long-acting co-carried hydrogel pharmacokinetics investigation
In order to verify that the long-acting co-carried hydrogel has a slow release effect, the drug release behavior of the preparation in vitro is examined.
The sustained release effect of the preparation TRE in the rat body was examined by using the long-acting co-carried hydrogel prepared in example 7. 10 healthy male SD rats (200-250 g) were randomly divided into two groups of 5. The long-acting co-carried hydrogel preparation (group a) was injected into the left forearm of the rat by 0.2mL of each of the two, and the free TRE solution (group B) was injected into the left forearm of the rat by 0.2mL of each of the two, and after administration, 0.02, 0.4, 0.08, 0.17, 0.25, 0.33, 0.5, 1, 2, 3, 5, 7, 10, and 15 days of orbital bleeding was performed by 0.5mL, and the supernatant was collected by centrifugation at 5,000g×5min to obtain plasma samples. 100. Mu.L of plasma sample is taken, 20. Mu.L of acetic acid and 300. Mu.L of methanol are added, the mixture is centrifuged for 2min after vortex, the centrifugal strength is 15,000rpm multiplied by 10min, the supernatant is taken as a sample to be detected, and TRE is quantitatively detected by a triple four-level rod-liquid chromatography-mass spectrometry system.
The pharmacokinetic curve of the TRE in the rat body in the long-acting co-carried hydrogel is shown in figure 17, the pharmacokinetic parameters are shown in table 1, the TRE in the intramuscular injection long-acting co-carried hydrogel can be released continuously for 15 days, the half life is obviously improved compared with the non-preparation form (free TRE solution) of intramuscular injection, the half life is 5.11 times of that of the non-preparation form (free TRE solution), the blood concentration is smaller, the medication safety of the TRE is improved, and the side effects of the organism are avoided.
The sustained release effect of the preparation IL-2 and free IL-2 solution in rats was examined by using the long-acting co-carried hydrogel prepared in example 7 and the IL-2plex prepared in example 5. 99 healthy male SD rats (200-250 g) were randomly divided into 3 groups of 33. The rats were intramuscular injected with 0.2mL of the long-acting co-loaded hydrogel formulation (group A), 0.2mL of IL-2plex (group B), and 0.2mL of the free IL-2 solution (group C) on day 0, respectively. Rats were dissected 0.04, 0.08, 0.17, 0.33, 0.5, 1, 2, 4, 7, 10, 15 days after dosing (n=3 at each time point), axillary lymph nodes were weighed, 100mg were prepared after tissue grinding, and IL-2 was detected with ELISA kit. The distribution of IL-2 in the long-acting co-loaded hydrogel in lymph nodes in rats is shown in FIG. 18, and the result shows that the IL-2 in the long-acting co-loaded hydrogel reaches C on day 4 max Sustained release for 15 days, the half-life period is obviously improved compared with that of intramuscular injection non-preparation form (free IL-2 solution) and is 2.38 times of that of the latter, and a stable concentration level is maintained in lymphatic circulation, so that the sustained release effect is shown.
TABLE 1 Long-acting Co-carried hydrogel pharmacokinetic parameters in rats
All data are expressed in mean±sd, n=5. Comparison to the free TRE group: ns indicates no significant difference, P <0.05, P <0.01, P <0.001.
TABLE 2 Long-acting Co-carried hydrogel pharmacokinetic parameters of lymph in rat
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All data are expressed in mean±sd, n=5. Comparison with the free IL-2 group: ns indicates no significant difference, P <0.05, P <0.01.
Example 11: therapeutic effects of long-acting co-loaded hydrogels in vivo in animal models
MCT 378mg
ITG 12mL
The long-acting co-carried hydrogel prepared in example 7 was used to examine the long-acting therapeutic effect of the formulation in MCT-induced pulmonary artery high pressure model rats.
MCT-induced pulmonary arterial hypertension rat model construction: MCT is dissolved in dimethyl sulfoxide solvent, after the MCT is fully dissolved, 2% MCT solution (dosage is 60 mg/kg) is subcutaneously injected into the neck and the back of a rat, and after molding, the subsequent experiment is carried out for four weeks. The model rats were randomly grouped and co-divided into: the number of rats in each group is 6, and the number of normal saline group, free TRE group, free IL-2 group, physical mixture group, ITG preparation group and ITG high dose group are six. Intramuscular administration was performed once for seven days, 3 times in total, depending on the group. The mean pulmonary artery pressure and right heart hypertrophy index were measured after the end of the dosing cycle.
The average pulmonary artery pressure is measured by a catheter method to evaluate the efficacy of the preparation in treating pulmonary artery hypertension. After the end of the dosing cycle, rats were anesthetized and fixed on a rat fixation plate. The trachea cannula is connected into a breathing machine for fixation. Left chest skin preparation, finding the strongest heart beat after skin preparation, separating and ligating surface ribs, avoiding cutting intercostal arteries. After the pericardium is carefully cut, the main trunk of the pulmonary artery is found, and then the main trunk of the pulmonary artery is penetrated by a puncture needle at the conical part of the pulmonary artery and is quickly connected with an electrocardiograph monitor. After stable mean pulmonary artery pressure waveforms and values were observed on the electrocardiograph monitor, the data were recorded. FIG. 19 is a graph showing the in vivo pulmonary hypertension resistance results of the long-acting co-loaded hydrogel in animal models, wherein the free TRE group, the free IL-2 group and the physical mixture group have a certain effect on reducing the average pulmonary hypertension, and compared with the physical mixture group, the ITG group has a more remarkable depressurization effect, so that the ITG can effectively play the synergic effect of the TRE and the IL-2 and reduce the pulmonary hypertension.
The anti-inflammatory effect of the preparation is evaluated by measuring the expression of inflammatory factor IL-6 in lung tissues of mice of the administration model by ELISA kit. After the end of the administration period, the model rats were sacrificed, the rat lung tissue was isolated, and the lung tissue was homogenized by adding an appropriate amount of physiological saline. Centrifuging for 1,000Xg/10 min, and collecting supernatant. Inflammatory factor expression was detected using the IL-6ELISA kit according to the instructions. FIG. 20 is a graph showing the anti-inflammatory results of ITG in animal models according to the present invention, showing that ITG group significantly reduced IL-6 expression and exerted anti-inflammatory effects compared to the physical mixture group.
The anti-pulmonary artery reconstruction effect of the preparation is evaluated by adopting an immunostaining method. As a pulmonary artery vessel-specific marker, increased expression of actin alpha indicates proliferation of pulmonary artery smooth muscle cells and remodeling of pulmonary vessels. FIG. 21 is a graph showing the results of anti-vascular remodeling of ITG in animal models according to the present invention, wherein compared with physiological saline, the physical mixture of free TRE group and free IL-2 group has no significant effect on actin alpha expression, while ITG significantly reduces actin alpha expression, indicating that ITG can effectively inhibit proliferation of pulmonary artery smooth muscle cells, and inhibit pulmonary vascular remodeling.

Claims (8)

1. A long-acting co-carried hydrogel is characterized in that a biological macromolecular drug phospholipid compound and a micromolecular chemical drug are co-carried in an in-situ temperature-sensitive hydrogel, and an in-situ hydrogel skeleton is poloxamer.
2. The long-acting co-carrier hydrogel of claim 1, wherein the small molecule drug is a pulmonary vasodilating drug comprising treprostinil sodium, treprostinil, epoprostenol, iloprost, beraprost, sildenafil, vardenafil, tadalafil, revampat, bosentan, ambrisentan, or macitentan.
3. The long-acting co-loaded hydrogel of claim 1, wherein the biopolymer drug comprises plasma proteins such as IL-2, IL-3, IFN- α, IL-10, IL-11, IL-12, IL-15, IFN- α, IFN- β, TNF- α, enkephalin, thymus peptide, pancreas, auxin, thyrotropin, insulin, relaxin, albumin, clotting factors, antithrombin, plasminogen or globulin.
4. The long-acting co-supported hydrogel of claim 1, wherein the phospholipid is a generic term for phosphate-containing lipid materials comprising lecithin, cephalin, inositol phospholipid, and phosphatidic acid.
5. The method for preparing the long-acting co-supported hydrogel according to any one of claims 1 to 4, wherein:
the molar ratio of the small molecular medicine to the biological macromolecular medicine is 8000:1-500:1; the mass ratio of the biological macromolecular drug to the phospholipid is 1:200 to 1:2000; the mass percentage concentration of the poloxamer P407 in the in-situ hydrogel skeleton is 15% -20%;
1) Dissolving a biological macromolecular drug in phosphate buffer with the pH value of 7.4 to obtain a biological macromolecular drug solution;
2) Adding the biological macromolecule medicine solution and phospholipid into an organic reagent, and stirring and dissolving at room temperature;
3) Removing residual reaction reagent by rotary evaporation at 45 ℃ by adopting a solvent evaporation method;
4) Adding 4mL of PBS 7.4 solution to dissolve in the steps, and performing rotary evaporation at room temperature under reduced pressure for 40min;
5) Ultrasonic treatment is carried out on the reaction solution for 10min under the ice bath condition, so as to obtain the biological macromolecule medicine phospholipid complex; the ultrasonic power of the probe is 80-200W;
6) In the stirring reaction condition, poloxamer P407 and small molecular medicine are added into the biological macromolecular medicine phospholipid complex solution, and the long-acting co-carried hydrogel is obtained by ice bath stirring.
6. The method for preparing the long-acting co-supported hydrogel according to claim 5, wherein the method comprises the following steps:
the molar ratio of the small molecular medicine treprostinil sodium to the biological macromolecular medicine IL-2 is 8000:1-500:1; the mass ratio of the biological macromolecular drug IL-2 to the lecithin is 1:200 to 1:2000; the mass percentage concentration of the poloxamer P407 in the in-situ hydrogel skeleton is 15% -20%;
1) Dissolving IL-2 in phosphate buffer solution with pH of 7.4 to obtain IL-2 medicine solution;
2) Adding IL-2 solution and phospholipid into 3mL of organic reagent, stirring at room temperature for dissolution, wherein the organic solvent comprises methanol, chloroform, tetrahydrofuran and diethyl ether; the stirring dissolution reaction time is 0.25-2h
3) Removing residual reaction reagent by adopting a solvent evaporation method and rotary evaporation at 45 ℃ for 1h;
4) Adding 4mL of PBS 7.4 solution to dissolve in the steps, and performing rotary evaporation at room temperature under reduced pressure for 40min;
5) Ultrasonic treatment is carried out on the reaction solution for 10min under the ice bath condition, so that IL-2 phospholipid complex IL=2plex can be obtained;
6) Adding poloxamer P407 and 2mg small molecule medicine treprostinil sodium into the IL-2 phospholipid complex solution in stirring reaction conditions, and stirring in an ice bath for 12 hours to obtain IL-2 phospholipid complex/TRE in-situ hydrogel; the mass percentage concentration of the poloxamer P407 is 15% -20%.
7. The method for preparing the long-acting co-supported hydrogel according to claim 6, wherein:
1) Treprostinil sodium to IL-2 molar ratio is 2000:1;
2) The organic solvent in the step 2) is diethyl ether as a reaction solvent;
3) The stirring dissolution reaction time in the step 2) is 1h;
4) The ultrasonic power of the probe in the step 5) is 150W;
5) The poloxamer P407 mass percentage concentration in the step 6) is 17%.
8. The use of a long-acting co-carried hydrogel according to any one of claims 1-4 in the preparation of a pulmonary arterial hypertension drug.
CN202311494988.7A 2023-11-10 2023-11-10 Long-acting co-carried hydrogel preparation for pulmonary arterial hypertension treatment, preparation method and application Pending CN117503690A (en)

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