CN115364055A

CN115364055A - Nitric oxide and drug loaded microvesicle and preparation method thereof

Info

Publication number: CN115364055A
Application number: CN202210875604.5A
Authority: CN
Inventors: 任杰; 邱晨; 卫洪波; 杨文德; 尹庭辉; 张薇; 郑博文
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2022-07-25
Filing date: 2022-07-25
Publication date: 2022-11-22

Abstract

The invention belongs to the field of biological medicines, and particularly relates to nitric oxide and medicine loaded microvesicles and a preparation method thereof. The preparation method comprises the steps of preparing a drug-loaded liposome; and introducing mixed gas containing nitric oxide into the drug-loaded liposome, and oscillating to prepare the microbubble loaded with nitric oxide and the drug. The method disclosed by the invention can successfully prepare the microvesicle loaded with NO and the medicament at the same time, the obtained microvesicle can not generate toxicity to target cells, and the controllable targeted release of the medicament and nitric oxide can be realized, thereby reducing the systemic toxic and side effects. Thereby having good clinical application prospect in the treatment field of the nervous erectile dysfunction.

Description

Nitric oxide and drug loaded microvesicle and preparation method thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to nitric oxide and medicine loaded microvesicles and a preparation method thereof.

Background

Erectile Dysfunction (ED) caused by nerve injury is a common complication after rectal cancer operation, and no effective treatment method exists at present, so that the postoperative life quality of a patient is seriously affected. Penile erection is closely related to the relaxation degree of smooth muscle and the filling degree of blood vessels of the penis, and the concentration of cyclic guanosine monophosphate (cGMP) plays a positive correlation regulation role. Under the stimulation of sexual activity, nitric Oxide (NO) released by cholinergic nerve cell terminals, endothelial cells and the like activates soluble guanylate cyclase (sGC) to convert guanosine triphosphate into cGMP, so that the concentration of cpcmp in vascular smooth muscle cells is increased, and blood is filled into penis to produce erection. On the other hand, the presence of erectile-inhibitory factors in the body, such as the presence of phosphodiesterase type 5 (PDE 5) in tissues, can cause cGMP degradation and thus the inhibition of an erection. Sildenafil (Sildenafil) is a type 5 phosphodiesterase inhibitor (PDE 5 i) represented by the most promising drug for ED with nerve injury at present, facilitating erection by inhibiting degradation of cGMP by PDE 5.

PDE5i, as a first-line drug for treating ED, depends on cGMP concentration, and cGMP pathway initiation depends on NO activation, so that after administration, it will take effect only after effective stimulation and intact nerve and blood vessel pathways, which is the main reason why administration is ineffective. For the ED patient, the NO-cGMP pathway is disturbed due to the inhibition of self NO production by the degradation of the nitrated nerves innervating the cavernous smooth muscle and vascular system, which results in poor therapeutic effect of PDE5 i. Besides, PDE5i is currently administered orally, with limited concentrations of drug in the target organ. Therefore, PDE5i is less effective in patients with nerve-damaged ED due to the low drug concentration in the ED-damaged area caused by the disorder of NO production and the limitation of oral administration; in addition, the toxic and side effects of continuous administration of PDE5i on the whole body organs also limit further clinical application.

With regard to the formulation of sildenafil, in addition to the oral administration commonly adopted, chinese patent application CN103429247A discloses topical delivery of sildenafil-containing compositions by transdermal administration; chinese patent application CN101043874A discloses a preparation method of sildenafil liposome, but the active ingredient used is sildenafil citrate, which is a water-soluble active ingredient, and the prepared liposome is administered by means of topical application, both of which can result in limited concentration of sildenafil delivered to the lesion area.

In recent years, with the continuous development of the field of targeted therapy and drug delivery, the microbubble-targeted drug delivery (UTMD) effect of ultrasound mediated drug-loaded microbubbles is gradually applied as a drug delivery system. The UTMD is characterized in that acoustic microbubbles are used for encapsulating drugs or therapeutic gases, and ultrasonic irradiation is carried out on a target area after intravenous injection, so that the effects of slow release and targeted drug delivery or therapeutic gases can be realized: under ultrasonic irradiation, ultrasonic waves generate an inertial cavitation effect, namely, alternating pressure generated by ultrasonic irradiation with certain intensity can form micro bubbles and cause the micro bubbles to generate 'explosion', so that drug-loaded micro bubbles are cracked to release drugs or gas and simultaneously promote cell membranes of surrounding tissues to be open, and the drugs or gas outside cells are further promoted to enter cells. UTMD facilitates high local concentrations of therapeutic drugs or gases in the target organ while maintaining low systemic concentrations, enabling accurate delivery of the target area of the lesion.

Disclosure of Invention

In one aspect, the present application provides a microbubble comprising: a central region and a housing; the housing includes: sildenafil and liposomes; the central region includes: a gas comprising nitric oxide.

In some embodiments, the liposome to sildenafil mass ratio is 22: (0.5-2.5); in some embodiments, the liposome to sildenafil mass ratio is 22: (0.5-2); in some embodiments, the liposome to sildenafil mass ratio is 22:2.

in some embodiments, the liposome comprises a lipid comprising one or more of a phospholipid, cholesterol; in some embodiments, the lipid comprises a phospholipid and cholesterol.

In some embodiments, the phospholipid comprises one or more of DPPC, DSPE-PEG2000, DPPA; in some embodiments, the phospholipid is a combination of DPPC, DSPE-PEG2000, DPPA; in some embodiments, the mass ratio of DPPC, DSPE-PEG2000, DPPA, cholesterol is 8 (1-2) to 1; in some embodiments, the mass ratio of DPPC, DSPE-PEG2000, DPPA, cholesterol is 8.

In some embodiments, the nitric oxide containing gas comprises 1 to 100% by volume of nitric oxide; in some embodiments, the nitric oxide is 20 to 100% by volume.

In some embodiments, the gas further comprises an inert gas; in some embodiments, the inert gas is selected from one or more of helium, neon, argon, sulfur hexafluoride, and fluorocarbon; in some embodiments, the inert gas is perfluoropropane and/or sulfur hexafluoride; in some embodiments, the inert gas is perfluoropropane; in some embodiments, the nitric oxide containing gas has a volume percentage of nitric oxide to perfluoropropane of 4.

In some embodiments, the microbubbles have a particle size of 1100 to 1600nm.

In a specific embodiment, the microbubbles have a particle size of 1361 ± 208.5nm.

In one aspect, the present application provides a method for preparing the microvesicle, comprising the following steps:

s1, preparing a shell wrapping a hydration liquid by a thin film-hydration method;

s2, introducing gas containing nitric oxide into the shell wrapped with the hydration liquid prepared in the step S1 to prepare a mixture; the mixture was shaken to prepare the microbubbles.

In some embodiments, the step S1 comprises the steps of:

s11, heating and uniformly mixing the lipid, the sildenafil and the chloroform;

s12, performing rotary evaporation on the solution prepared in the step S11 until chloroform is completely evaporated to form a lipid film containing sildenafil;

s13, adding a hydration liquid into the lipid film containing the sildenafil prepared in the S12, and hydrating by a constant-temperature shaking table to form a shell wrapping the hydration liquid.

In some embodiments, the heating temperature in step S11 is 40 to 80 ℃; in some embodiments, the hydration solution is selected from one or more of double distilled water, sodium chloride, or phosphate buffered saline, PBS; in some embodiments, the hydration solution is selected from phosphate buffered saline PBS; in some embodiments, in step S12, the temperature of the rotary evaporation is 25 to 80 ℃ and the rotation speed is 60 to 240rpm; in some embodiments, the rotary evaporation is performed under negative pressure conditions; in some embodiments, in step S13, the hydration temperature is 25 to 80 ℃, the rotation speed of the shaker is 50 to 250rpm, and the hydration time is 10min to 60min.

In some embodiments, the step S2 comprises the steps of:

s21, introducing gas containing nitric oxide into the shell wrapping the hydration liquid prepared in the step S1 under a vacuum condition, and oscillating to obtain microbubble suspension;

s22, carrying out centrifugal washing on the microbubble suspension prepared in the step S21, and carrying out heavy suspension by using PBS to obtain the microbubbles.

In some embodiments, the oscillation frequency in step S21 is 40 to 80Hz, preferably 70Hz; the oscillation time is 20-100s, preferably 50s; in some embodiments, the rotation speed of the centrifugal washing in step S22 is 500 to 3000rpm, preferably 2000rpm, and the centrifugation time is 1 to 20min, preferably 5min; in some embodiments, the centrifugation wash in step S22 is 2-5 times; in some embodiments, after the centrifugal washing, before resuspension with PBS, a step of removing the lower layer liquid to remove liposome debris is further included.

In one aspect, the application provides the use of said microvesicles for the preparation of a medicament for the treatment of erectile dysfunction; in some embodiments, the is neuro-impaired erectile dysfunction.

In one aspect, the present application provides a medicament for the treatment of erectile dysfunction comprising said microvesicles.

In some embodiments, the medicament is formulated for intravenous administration, subcutaneous administration, intramuscular administration, intracerebroventricular administration, intrathecal administration, or transdermal administration; in some embodiments, the medicament is formulated for intravenous administration.

In some embodiments, the medicament is formulated for UTMD-mediated administration; in some embodiments, the UTMD is conditioned at 0.5 to 1.5MHz, a pulse repetition frequency of 0.5 to 1.5kHz, a sound pressure of 300 to 700kPa, a duty cycle of 0.1 to 1%, and a time of 5 to 15s.

In a specific embodiment, the UTMD conditions are 1MHz, pulse repetition frequency 1kHz, sound pressure 500kPa, duty cycle 0.5%, time 10s.

According to the invention, sildenafil and NO are encapsulated in liposome microbubbles, the target drug release is achieved by combining the intravenous injection target area and the ultrasonic cavitation effect, and the curative effect of sildenafil is expected to be improved and the toxic and side effects are reduced. Chinese patent application CN110974844A discloses a preparation method of NO-loaded targeted microvesicles and is used in the treatment field of heart transplant rejection, but at present, in the field of ED treatment, especially nerve injury ED treatment, such a comprehensive microvesicle system and administration mode simultaneously loaded with therapeutic gas (NO) and therapeutic drug (sildenafil) have not been reported yet.

In one particular embodiment of the present application, liposome microbubbles loaded with both NO and drug can be successfully prepared.

In a specific embodiment of the present application, the liposome microbubble loaded with nitric oxide and the drug obtained by the present invention has high safety and does not generate toxicity to target cells or target organs.

In a specific embodiment of the application, the liposome microbubble loaded with nitric oxide and a medicament obtained by the invention can release nitric oxide and the medicament by passively targeting the microbubble and ultrasonically breaking the microbubble, so that the aim of controllably and targetedly releasing nitric oxide and the medicament is fulfilled, the nitric oxide and the medicament can be efficiently absorbed by target cells and high-concentration NO is generated, the curative effect of the medicament is improved, and systemic toxic and side effects are reduced;

in a specific embodiment of the application, the microvesicle loaded with nitric oxide and sildenafil obtained by the invention successfully realizes the treatment of the neurogenic erectile dysfunction, has high safety in the treatment process and has good clinical application prospect.

Drawings

FIG. 1 shows MB in example 1 and comparative example 2 _Sd-NO (left in FIG. 1A) and MB- _Sd Appearance of liposomes (FIG. 1A, right), MB _Sd-NO And MB- _Sd Liposome microscopy (FIG. 1B).

FIG. 2 shows the Cavernous Smooth Muscle Cells (CSMCs) of example 4 (FIG. 2A) with MB added _Sd-NO Cell viability profiles between groups after 4h or 8h treatment with or without UTMD (fig. 2B, 2C).

FIG. 3 shows the CSMCs in combination with MB in example 5 _Sd-NO Fluorescence profiles of drug delivery in CSMCs after co-incubation without or after UTMD treatment, respectively (fig. 3A, 3B), where Coumarin (Coumarin) is a fluorescent indicator mimicking sildenafil loading.

FIG. 4 shows the CSMCs in combination with MB in example 5 _Sd-NO Fluorescence plots of NO content in CSMCs after co-culture, without or after UTMD treatment, respectively (FIGS. 4A, 4B).

Figure 5 is a graph of the effect of microbubble in vitro ultrasound contrast imaging at different NO volume percentages for example 6.

FIG. 6 is a graph of the therapeutic effect of NO and sildenafil loaded microvesicles of example 7 on rat ED.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Co-carried microbubbles and MB in the present application _Sd-NO "," NO-loaded and sildenafil-loaded microvesicles "may be used interchangeably. In the present application, the "novel microbubbles" are all referred to as "microbubbles loaded with NO and sildenafil" unless otherwise specified.

Example 1 Microvesicles (MB) loaded with NO and sildenafil _Sd-NO ) Preparation of (2)

S1: the sildenafil-loaded liposome is prepared by taking phospholipid and sildenafil as membrane materials and adopting a film-hydration method, and comprises the following steps:

1) 16mg of DPPC, 2mg of DPPA, 2mg of DSPE-PEG2000, 2mg of Cholesterol, 2mg of sildenafil were placed in a round bottom flask, and 4mL of chloroform was added. The water bath was heated to 50 ℃ to completely dissolve in chloroform.

2) The solution obtained in step 1) was rotary evaporated by means of a rotary evaporator (IKA, RV 10digital V) until the chloroform was completely evaporated. Wherein, the parameter setting of rotatory evaporimeter is as follows: the rotary evaporation temperature is 50 ℃, and the rotating speed is 120rpm. Chloroform was completely evaporated by negative pressure evaporation to form a thin film of phospholipids at the bottom of the round bottom flask.

3) Adding 4mL of Phosphate Buffered Saline (PBS) hydration solution into the phospholipid film obtained in the step 2), and hydrating for 20min at constant temperature in a shaker at 60 ℃ and 100rpm to obtain liposome solution.

S2: liposome Microbubble (MB) carrying NO and sildenafil _Sd-NO ) The preparation method specifically comprises the following steps:

under vacuum condition, perfluoropropane (C) ₃ F ₈ ) And Nitric Oxide (NO) in a volume percentage4 ratio of 1 into the liposome suspension continuously, and preparing MB entrapping NO and sildenafil by horizontal mechanical oscillation (70Hz, 50s) _Sd-NO Then, the cells were washed by centrifugation (2000rpm, 3min,3 times), the lower layer liquid was aspirated by a needle to remove liposome debris and the like, and the upper layer microbubbles were resuspended in 4ml of Phosphate Buffered Saline (PBS). Then the microvesicle loaded with NO and sildenafil, MB for short, can be obtained _Sd-NO 。

Comparative example 1 NO-loaded Microvesicles (MB) _NO ) Preparation of

S1: the liposome is prepared by a film-hydration method by taking phospholipid as a film material, and the method comprises the following steps:

1) 16mg DPPC, 2mg DPPA, 2mg DSPE-PEG2000, 2mg Cholesterol powder was placed in a round bottom flask and 4mL chloroform was added. The water bath was heated to 50 ℃ to completely dissolve in chloroform.

2) The solution obtained in step 1) is rotary evaporated by a rotary evaporator (IKA, RV 10digital V) until the chloroform is completely evaporated. Wherein, the parameter setting of rotary evaporator is as follows: the rotary evaporation temperature is 50 ℃, and the rotating speed is 120rpm. Chloroform was completely evaporated by negative pressure evaporation to form a thin film of phospholipids at the bottom of the round bottom flask.

3) Adding 4mL of Phosphate Buffered Saline (PBS) hydration solution into the phospholipid film obtained in the step 2), and hydrating for 20min in a shaker at the constant temperature of 60 ℃ and 100rpm to obtain a liposome solution.

S2: NO-loaded liposome microbubble (MB for short) _NO ) The preparation method specifically comprises the following steps:

under vacuum condition, perfluoropropane (C) ₃ F ₈ ) And Nitric Oxide (NO) mixed gas is continuously injected into the liposome suspension in a ratio of 4 volume percent to 1, and the NO-coated MB prepared by mechanical oscillation _NO Then, the cells were washed by centrifugation (2000rpm, 3min,3 times), the lower layer liquid was aspirated by a needle to remove liposome debris and the like, and the upper layer microbubbles were resuspended in 4ml of Phosphate Buffered Saline (PBS). The microbubble loaded with NO, MB for short, can be obtained _NO 。

Comparative example 2 sildenafil-loaded Microvesicles (MB) _Sd ) Preparation of (2)

1) 16mg of DPPC, 2mg of DPPA, 2mg of DSPE-PEG2000, 2mg of Cholesterol, 2mg of sildenafil were placed in a round bottom flask, and 4mL of chloroform was added. The water bath was heated to 50 ℃ to dissolve it completely in chloroform.

2) The solution obtained in step 1) of preparation S11 was rotary evaporated by a rotary evaporator (IKA, RV 10digital V) until the chloroform was completely evaporated. Wherein, the parameter setting of rotatory evaporimeter is as follows: the rotary evaporation temperature is 50 ℃, and the rotating speed is 120rpm. Chloroform was completely evaporated by negative pressure evaporation to form a thin film of phospholipids at the bottom of the round bottom flask.

S2: sildenafil-loaded liposome microbubble (abbreviated as MB) _Sd ) The preparation method specifically comprises the following steps:

under the vacuum condition, C is added ₃ F ₈ Continuously injecting into liposome suspension, and mechanically oscillating to obtain sildenafil-coated MB _Sd Then, the cells were washed by centrifugation (2000rpm, 3min,3 times), the lower layer liquid was aspirated by a needle to remove liposome debris and the like, and the upper layer microbubbles were resuspended in PBS. The micro-bubble loaded with sildenafil, MB for short, can be obtained _Sd 。

Comparative example 3 control Microvesicles (MB) _Con ) Preparation of

S1: the liposome is prepared by taking phospholipid as a membrane material and adopting a thin film-hydration method, and the preparation method specifically comprises the following steps:

1) 16mg DPPC, 2mg DPPA, 2mg DSPE-PEG2000, 2mg Cholesterol phospholipid powder was placed in a round bottom flask and 4mL chloroform was added. The water bath was heated to 50 ℃ to dissolve it completely in chloroform.

2) The solution obtained in step 1) of preparation S11 was rotary evaporated by a rotary evaporator (IKA, RV 10digital V) until the chloroform was completely evaporated. Wherein, the parameter setting of rotatory evaporimeter is as follows: the rotary evaporation temperature is 50 ℃, and the rotating speed is 120rpm. Chloroform was completely evaporated by negative pressure evaporation and a thin film of phospholipid was formed at the bottom of the round bottom flask.

S2: liposome microbubbles (abbreviated as MB) _Con ) The preparation method specifically comprises the following steps:

under the vacuum condition, C is added ₃ F ₈ Continuously injecting into liposome suspension, and mechanically oscillating to obtain sildenafil-coated MB _Sd Then, the cells were washed by centrifugation (2000rpm, 3min,3 times), the lower layer liquid was aspirated by a needle to remove liposome debris and the like, and the upper layer microbubbles were resuspended in Phosphate Buffered Saline (PBS). Obtaining the no-load micro-bubbles of the control group, MB for short _Con 。

Example 2 Structure and characterization of NO and sildenafil loaded microvesicles

Detection of MB prepared in example 1 by dynamic light scattering _Sd-NO Has a particle diameter of 1361 + -208.5 nm.

Example 3 MB _Sd-NO (encapsulation efficiency/drug Loading) measurement

Loading 0.5mg,1mg,1.5mg,2mg sildenafil into liposome to obtain MB _Sd-NO After lyophilization, the drug loading (DLC%) and the encapsulation efficiency (DLE%) were measured with an ultraviolet spectrophotometer (table 1).

TABLE 1

Example 4 cytotoxicity detection of ultrasound microvesicles

In this example, MB was detected by CCK8 kit _Sd-NO The effect on apoptosis was followed to evaluate whether the novel microvesicles co-loaded with sildenafil and NO were cytotoxic. The specific method comprises the following steps:

1) 500 μ L of MB prepared according to example 1 _Sd-NO A suspension of microbubbles;

2) Will have a density of 2X 10 ⁷ one/mL of Cavernous Smooth Muscle Cells (CSMCs) Cells were plated in a six-well plate;

3) Respectively convert MB into _Sd-NO Diluting the microbubble suspension by 5, 10, 50, 100, 500, 1000 and 2000 times by using PBS, correspondingly adding 100 mu L/hole microbubble suspension diluent into CSMCs cells in a six-hole plate, and continuously culturing for 4h and 8h at 37 ℃ after ultrasonic mediated drug-loaded microbubble targeted drug release (UTMD) or NO UTMD (NO UTMD), wherein the ultrasonic condition is 1MHz in frequency, 1kHz in pulse repetition frequency, 500kPa in sound pressure, 0.5% in duty ratio and 10s in time;

4) The cell viability of different groups of CSMCs was assayed according to the protocol of the CCK8 assay kit (Biyun, C0037).

The results of the cytotoxicity assay are shown in fig. 2, and show that there is NO difference in the cell activity rate between the groups regardless of whether the cells are subjected to UTMD, compared to the control group of NO UTMD, which indicates that the novel microvesicles have NO toxic effect on the cells. In addition, the ultrasonic intensity used in the experiment is proved to have no killing effect on CSMCs cells.

Example 5 detection of delivery Effect of NO and sildenafil-Supported microvesicles

To determine whether the co-loaded microvesicles are more readily taken up by CSMCs under UTMD and accurately release NO to CMSCs, the following experiment was performed:

1. experiment grouping

Ctrol group: without addition of MB _Sd-NO Simple culture of the microvesicles for CSMCs;

UTMD group: and MB _Sd-NO CSMCs incubated with microbubbles are subjected to ultrasound mediated drug-loaded microbubble targeted drug release (UTMD), wherein the ultrasound conditions are 1MHz, the pulse repetition frequency is 1kHz, the sound pressure is 500kPa, the duty ratio is 0.5%, and the time is 10s;

NO UTMD group: and MB _Sd-NO Microvesicles co-incubated CSMCs, without UTMD.

2. Experimental methods

1) The procedure was as in 1) and 2) of example 3, first dividedSeparately preparing MB _Sd-NO Microbubble suspension and planting of CMSCs followed by MB _Sd-NO After the microbubbles are diluted by 100 times, adding 100 mu L/hole of microbubble suspension diluent into the CSMCs, and incubating the diluted solution and the CSMCs for 1h at 37 ℃;

2) After UTMD, NO content analysis was performed by following the method described in the instruction of the fluorescent probe DAF-FM DA (Biyuntian, S0019) for nitric oxide.

3) The procedure was as in 1) and 2) of example 4, first preparing MB, respectively _Sd-NO Microbubble suspension and planting of CMSCs followed by MB _Cou-NO Diluting the microvesicles by 100 times, adding 100 mu L/hole microvesicle suspension diluent into the CSMCs, and incubating the microvesicles and the CSMCs for 1h at 37 ℃;

4) After UTMD, the cell green fluorescence intensity is observed under a confocal microscope to indirectly reflect the cell delivery efficiency of the medicine;

as shown in fig. 3 and 4, the results showed that CSMCs cells had a significantly increased NO content after UTMD compared to the non-treated and non-UTMD experimental groups; meanwhile, the coumarin is used for simulating sildenafil drug loading to prompt that the drug can better enter CSMCs after UTMD, and the novel microbubble carrying sildenafil and NO can accurately release NO and the drug to CSMCs after UTMD, so that a foundation is laid for the novel microbubble to exert curative effect, and the preparation method of the microbubble MB carrying nitric oxide and sildenafil is highlighted _Sd-NO The advantage of (1).

EXAMPLE 6 Effect of different NO content on NO and sildenafil loaded microvesicles (concentration, quality, effect, stability, etc.)

To demonstrate the difference between NO and C ₃ F ₈ The volume percentage of (a) to the stability of the targeted lipid microbubble, respectively selecting different NO and C ₃ F ₈ Volume percent of (3) preparation of MB _Sd-NO The rest of the procedure was the same as in example 1.

Specifically, the volume percentage of NO is respectively 0%, 20%, 40%, 60%, 80% and 100%, and the residual volume is C ₃ F ₈ Mixed gas of (2) to prepare MB _Sd-NO For MB by means of ultrasound contrast imaging _Sd-NO Assays for in vitro imaging were performed. The determination method comprises the following specific steps: diluting with PBS 1500 times, and adding special 2% agarUltrasonic contrast imaging is carried out in the lipoid candy mould, and continuous observation is carried out for 1h. In this embodiment, the GE LOGIQ E20 clinical ultrasound diagnostic system and the ML6-15 linear array probe are used for ultrasound contrast imaging, and experimental results show that the volume percentage is 20%, and the imaging effect and stability of microbubbles are both optimal (fig. 5).

Example 7 therapeutic Effect of NO and sildenafil loaded microvesicles on rat ED

To determine whether animal-level co-carried microvesicles would have a therapeutic effect on ED under UTMD, the following experiments were performed:

the experimental method comprises the following steps:

1) Experimental animals: SD (Sprague-Dawley) rats, SPF grade, male, body weight 250-300g,10 weeks old, provided by the center of laboratory animals, guangdong province. The living environment of the rat is a constant-temperature sterile cage with the indoor humidity of 30% -50%, and sterilized water and food are provided for the rat; modeling: taking male SD rats of 10 weeks old, performing intraperitoneal injection anesthesia with pentobarbital (35 mg/kg), sterilizing, taking a midline incision of the abdomen, entering the abdominal cavity, and fully exposing the bladder and two lateral prostates. The fascial structure between the left prostate and the spermatic cord is separated bluntly, and the white polypod-like structure attached to the surface of the dorsal lobe of the prostate is visible after the left prostate is turned upwards and backwards, which is the pelvic main ganglion (MPG). MPG has about 2-3 thicker branches, one of which is the Cavernous Nerve (CN) when moving inwards and downwards to the penis;

2) Carefully dissociate CN, and complete bilateral CN injury (BCNI) procedure with vascular forceps for 2min at 5mm distance from MPG. The search pattern and damage pattern of the right CN are the same as the left. After the operation is finished, suturing the abdominal incision layer by layer, and placing the abdominal incision in a warm cushion for resuscitation after the incision is disinfected again; grouping 5 groups: 1) Sham group (Sham group); 2) Control group (BCNI group); 3) Sildenafil monotherapy (Sildenafil group); 4) Sildenafil and a new NO microbubble group (Sildenafil + NO-MBs group); 5) Sildenafil and NO novel microbubbles + UTMD group (Sildenafil + NO-MBs + UTMD group) are carried together;

evaluation of erectile function: rats were anesthetized and CN exposed as described above. The skin of the penis of a rat is incised, the auxiliary fascia structure is fully cut off, the corpus cavernosum and the penis dorsal vein on two sides and the penis artery on two sides are exposed, a No. 23 butterfly needle is wetted by 100U/ml heparin sodium in advance and is inserted into the right corpus cavernosum or the right penis artery, and the other end of the butterfly needle is connected to an electrophysiological collection system. And designing electrical stimulation parameters of CN, wherein the stimulation parameters are 1.5mA and 20Hz, the pulse width is 0.2ms, and the duration time is 50s. After the instrument is connected, placing a bipolar electric stimulation head of the electric stimulation rod at the CN position, and recording the intracavernosal pressure (ICP); the skin was cut straight along the midline of the neck, and the length was about 3-5cm. Exposing a left stiff common artery, ligating a silk thread at the far-center end, clamping an artery at the near-center end by an artery clamp, obliquely cutting a small opening between the two ends, inserting a PE-50 catheter filled with 100U/ml heparin sodium into the artery, connecting the other end of the catheter to an electrophysiological acquisition system, recording Mean Arterial Pressure (MAP), and evaluating erectile function by calculating ICP/MAP ratio (figures 6A, 6B and 6C).

Claims

1. A microbubble, comprising: a central region and a shell;

the housing includes: sildenafil and liposomes;

the central region includes: a gas comprising nitric oxide.

2. The microbubble of claim 1, wherein the liposome to sildenafil mass ratio is 22: (0.5-2.5);

preferably, the mass ratio of the liposome to sildenafil is 22: (0.5-2);

preferably, the mass ratio of the liposome to the sildenafil is 22:2;

preferably, the liposomes comprise lipids comprising one or more of phospholipids, cholesterol;

preferably, the lipids include phospholipids and cholesterol;

preferably, the phospholipid comprises one or more of DPPC, DSPE-PEG2000, DPPA;

preferably, the phospholipid is a combination of DPPC, DSPE-PEG2000 and DPPA;

preferably, the mass ratio of DPPC, DSPE-PEG2000, DPPA and cholesterol is 8 (1-2) to 1;

preferably, the mass ratio of DPPC, DSPE-PEG2000, DPPA and cholesterol is 8.

3. The microbubble of claim 1, wherein the nitric oxide containing gas comprises from 1% to 100% by volume of nitric oxide;

preferably, the volume percentage of the nitric oxide is 20-100%;

preferably, the gas further comprises an inert gas;

preferably, the inert gas is selected from one or more of helium, neon, argon, sulfur hexafluoride and fluorocarbon;

preferably, the inert gas is perfluoropropane and/or sulfur hexafluoride;

preferably, the inert gas is perfluoropropane;

preferably, the volume percentage of nitric oxide to perfluoropropane in the nitric oxide-containing gas is 4;

preferably, the particle size of the microbubbles is 1100 to 1600nm.

4. A method for preparing the microvesicles according to any of claims 1 to 3, comprising the steps of:

5. The method of claim 4, wherein the step S1 comprises the steps of:

s11, heating and uniformly mixing the lipid, the sildenafil and the chloroform;

s13, adding a hydration liquid into the lipid film containing the sildenafil prepared in the S12, and hydrating by using a constant-temperature shaking table to form a shell wrapping the hydration liquid;

preferably, the heating temperature in the step S11 is 40-80 ℃;

preferably, the hydration liquid is selected from one or more of double distilled water, sodium chloride or Phosphate Buffered Saline (PBS);

preferably, the hydration solution is selected from phosphate buffered saline PBS;

preferably, in the step S12, the temperature of the rotary evaporation is 25-80 ℃, and the rotating speed is 60-240rpm;

preferably, the rotary evaporation is carried out under negative pressure conditions;

preferably, in step S13, the hydration temperature is 25-80 ℃, the rotation speed of the shaker is 50-250rpm, and the hydration time is 10-60 min.

6. The method of claim 4, wherein the step S2 comprises the steps of:

s21, introducing gas containing nitric oxide into the shell wrapped with the hydration liquid prepared in the step S1 under a vacuum condition, and oscillating to obtain microbubble suspension;

s22, carrying out centrifugal washing on the microbubble suspension prepared in the step S21, and carrying out resuspension by PBS (phosphate buffer solution) to obtain microbubbles;

preferably, the oscillation frequency in the step S21 is 40-80Hz, preferably 70Hz; the oscillation time is 20-100s, preferably 50s;

preferably, the rotation speed of the centrifugal washing in the step S22 is 500-3000rpm, preferably 2000rpm, and the centrifugal time is 1-20min, preferably 5min;

preferably, the centrifugal washing in step S22 is performed 2 to 5 times;

preferably, after the centrifugal washing, before the resuspension with PBS, a step of removing the lower layer liquid to remove the liposome debris is further included.

7. Use of microvesicles according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of erectile dysfunction;

preferably, the is neuro-impaired erectile dysfunction.

8. A medicament for the treatment of erectile dysfunction comprising a microvesicle according to any one of claims 1 to 3.

9. The medicament of claim 8, wherein the medicament is formulated for intravenous administration, subcutaneous administration, intramuscular administration, intracerebroventricular administration, intrathecal administration, or transdermal administration;

preferably, the medicament is formulated for intravenous administration.

10. The medicament of claim 8, wherein the medicament is formulated for UTMD-mediated administration;

preferably, the UTMD conditions are 0.5-1.5MHz, pulse repetition frequency 0.5-1.5kHz, sound pressure 300-700kPa, duty cycle 0.1-1%, time 5-15s.