Method for accurately adjusting targeted drug delivery in small animals on basis of supergravity technology

Description

Method for precisely adjusting drug targeting delivery for small animals based on high gravity technology TECHNICAL FIELD

[0001] The present application belongs to the technical field of targeted drug delivery, and particularly relates to a method for precisely adjusting drug targeting delivery for small animals in vivo based on high gravity technology. BACKGROUND

[0002] Targeted drug delivery is a novel drug delivery mode that selectively and preferentially accumulates therapeutic drugs at target sites and reaches a therapeutic concentration, while preventing entry into non-target sites. Conventional targeted drug delivery methods include passive drug targeting, which achieves delivery by acting on the action site of a target receptor through chemical modification and biological modification of molecules; active drug targeting, which, unlike passive targeting, mainly involves interactions between specific biological systems, such as ligand-receptor, antigen-antibody, enzyme-substrate, etc. In active targeting, there is also a way to achieve specific delivery through stimulus-responsive nanocarriers; in addition to the above two main targeted delivery methods, there are also combination positioning delivery and physical targeting positioning. However, some of the above targeted drugs themselves and carriers exhibit different toxicities and have certain side effects, although these side effects can sometimes be alleviated by supplementing drugs, but usually result in a narrow therapeutic window and greatly reduced therapeutic effect. In drug targeting design, most drugs are not easy to modify on the surface of the drug through chemical and biological means, so the off-target probability is greatly increased, causing off-target toxicity risk. In addition, for the blood organ barriers of the biological body itself, including the blood-brain barrier, blood-testis barrier, blood-placenta barrier, etc., most targeted drugs cannot pass through due to problems such as drug or encapsulated material size. At the same time, for the current physical field means of targeted positioning delivery, the penetration depth of ultrasound is limited, and scattering and diffusing phenomena easily occur when encountering cavities in the body, while the magnetic field method has high penetration depth, but requires the assistance of large equipment such as MRI and the intervention of contrast agents, has low magnetic focusing degree, low positioning resolution, and great limitations in precise control of magnetic fields to achieve physical field directional regulation. Therefore, there are certain limitations in using the above biochemical methods and physical means for targeted delivery of blood organ barriers, organs, and sub-organs. Therefore, there is an urgent need for a non-contact physical field method that can precisely regulate drug targeting and barrier penetration in vivo.

[0003] The supergravity technology refers to a technology of increasing the gravity field acting on an object or an environment to more than 9.8 m / s2. In planetary bodies, the gravity field environment of some planets is a supergravity field, such as Jupiter, red dwarf stars and the like. The supergravity technology is also widely used in the fields of industry and national defense, such as 5g (g=9.8 m / s2) in the process of rocket launch; 10g in the process of fighter plane flight; and several hundred or even tens of thousands of times of g in the process of chemical separation and operation of turbine engines. The supergravity technology can be realized in two ways, i.e., a linear way or a rotating way. Compared with the linear way requiring a large space, the rotating way of creating a supergravity environment by a centrifuge has high space utilization, is safe and reliable, and has adjustable parameters and low energy consumption. Therefore, the centrifuge becomes the main technical means for creating a supergravity environment. The supergravity technology not only has the characteristics of accurately adjusting the size and direction of the gravity field, but also can globally and non-contactly adjust different substances. The increase of the gravity field strength increases the volume force of the substances, so that the self-weight stress and stress gradient of different substances in the supergravity environment are increased, and the size effect is strengthened. In addition, the supergravity also increases the driving force of the relative motion of different substances, and strengthens the separation effect. In addition, the supergravity also accelerates the motion and migration speed between substances, and has a time compression effect. In summary, the supergravity technology has the advantages of in-vivo adjustment, non-contact directional control and the like, and is expected to accurately adjust the in-vivo blood flow environment by a mechanical method from the time and space dimensions, to affect the barrier permeability, and finally realize the target enrichment and delivery of drugs that are difficult to target organs. SUMMARY

[0004] The application provides a method for accurately adjusting drug target delivery of small animals based on supergravity technology, which precisely controls the blood flow environment in the bodies of multiple small animals by a non-contact three-dimensional directional supergravity field, and adjusts the barrier state of multiple blood organs in the body, the position and efficiency of drug target delivery in the organs of small animals by a novel force-chemistry-biology coupling effect, so as to make up for the deficiencies of the previous pure biochemical modification of drugs to adjust the organ targeting and break through the blood organ barrier, and solve the problems in the above background technology.

[0005] To achieve the above object, the application provides the following technical scheme.

[0006] A method for accurately adjusting drug target delivery of small animals based on supergravity technology, comprising the following steps:

[0007] S1: constructing a supergravity field applying device;

[0008] S2: manufacturing a supergravity three-dimensional directional small animal intelligent fixing device;

[0009] The three-dimensional directional small animal intelligent fixing device comprises a small animal fixing device made of transparent material and a disc; a connecting block matching the supergravity field applying device and the small animal fixing device in the S1 step is manufactured; and an electric push rod for accurately controlling the height of the disc is fixed on the connecting block.

[0010] S3: assembly of the supergravity field applying device and the three-dimensional directional small animal intelligent fixing device, and design and integration of the intelligent monitoring equipment;

[0011] The three-dimensional directional small animal intelligent fixing device in the S2 step is assembled to the supergravity field applying device in the S1 step through corresponding position connection.

[0012] The small animal fixing device is placed at the heart position and integrated with a flexible electromyography patch, and a T-shaped / K-shaped / J-shaped flexible thermocouple temperature detection patch is placed at the abdominal cavity position, and the patch is integrated into the upper computer software through RS232 / USB / LAN / GPIB and other instrument signal serial ports for integrated monitoring and control.

[0013] A high-definition CCD camera system is integrated on the front of the supergravity field applying device in the S1 step, corresponding to the position of the small animal fixing device, an optical fiber measurement system is hung in the middle of the device, a black box frame for in vivo fluorescence imaging is integrated on the outer frame of the device, and a photoelectric detection and sensing module for in vivo fluorescence imaging is integrated above the small animal fixing device in the S2 step.

[0014] S4: preparation of drugs for treating various diseases and administration of the drugs to small animals;

[0015] The drugs for treating various diseases are prepared, and then the specific drugs required are released into the small animals.

[0016] S5: target regulation and analysis of the directional supergravity technology for the spatiotemporal precise delivery of drugs in small animals;

[0017] The small animal to which the drug is administered in the S4 step is anesthetized, and then the pre-anesthetized small animal is placed in the fixing device cabin in the S2 step, and the small animal is fixed at the heart position with the flexible electromyography patch and at the abdominal cavity position with the thermocouple temperature detection patch.

[0018] According to the required target requirements, the supergravity field applying speed and the time of applying supergravity in the S1 step are adjusted, and the three-dimensional applying angle of the intelligent fixing device in the S2 step is adjusted.

[0019] After the supergravity applying is completed, according to the pharmacokinetics of different drugs, the small animal is placed for a certain period of time and then in vivo imaging is performed to show the actual enrichment of the labeled drug.

[0020] Subsequently, the experimental small animals are dissected and specific target organs are extracted, then the extracted organs are dissected and prepared into single cell suspension, and finally the flow cytometry is used for flow cytometry analysis of cell target enrichment rate and the like, and single cell omics sequencing is used to obtain the spatial distribution of the drug in the specific organ and the further determination of the specific cell group in the target organ.

[0021] Preferably, the supergravity field applying device is realized by a rotating acceleration type; the rotating acceleration type includes a force receiving component rotating drum and a main shaft, and the main shaft is driven by a motor.

[0022] Preferably, the supergravity field applying device is realized by a linear acceleration type.

[0023] Preferably, the three-dimensional directional small animal intelligent fixing device in the S2 step is extended and stacked up and down to meet the demand of high-throughput experiment.

[0024] Preferably, the drug preparation in S4 includes:

[0025] Nucleic acid drugs - the microfluidic chip is used to combine the buffer containing nucleic acid and nanoliposomes through charge to form nanoliposomes wrapping nucleic acid drugs;

[0026] Inorganic drugs - chemical reactions are used to convert different inorganic compounds into drug molecules, or biological bodies and enzyme reactions are used to synthesize drug molecules;

[0027] Organic drugs - organic chemical reactions are used to synthesize organic molecules to realize drug preparation;

[0028] Nanodrugs - the preparation is realized through nanotechnology design of particle size, structure and material.

[0029] Compared with the prior art, the beneficial effects of the present application are:

[0030] 1. The present application can precisely regulate the blood flow environment in the bodies of multiple small animals through a non-contact three-dimensional directional supergravity field, and regulate the state of various blood organ barriers in the body, the position of organ targeted delivery of the drug in the body of the small animal and the efficiency of the drug in the body of the small animal through a novel force-chemistry-biology coupling effect, thereby making up for the deficiencies of the previous simple biochemical modification of the drug to regulate organ targeting and breakthrough of the blood organ barrier.

[0031] 2. The present application can be combined with any existing drug delivery method that has a demand, and the drug type is not limited, and can be combined with various drug administration methods (tail vein injection, intraperitoneal injection, intramuscular injection and atomization, etc.), and the coupling logic control of physical field regulation first and chemical modification-biological targeting later can be quickly realized.

[0032] 3、The centrifugal generated supergravity physical field regulation means is a non-invasive method, which is safe and non-toxic to small animals and has excellent biological safety.

[0033] 4、The application has simple structure, can be combined with laser speckle equipment, live imaging equipment, optical fiber infrared monitoring equipment and the like, can realize real-time observation of the change of blood flow environment, in-vivo visualization of drug delivery, real-time tracking and the like.

[0034] 5、The application has simple operation, good universality, and the size and number of the three-dimensional adjustable small animal fixer and the integrated intelligent vital sign monitoring device can be expanded arbitrarily, so that high-throughput, large-scale experiments, analysis and production can be easily carried out and the application can be easily integrated with various analysis and screening platforms. BRIEF DESCRIPTION OF DRAWINGS

[0035] Fig. 1 is a structural schematic diagram of a centrifugal rotation type supergravity application device in the embodiment of the application;

[0036] Fig. 2 is a structural schematic diagram of a centrifugal rotation type supergravity application device in the embodiment of the application;

[0037] Fig. 3 is a structural schematic diagram of a connecting piece of a small animal intelligent fixer and a supergravity application device in the embodiment of the application;

[0038] Fig. 4 is a structural schematic diagram of a structural piece fixed to a disc under a small animal fixer in the embodiment of the application;

[0039] Fig. 5 is a structural diagram of a disc fixed to a small animal fixer in the embodiment of the application, when the length (Z axis) of the pneumatic rod is adjusted to be 0;

[0040] Fig. 6 is a manufacturing structural diagram of a small animal fixer in the embodiment of the application;

[0041] Fig. 7 is a manufacturing structural diagram of a small animal fixer in the embodiment of the application;

[0042] Fig. 8 is a structural diagram of an assembled three-dimensional supergravity adjustable direction small animal intelligent fixer in the embodiment of the application;

[0043] Fig. 9 is a structural diagram of three-dimensional directional small animal intelligent fixers stacked in three layers to achieve high-throughput experiments in the embodiment of the application;

[0044] Fig. 10 is a structural diagram of a small animal fixer (fixing cabin) to which a flexible myoelectric sensor and a K-type flexible thermocouple patch are added in the embodiment of the application;

[0045] Fig. 11 is a fluorescence diagram of brain tissue of a live body and a fluorescence intensity quantitative statistical diagram thereof in the embodiment of the application. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be apparently and completely described in combination with the drawings of the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative work fall within the protection scope of the present application.

[0047] Please refer to FIGS. 1-11, the present application provides a method for precisely adjusting drug targeted delivery of small animals based on high gravity technology, comprising the following steps:

[0048] S1: Constructing a "rotating disc centrifugal" type high gravity field applying device. In the embodiment, the high gravity field applying mode is realized by the construction of rotating disc centrifugal type. The overall structure is shown in FIGS. 1-2. In the embodiment, the carbon steel and alloy steel material is processed by the numerical control machine tool five-axis to prepare the force components of the high gravity device, i.e. the rotating drum and the main shaft 1. In the embodiment, the motor mainly adopts the carbon brushless motor, which is then driven by the frequency conversion induction motor, and the power and transmission components of the high gravity device are controlled by the microcomputer, to accurately control the rotating speed of the rotor and the rotating disc (the step high gravity size adjustment of 0.01 g can be realized) and the applying time (1 ms step can be realized, and the maximum single time is 120 h), to realize the programmable automatic control. After the main part of the "rotating disc centrifugal" type high gravity field applying device is completed, the alloy steel material is processed by the numerical control machine tool five-axis to manufacture the placement module for the subsequent fixed CCD monitoring system 2, which is welded to the corresponding position of the rotating disc 4. Then, the device fixing structure 3 for fixing the subsequent experimental devices on the rotating disc is manufactured by the numerical control machine tool five-axis processing of the alloy steel material. Finally, the protection frame 5 of the high gravity centrifugal device is manufactured and processed by the wire cutting flexible metal iron sheet, and is fixed to the corresponding position according to the size of the rotating radius of the rotating disc;

[0049] S2: Processing and manufacturing of three-dimensional supergravity adjustable direction small animal intelligent fixing device. First, through light curing printing, a three-dimensional directional small animal fixing device is manufactured by matching the configuration of the supergravity field applying device manufactured in step S1 and the connection block. As shown in FIG. 3, the ring-shaped connection part 6 of the connection block is matched with the device fixing structure 3 in S1 and is assembled and fixed by pressure. The first connection hole 7 of the connection block is an M8 threaded structure for subsequent assembly and fixation with the small animal fixer and the disc. Then, a structure for fixing the small animal fixer and the disc is manufactured by light curing printing, and the structure is shown in FIG. 4. The second connection hole 8 of the structure is a threaded structure matched with the first connection hole 7, and the two can be assembled by M8-M10 screws. The pneumatic telescopic rod 9 on the structure can be hinged with the mouse fixer and the underlying flat disc to realize the freedom in the Z direction. Then, another electric push rod with precise control is connected behind the disc to realize the positioning after the quantitative control of the Z direction of the disc.

[0050] Then, a disc structure for fixing the small animal fixer is manufactured by the same manufacturing method, and the structure is shown in FIG. 5. The disc 10 is the specific structure of the disc when the pneumatic telescopic rod 9 is adjusted to 0. The threaded holes in the circular array on the disc can be used for subsequent adjustment and fixation of the small animal fixer at different angles. Finally, the small animal fixer is processed by CNC numerical control milling using high-transparency acrylic. The specific structure is shown in FIGS. 6-7. The threaded hole 11 of the small animal fixer is a threaded hole matched with the threaded hole in the circular array of the disc 10. The buckle 12 is used to fix the head of the small animal after the small animal is put in;

[0051] S3: Assembly of the super gravity field applying device and the three-dimensional directional small animal intelligent fixing device, and design and integration of the intelligent monitoring equipment. The three-dimensional directional small animal intelligent fixing device in step S2 is assembled through threaded connection at corresponding positions, and the specific structure is shown in FIGS. 6-7. The three-dimensional directional small animal intelligent fixing device is installed into the connecting structure of the super gravity field applying device manufactured in step S1. In this embodiment, the three-dimensional directional small animal intelligent fixing device in step S2 is expanded and stacked in three layers to meet the requirement of high-throughput experiment in this embodiment, and the structure is shown in FIGS. 8-9. Then, when the small animal is placed in the rotatable fixer on the top layer of each three-dimensional directional small animal intelligent fixing device, a flexible electromyography patch 13 is integrated at the heart position of the small animal, and a K-type flexible thermocouple temperature detection patch 14 is placed at the abdominal cavity position of the small animal. The data are collected and integrated into the self-programmed host computer software through the RS232 signal serial port for integrated monitoring and control. Then, the CCD camera system for high-definition quantitative UV to NIR and 1000HZ high-frequency shooting is loaded on the front surface of the super gravity field applying device at the position corresponding to the small animal fixing device to record the experimental state of the small animal, etc.

[0052] S4: Preparation of drugs for breaking through the blood-brain barrier to treat brain diseases and administration in small animals. First, fluorescently labeled cy5-mRNA was obtained by chemical synthesis, and dissolved in a 0.05M sodium citrate buffer with a pH of 4.0 to obtain a cy5-mRNA buffer with a concentration of 1200 ng / μl. Then, using a precision balance, anhydrous ethanol was weighed and added, and shaken to obtain ionized lipids (MC3) with a volume of 1 ml and a concentration of 650 g / mol, phospholipids (DSPC) with a volume of 1 ml and a concentration of 790 g / mol, cholesterol (CHOL) with a volume of 1 ml and a concentration of 390 g / mol, and amphiphilic phospholipid PEG conjugates (DMPE-PEG2000) with a volume of 1 ml and a concentration of 2700 g / mol. Then, MC3:DSPC:CHOL:DMPE-PEG2000 was dissolved in anhydrous ethanol at a molar ratio of 40:20:35:5 to obtain 500 μl of a nanoliposome alcohol phase with a concentration of 15 mM. Then, 300 μl of the cy5-mRNA buffer and 100 μl of the nanoliposome alcohol phase were drawn into two sterile syringes, respectively, and placed on two different single-channel injection pumps. The flow rate of the cy5-mRNA buffer was set to 0.9 ml / min, and the flow rate of the nanoliposome alcohol phase was set to 0.3 ml / min. The two syringes were connected to the microfluidic chip through a capillary and a steel needle, and the injection pumps were started to push all the solutions in the syringes into the microfluidic chip. The other end collected the cy5-mRNA- wrapped nanoliposomes. The collected cy5-mRNA-wrapped nanoliposomes were transferred to a dialysis bag and placed in 500 ml of a 1% concentration of PBS solution for dialysis for 12 hours. 200 μl of the cy5-mRNA-wrapped nanoliposomes after dialysis for 12 hours were injected into C57BL / 6J mice through the tail vein;

[0053] S5: Directional hypergravity technology for in vivo drug spatiotemporal precise brain targeting delivery regulation and analysis of C57BL / 6J mice. First, the C57BL / 6J mice injected with 200 μl of cy5-mRNA nanoliposomes in the tail vein in step S4 were anesthetized with isoflurane, and then the pre-anesthetized C57BL / 6J mice were placed in the fixed device cabin in step S2 as shown in Figure 7. When placing the C57BL / 6J mice, the heart position of the C57BL / 6J mice was fixed with the flexible electromechanical recording patch, and the abdominal cavity was fixed with the thermocouple temperature detection patch. Then the rotation speed of the hypergravity field application device in step S1 was set to 8g, the application time of the hypergravity field was 1 minute, the three-dimensional application angle of the intelligent fixing device in step S2 was set to 0 degrees in the z-axis direction and 180 degrees in the xy plane. The above operation was set as one cycle, two cycles of hypergravity field were applied, and 5 minutes of static operation was applied between the two cycles to maximize the breakthrough of the blood-brain barrier and achieve brain targeting delivery. After applying two cycles of hypergravity field, the C57BL / 6J mice were placed for 3 hours before brain live imaging. The brain live imaging results are shown in Figure 11, and the cy5-mRNA enrichment of the experimental group is 4-5 times that of the blank group and the control group. Then the C57BL / 6J mice were dissected and their brain tissues were extracted, the brain tissues were dissociated and prepared into single cell suspension by using the kit, and finally the flow cytometry was used for flow analysis to obtain the cell targeting enrichment rate of 80%. The above examples fully demonstrate that the methodology can actually achieve the breakthrough of the blood organ barrier during in vivo drug delivery, and completely non-invasively achieve precise delivery of organs or lesions.

[0054] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for precise adjustment of drug targeting delivery in small animals based on high gravity technology, characterized in that, The method comprises the following steps: S1: constructing a supergravity field applying device; S2: manufacturing a supergravity three-dimensional directional small animal intelligent fixing device; The three-dimensional directional small animal intelligent fixing device comprises a small animal fixer made of transparent material and a disc; A connecting block matching the supergravity field applying device and the small animal fixer in S1 is manufactured, and an electric push rod for accurately controlling the height of the disc is fixed on the connecting block; S3: assembling the supergravity field applying device and the three-dimensional directional small animal intelligent fixing device, and designing and integrating an intelligent monitoring device; The three-dimensional directional small animal intelligent fixing device in S2 is assembled to the supergravity field applying device in S1 through corresponding position connection; A flexible electromyography patch is integrated at the heart position of the small animal fixer, and a T / K / J type flexible thermocouple temperature detection patch is placed in the abdominal cavity, and the two are integrated and monitored and controlled through RS232 / USB / LAN / GPIB instrument signal serial ports and an upper computer software; A high-definition CCD camera system is integrated on the front of the supergravity field applying device in S1 corresponding to the position of the small animal fixer, an optical fiber measurement system is hung in the middle of the device, a black box frame for in vivo fluorescence imaging is integrated on the outer frame of the device, and a photoelectric detection and sensing module for in vivo fluorescence imaging is integrated above the small animal fixer in S2; S4: preparation of drugs for treating various diseases and administration of the drugs to small animals; The drugs for treating various diseases are prepared, and then the specific drugs required are released into the small animals; S5: target regulation and analysis of the time-space accurate delivery of drugs in small animals by directional supergravity technology; The small animal to which the drugs are administered in S4 is anesthetized, and then the pre-anesthetized small animal is placed in the fixing device cabin in S2, and the small animal is fixed with the flexible electromyography patch at the heart position and the thermocouple temperature detection patch in the abdominal cavity; According to the required target requirements, the supergravity field applying speed and the time of applying supergravity in S1 are adjusted, and the three-dimensional applying angle of the intelligent fixing device in S2 is adjusted; After the supergravity application is completed, according to the pharmacokinetics of different drugs, the small animal is placed for a certain period of time and then in vivo imaging is performed to show the actual enrichment of the labeled drugs; Then the experimental small animal is dissected and the specific target organ is extracted, and then the dissected organ is dissociated and prepared into a single cell suspension, and finally the flow cytometry is used for flow cytometry analysis of the cell target enrichment rate and the single cell omics sequencing is used to obtain the spatial distribution of the drugs in the specific organ and the further determination of the specific cell groups in the target organ.

2. The method for precisely regulating the targeted delivery of drugs for small animals based on the supergravity technology according to claim 1, characterized in that, The supergravity field applying device is realized by rotation acceleration; It comprises a force receiving component rotating drum and a main shaft, and the main shaft is driven by a motor.

3. The method for precisely regulating the targeted delivery of drugs for small animals based on high gravity technology according to claim 1, characterized in that, The supergravity field applying device is realized by linear acceleration.

4. The method for precisely regulating the targeted delivery of drugs for small animals based on the supergravity technology according to claim 1, characterized in that, The three-dimensional directional small animal intelligent fixing device in S2 is expanded and stacked up and down to meet the demand of high-throughput experiment.

5. The method for precisely regulating the targeted delivery of drugs for small animals based on high gravity technology according to claim 1, characterized in that, The drug preparation in S4 comprises: Nucleic acid drugs - the buffer containing nucleic acids and nanoliposomes are combined by charge to form nanoliposomes wrapping nucleic acid drugs by using a microfluidic chip; Inorganic drugs - their preparation involves chemical reactions to convert different inorganic compounds into drug molecules, or synthesis of drug molecules using biological and enzymatic reactions; Organic drugs - their preparation involves synthesis of organic molecules through organic chemical reactions to achieve drug preparation; Nanopharmaceuticals - their preparation involves nanotechnology to design particle size, structure, and materials.

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