A delivery carrier for targeted pituitary drug delivery and its preparation method and application

Abstract

The present invention discloses a delivery carrier for targeted pituitary drug delivery and its preparation method and application. The present invention designs a carrier that can target the pituitary for drug delivery, and treats patients with spinocerebellar disorders, hypothalamic hypothyroidism, congenital single thyrotropin-releasing hormone (TRH) deficiency, non-alcoholic fatty liver disease (NAFLD), etc. The present invention modifies TRH (TRH-PEG-LP) on the surface of a PEGylated lipid carrier (PEG-LP), injects the TRH-PEG-LP carrier loaded with a near-infrared fluorescent dye (DiR) into the mouse body through the tail vein for 2 hours, takes the pituitary tissue, and observes the color development of the fluorescent dye in the pituitary of the mouse under a small animal imager (Ex / Em: 748 / 780nm), and finds that the carrier modified by TRH can target pituitary tissue well. TRH modification on the surface of the PEGylated lipid carrier can play a role in targeted delivery, and TRH can bind to the TRH receptor specifically expressed on the surface of the pituitary cells, thereby realizing targeted drug delivery of the pituitary.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery, and in particular relates to a delivery carrier for targeted pituitary drug delivery, and a preparation method and application thereof. Background Art

[0002] The pituitary gland is located on the ventral side of the hypothalamus, in the middle cranial fossa and the pituitary fossa above the sphenoid body, and is covered with a tough dura mater. It is difficult for systemic administration to accumulate in the pituitary tissue to reach an effective concentration. The treatment of pituitary diseases mainly relies on surgical treatment and drug therapy. Due to the rich blood vessels and nerves around the pituitary gland, the surgical risk is high, and there are risks of infection and cerebrospinal fluid leakage after surgery, and the prognosis is poor. During drug treatment, the drug is non-specifically distributed in the body, and high-dose systemic administration is required to ensure the drug concentration in the pituitary tissue and ensure the therapeutic effect of pituitary diseases. However, the accumulation of drugs in healthy tissues can cause a series of adverse reactions.

[0003] In order to improve the therapeutic efficacy of drugs in pituitary tissue and reduce the distribution and side effects in healthy tissues, a possible solution is stereotaxic injection of drugs into the pituitary gland. However, there is a high risk of damaging brain nerves and blood vessels during stereotaxic injection of the pituitary gland. Therefore, how to safely and effectively increase the accumulation of drugs in pituitary tissue and reduce toxic side effects on healthy organs without invasiveness is the key to treating pituitary and related diseases.

[0004] Although there have been studies on pituitary-targeted drug delivery, with carriers involving liposomes, nanoparticles, and engineered recombinant proteins, most of the experiments were conducted using tumor-bearing mice or in vitro cells, and the accumulation of carriers in pituitary tissue has not been truly observed. Currently, only one literature has been retrieved, which uses an in situ gel to administer the drug through the nasal cavity to treat pituitary adenomas, but the tissues observed are biased towards the brain, and the results of the pituitary accumulation are not particularly clear. The reasons for these problems are that the incidence and malignancy of pituitary tumors are lower than those of other malignant tumors, such as lung cancer, breast cancer, and liver cancer; and the pituitary gland is relatively small in shape and size and is located in the pituitary fossa. There are certain technical difficulties in reproducing in situ tumors and in vivo imaging, resulting in a relative lag in pituitary-targeted drug delivery research. Summary of the invention

[0005] The present invention provides a delivery carrier for delivering drugs targeted to the pituitary gland and a preparation method thereof, so as to solve the problems in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A delivery vector for targeted pituitary drug delivery, wherein MPEG-2000-DSPE and DSPE-PEG-TRH are embedded on the phospholipid bilayer of the liposome, and TRH is located on the surface of the polyethylene glycol (PEG) lipid delivery vector. The delivery vector can load both fat-soluble drugs and water-soluble drugs.

[0008] Furthermore, the structure of the delivery vector is as follows:

[0009]

[0010] A method for preparing a delivery vector for targeted pituitary drug delivery comprises the following steps:

[0011] S1. Preparation of PEGylated liposomes (PEG-LP):

[0012] PEG-LP was prepared by thin film hydration ultrasound method. A mixture of 56.94 mg hydrogenated soybean phosphatidylcholine (HSPC), 19.14 mg cholesterol and 9.6 mg MPEG-2000-DSPE (molar ratio 62.5:37.5:0.4) was accurately weighed and dissolved in 6 mL chloroform. The mixture was then rotary evaporated at 55°C for 1 h to form a lipid film. At the end of rotary evaporation, 99.999% high-purity nitrogen was used to blow away trace chloroform (repeated 3 times). The dried lipid film was then hydrated with 6 mL normal saline for 10 to 15 min and ultrasonically treated at 4°C for 3 to 4 cycles (3 s pause for 2 s for each ultrasonic treatment). Small unilamellar vesicles were formed in this process. The solution was passed through polycarbonate membranes with pore sizes of 200 nm, 100 nm and 50 nm respectively using an extruder to control the particle size of the nanocarrier. The average particle size of the nanodrug delivery system was monitored by a Brookhaven particle size potential analyzer, and it was observed that the particle size of the carrier could be controlled between 50 and 80 nm. nm range, which facilitates the vector to cross the vascular endothelial barrier and reach the pituitary tissue.

[0013] S2. Preparation of thiolated TRH:

[0014] 1.7 mg TRH and 6.5 mg Traut's reagent were accurately weighed and dissolved in 2.5 mL HEPES buffer (25 mM HEPES, 140 mM NaCl, pH 8.0) at a molar ratio of 1:10. The mixture was reacted at room temperature, protected from light and under inert nitrogen for 1 h to obtain a thiolated TRH solution.

[0015] S3. Preparation of TRH-coupled DSPE-PEG micelles:

[0016] 2.5 mg of DSPE-PEG maleimide (DSPE-PEG-Mal) was accurately weighed, and 1.5 mL of degassed HEPES buffer (pH 8.0) was added, dissolved at 60°C, micelles were formed, and then cooled to room temperature to obtain freshly prepared DSPE-PEG-Mal micelles; then the thiolated TRH solution prepared in step S2 was added to the freshly prepared DSPE-PEG-Mal micelles, and the mixture was reacted overnight at room temperature and protected from light under magnetic stirring under inert nitrogen protection to obtain TRH-coupled DSPE-PEG micelles;

[0017] The TRH-coupled DSPE-PEG micelles were concentrated in an Amicon Ultra-0.5 centrifugal filter device (UFC5003Millipore), and the unreacted Traut reagent, TRH, and thiolated TRH were filtered out using an ultrafiltration centrifuge tube with a size cutoff of 3000 Da, and the micelles formed by TRH-coupled DSPE-PEG were retained; the coupled DSPE-PEG-TRH was identified using ultraviolet spectrophotometry. The concentration of TRH in the solution was determined by the BCA method.

[0018] S4. Preparation of TRH modified liposomes (TRH-PEG-LP):

[0019] The TRH-coupled DSPE-PEG micelles prepared in step S3 were added to the PEGylated liposomes prepared in step S1, so that the molar ratio of TRH to the phospholipids in the liposomes was 1:1000, and incubated at 60° C. for 1 h to obtain TRH-modified liposomes (TRH-PEG-LP).

[0020] The chemical synthesis formula of DSPE-PEG-TRH is as follows:

[0021] (1) TRH reacts with Traut's reagent to generate free thiol groups:

[0022]

[0023] (2) TRH modified with thiol groups reacts with DSPE-PEG-Mal to generate DSPE-PEG-TRH:

[0024]

[0025] The delivery carrier is used in the preparation of a drug, wherein the drug is a drug for treating spinocerebellar disorders, a drug for treating hypothalamic hypothyroidism, a drug for treating congenital isolated thyrotropin-releasing hormone (TRH) deficiency, or a drug for treating non-alcoholic fatty liver disease (NAFLD).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention designs a carrier that can target the pituitary gland for drug delivery, and specifically treats patients with spinocerebellar disorders, hypothalamic hypothyroidism, congenital TRH deficiency, non-alcoholic fatty liver disease (NAFLD), etc. The present invention modifies the surface of a PEG lipid carrier (TRH-PEG-LP) with thyrotropin-releasing hormone (TRH), injects the TRH-PEG-LP carrier loaded with infrared fluorescent dye (DiR) into the mouse through the tail vein for 2 hours, takes the pituitary tissue, and observes the color development of the fluorescent dye in the pituitary gland of the mouse under a small animal imager, and finds that the carrier can target the pituitary tissue well. TRH modification on the surface of the PEGylated lipid carrier can play a role in targeted delivery, and TRH can bind to the TRH receptor specifically expressed on the surface of the pituitary cells, thereby achieving targeted drug delivery to the pituitary gland. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the structural formula of the delivery vector of the present invention;

[0029] Figure 2 is a flow chart of the preparation of the delivery vector in the present invention;

[0030] Figure 3 is a schematic diagram of the process of preparing the delivery vector in the present invention;

[0031] Figure 4 This is the UV spectrophotometric identification diagram of DSPE-PEG-TRH in the present invention;

[0032] Figure 5 This is a graph showing the efficiency of the PEG-LP vector modified with TRH (TRH-PEG-LP) and the PEG-LP vector without TRH modification in delivering the fluorescent dye DiR to the pituitary gland. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments. Example

[0034] A delivery carrier for targeted pituitary drug delivery, wherein MPEG-2000-DSPE and DSPE-PEG-TRH are embedded on the phospholipid bilayer of the liposome, TRH is located on the surface of the PEGylated lipid delivery carrier, and the delivery carrier can load both fat-soluble drugs and water-soluble drugs.

[0035] The structure of the delivery vector is shown in 1:

[0036]

[0037] Example 2

[0038] like Figure 2 and 3 As shown, a method for preparing a delivery vector for targeted pituitary drug delivery comprises the following steps:

[0039] S1. Preparation of PEGylated liposomes (PEG-LP):

[0040] PEG-LP was prepared by thin film hydration ultrasonic method. A mixture of 56.94 mg hydrogenated soybean phosphatidylcholine (HSPC), 19.14 mg cholesterol and 9.6 mg MPEG-2000-DSPE (molar ratio 62.5:37.5:0.4) was accurately weighed and dissolved in 6 mL chloroform. The mixture was then rotary evaporated at 55°C for 1 h to form a lipid film. At the end of the rotary evaporation, 99.999% high-purity nitrogen was used to blow away trace amounts of chloroform (repeated 3 times). The dried lipid film was then hydrated with 6 mL of normal saline for 10 to 15 min (10 min, 13 min or 15 min can be selected in different embodiments), and ultrasonic treatment was performed at 4°C for 3 to 4 cycles (3 s pause for 2 s for each ultrasonic treatment). Small unilamellar vesicles were formed in the process. The solution was extruded through pores of 200 nm and 100 nm, respectively. The particle size of the nanocarrier was controlled by polycarbonate membranes of 50 nm and 50 nm; the average particle size of the nanodrug delivery system was monitored by a Brookhaven particle size potential analyzer, and it was observed that the particle size of the carrier could be controlled in the range of 50 to 80 nm, which facilitated the carrier to cross the vascular endothelial barrier and reach the pituitary tissue.

[0041] S2. Preparation of thiolated TRH:

[0042] 1.7 mg TRH and 6.5 mg Traut's reagent were accurately weighed and dissolved in 2.5 mL HEPES buffer (25 mM HEPES, 140 mM NaCl, pH 8.0) at a molar ratio of 1:10. The mixture was reacted at room temperature, protected from light and under inert nitrogen for 1 h to obtain a thiolated TRH solution.

[0043] S3. Preparation of TRH-coupled DSPE-PEG micelles:

[0044] 2.5 mg of DSPE-PEG-Mal was accurately weighed, and 1.5 mL of degassed HEPES buffer (pH 8.0) was added, dissolved at 60°C, micelles were formed, and then cooled; then the thiolated TRH solution prepared in step S2 was added to the freshly prepared DSPE-PEG-Mal micelles, and the mixture was reacted overnight at room temperature in the dark under the protection of inert nitrogen gas to obtain TRH-coupled DSPE-PEG micelles;

[0045] Since thiolated TRH reacts with DSPE-PEG-Mal to generate TRH-coupled DSPE-PEG micelles, the substances that may exist in the final reaction system include: TRH, thiolated TRH, Traut reagent, DSPE-PEG-Mal, and DSPE-PEG-TRH. Therefore, the TRH-coupled DSPE-PEG micelles were concentrated in an Amicon Ultra-0.5 centrifugal filter device (UFC5003 Millipore). After repeated centrifugation and concentration for three times using an ultrafiltration centrifuge tube with a size cutoff value of 3000 Da, TRH with a molecular weight less than 3000 Da, thiolated TRH, unreacted Traut reagent, free DSPE-PEG-Mal, and DSPE-PEG-TRH were all centrifugally filtered out, and the micelles formed by TRH-coupled DSPE-PEG were retained; TRH-PEG-DSPE can be identified by ultraviolet spectrophotometry (such as Figure 4 As shown in Figure 2, the concentration of TRH in the solution was determined by BCA method. Therefore, most of the TRH-coupled DSPE-PEG micelles were retained by ultrafiltration centrifuge tube (molecular weight cutoff 3000 Da).

[0046] S4. Preparation of TRH modified liposomes (TRH-PEG-LP):

[0047] The TRH-coupled DSPE-PEG micelles prepared in step S3 were added to the PEGylated liposomes prepared in step S1, so that the molar ratio of TRH to the phospholipids in the liposomes was 1:1000, and incubated at 60° C. for 1 h to obtain TRH-modified liposomes (TRH-PEG-LP).

[0048] Specifically, the chemical synthesis formula of DSPE-PEG-TRH is as follows:

[0049] (1) TRH reacts with Traut's reagent to generate free thiol groups:

[0050]

[0051] (2) TRH modified with thiol groups reacts with DSPE-PEG-Mal to generate DSPE-PEG-TRH:

[0052]

[0053] The thiolated TRH was covalently coupled to the maleimide terminus of DSPE-PEG-Mal. The reaction between thiol and maleimide groups is one of the most efficient reactions in bioconjugation chemistry, resulting in a stable thioether bond. Example

[0054] Experimental: Small animal imaging showed accumulation of the vector in the pituitary tissue of mice.

[0055] Preparation of TRH-PEG-LP-DiR and PEG-LP-DiR:

[0056] The preparation of DiR-loaded liposomes was carried out by adding DiR chloroform solution during the preparation of PEG-LP, rotary evaporation, hydration and ultrasonic membrane to obtain DiR-loaded PEGylated liposomes, and the concentration of DiR in the liposomes was 30 μg / mL. TRH-PEG-LP-DiR was prepared by co-incubation of DiR-loaded PEGylated liposomes with TRH-coupled DSPE-PEG micelles, while the control group PEG-LP-DiR was prepared by co-incubation of DiR-loaded PEGylated liposomes with MPEG-2000-DSPE micelles of the same concentration as DSPE-PEG-TRH micelles.

[0057] Administration and observation of TRH-PEG-LP-DiR and PEG-LP-DiR: The drugs were administered at a dose of 300 μg / kg DiR and a volume of 10 mL / kg. Two hours after administration through the tail vein, the pituitary tissue of the mice was obtained and the color development of the fluorescent dye DiR in the pituitary of the mice was observed using the IVIS® Lumina III Small Animal In vivo Imaging System (Ex / Em: 748 / 780 nm) to evaluate the pituitary targeting properties of the TRH-PEG-LP vector.

[0058] like Figure 5 As shown, by comparing the efficiency of PEG-LP vector modified with TRH (TRH-PEG-LP) and PEG-LP vector without TRH modification in delivering fluorescent dye DiR to the pituitary, it was found through small animal imaging detection that the fluorescence intensity of TRH-PEG-LP as a vector in the pituitary was 1.55 times that of PEG-LP as a vector. After TRH modification, the targeting of the delivery vector in the pituitary can be significantly increased. Figure 5 In the figure, the dotted circle indicates the pituitary tissue.

[0059] As a preferred embodiment, the delivery vector is used in the preparation of a drug, wherein the drug is a drug for treating spinocerebellar disorders, a drug for treating hypothalamic hypothyroidism, a drug for treating congenital isolated thyrotropin-releasing hormone (TRH) deficiency, or a drug for treating non-alcoholic fatty liver disease (NAFLD).

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A delivery vector for targeting pituitary gland drug delivery, characterized in that: MPEG-2000-DSPE and DSPE-PEG-TRH are embedded in the phospholipid bilayer of the liposome, and TRH is located on the surface of the polyethylene glycol PEGylated lipid delivery carrier. The delivery carrier can load both fat-soluble drugs and water-soluble drugs. The structure of the delivery vector is as follows: 。 2. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 1, characterized in that: The following steps are involved: S1. Preparation of PEGylated liposomes PEG-LP: A mixture of hydrogenated soybean phosphatidylcholine HSPC, cholesterol, and MPEG-2000-DSPE was dissolved in chloroform and dried by rotary evaporation at 55°C for 1 h to form a lipid film. At the end of rotary evaporation, nitrogen was used to blow away trace amounts of chloroform. The dried lipid film was then hydrated with saline for 10 to 15 min and subjected to ultrasonic treatment at 4°C. The solution was passed through a polycarbonate membrane to control the particle size of the nanocarrier to be 50 to 80 nm. S2. Preparation of thiolated TRH: TRH and Traut's reagent were mixed and dissolved in HEPES buffer, and then reacted for 1 h at room temperature, in the dark and under the protection of inert nitrogen to obtain a thiolated TRH solution; S3. Preparation of TRH-coupled DSPE-PEG micelles: DSPE-PEG-Mal is added to degassed HEPES buffer, dissolved at 60°C, micelles are formed, and then cooled to room temperature to obtain freshly prepared DSPE-PEG-Mal micelles; then the thiolated TRH solution prepared in step S2 is added to the freshly prepared DSPE-PEG-Mal micelles, and the mixture is reacted overnight at room temperature and away from light under the protection of inert nitrogen gas to obtain TRH-coupled DSPE-PEG micelles; The TRH-coupled DSPE-PEG micelles were concentrated in a centrifugal filter device and filtered using an ultrafiltration centrifuge tube with a size cutoff of 3000 Da to remove impurities and retain the micelles formed by the TRH-coupled DSPE-PEG; S4. Preparation of TRH-PEG-LP modified liposomes: The TRH-coupled DSPE-PEG micelles prepared in step S3 were added to the PEGylated liposomes prepared in step S1, so that the molar ratio of TRH to the phospholipids in the liposomes was 1:1000, and incubated at 60° C. for 1 h to obtain TRH-PEG-LP modified liposomes.

3. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: In the step S1, the molar ratio of hydrogenated soybean phosphatidylcholine HSPC, cholesterol, and MPEG-2000-DSPE is 62.5:37.5:0.

4.

4. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: In the step S1, nitrogen gas is blown away to remove trace amounts of chloroform for three times.

5. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: In step S1, the ultrasonic treatment is performed for 3 to 4 cycles, with a 2 s pause after each 3 s ultrasonic treatment.

6. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: In the step S1, the solution is passed through polycarbonate membranes with pore sizes of 200 nm, 100 nm, and 50 nm respectively by an extruder to control the particle size of the nanocarrier.

7. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: In the step S2 for preparing thiolated TRH, the molar ratio of TRH to Traut's reagent is 1:10; the HEPES buffer includes 25 mM HEPES and 140 mM NaCl, and the pH of the HEPES buffer is 8.

0.

8. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: The pH of the HEPES buffer in step S3 is 8.

0.

9. The method for preparing a delivery carrier for targeted pituitary drug delivery according to claim 2, characterized in that: The chemical synthesis formula of DSPE-PEG-TRH is as follows: (1) TRH reacts with Traut's reagent to generate free thiol groups: ； (2) TRH modified with thiol groups reacts with DSPE-PEG-Mal to generate DSPE-PEG-TRH: 。 10. Use of the delivery vector according to claim 1 in the preparation of a drug, wherein the drug is a drug for treating spinocerebellar disorders, a drug for treating hypothalamic hypothyroidism, a drug for treating congenital isolated thyrotropin-releasing hormone deficiency, or a drug for treating non-alcoholic fatty liver disease.

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