Coenzyme Q10 nano-liposome capable of being endocytosed by neurons as well as preparation method and application of coenzyme Q10 nano-liposome

Abstract

The invention provides a coenzyme Q10 nano-liposome capable of being endocytosed by brain neurons as well as a preparation method and application of the coenzyme Q10 nano-liposome, and belongs to the technical field of biological medicines. According to the coenzyme Q10 nano-liposome and the preparation method thereof, polycarbonate filter membranes of 220 nm, 100 nm and 50 nm are adopted to sequentially extrude a solution, the coenzyme Q10 nano-liposome which has the particle size range of 30-50 nm, is not prone to melt collapse and can be endocytosed by neurons is prepared, and the preparation method is simple and efficient. According to the invention, nano-liposomes simultaneously wrapping 5nm gold and coenzyme Q10 are further prepared, distribution of the nano-liposomes can be traced through a transmission electron microscope, and whether the drug coenzyme Q10 enters extracellular gaps of brain cells and whether the drug coenzyme Q10 is endocytosed into neurons or not is verified. The coenzyme Q10 nano-liposome disclosed by the invention can penetrate through a blood brain barrier, enter extracellular gaps and are endocytosed by neurons to eliminate formaldehyde accumulated in the brain, so that a foundation is provided for research and development of drugs for brain diseases such as Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a coenzyme Q10 nanoliposome capable of being endocytosed by neurons, a preparation method and application thereof. BACKGROUND

[0002] In the world, the incidence of central nervous system (CNS) diseases is rising, but the development of CNS drugs is still very challenging, with high cost, long development cycle and high failure rate. CNS is highly protected by physiological barriers, especially the blood-brain barrier (BBB) and the extracellular space (ECS) of neurons of only 38-64 nm, which prevents drugs from reaching the target in the brain from the site of entry into the human body. The diameter of the drug also limits the endocytosis of the drug by neurons, and drugs with too large a diameter cannot enter the cytoplasm of neurons to exert the effect of rescuing the viability of neurons. It can be seen that the challenge of drug delivery to the brain is usually attributed to complex and highly regulated barriers, and the drug delivery system must penetrate the brain parenchyma to reach the target disease site. For example, the typical pathological feature of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aβ) in the 38-64 nm ECS to form senile plaques (SP). The accumulation of endogenous formaldehyde with age promotes the aggregation of Aβ, which can block the extracellular space and slow down or stop the drainage of interstitial fluid (ISF); cause disorders in neurotransmitter transmission, metabolic waste discharge, etc.; and induce neuronal death and memory disorders. Even drugs dissolved in interstitial fluid cannot reach damaged neurons, so they cannot exert a rescuing effect, and the blockage of ECS by senile plaques is a key reason why drugs cannot reach target cells. Therefore, it is crucial to prepare nanodrugs that can pass through ECS and BBB for the treatment of AD and other CNS diseases.

[0003] Liposomes are one of the world-recognized relatively safe nanomaterials, which can be used as efficient carriers for carrying drugs or other nanoparticles. A large number of studies have shown that drugs with a particle size of less than 100 nanometers can penetrate the blood-brain barrier (BBB). At present, although a large number of methods for preparing nanoliposomes have been reported, such as the thin film hydration method and the reverse evaporation method, the preparation methods are usually complex, and the nanoliposomes prepared by the methods are prone to collapse, with poor stability and drug efficacy. At the same time, the nanoliposomes prepared by the existing methods are also difficult to meet the particle size requirement of less than 38-64 nm and to pass through ECS, especially for coenzyme Q10, there is no related report on the preparation of nanoliposomes with a particle size of less than 50 nm and stable delivery to neurons in the brain. SUMMARY

[0004] In order to solve the problems in the prior art, the present application aims to provide a coenzyme Q10 nanoliposome capable of being endocytosed by neurons and a preparation method thereof.

[0005] The present application also aims to provide a nanoliposome simultaneously encapsulating nanogold and coenzyme Q10 and a preparation method thereof.

[0006] In order to achieve the above-mentioned aims, the present application provides the following technical solutions.

[0007] The present application provides a preparation method of a coenzyme Q10 nanoliposome, comprising the following steps:

[0008] Coenzyme Q10, soybean oil, egg yolk lecithin, polyethylene 12-hydroxy stearate and 95% ethanol are mixed to obtain a uniform solution; the uniform solution is added into glucose injection and mixed to obtain a mixed solution; the mixed solution is extruded 8-12 times using polycarbonate filter membranes with diameters of 220 nm, 100 nm and 50 nm in sequence to obtain a coenzyme Q10 nanoliposome solution.

[0009] Preferably, the coenzyme Q10, soybean oil, egg yolk lecithin, polyethylene 12-hydroxy stearate and 95% ethanol are mixed according to a mass ratio of (9-11):(9-11):(9-11):(13-17):(50-60).

[0010] Preferably, the mixing step of the uniform solution comprises: taking egg yolk lecithin in a container, sequentially adding 95% ethanol, soybean oil and polyethylene 12-hydroxy stearate, uniformly stirring under water bath heating, then adding coenzyme Q10, sealing, and uniformly stirring under water bath heating to obtain the uniform solution.

[0011] Preferably, the uniform solution and glucose injection are mixed according to a volume ratio of 1:(90-110).

[0012] The present application also provides a coenzyme Q10 nanoliposome prepared by the above-mentioned preparation method.

[0013] Preferably, the coenzyme Q10 nanoliposome has a particle size of 30-50 nm.

[0014] The application further provides a preparation method of the nano-liposome simultaneously wrapping nano-gold and coenzyme Q10, comprising the following steps: mixing coenzyme Q10, gold nanoparticles, soybean oil, egg yolk lecithin, polyethylene 12-hydroxy stearate and 95% ethanol to obtain a uniform solution; adding the uniform solution into glucose injection to mix uniformly to obtain a mixed solution; and extruding the mixed solution using polycarbonate filter membranes with diameters of 220 nm, 100 nm and 50 nm for 8-12 times respectively to obtain a nano-liposome solution simultaneously wrapping nano-gold and coenzyme Q10.

[0015] Preferably, the gold nanoparticles, coenzyme Q10, soybean oil, egg yolk lecithin, polyethylene 12-hydroxy stearate and 95% ethanol are mixed according to the mass ratio (5-7) : (9-11) : (9-11) : (9-11) : (13-17) : (50-60).

[0016] The application further provides the nano-liposome simultaneously wrapping nano-gold and coenzyme Q10 prepared by the above preparation method.

[0017] The application further provides application of the above coenzyme Q10 nano-liposome or the above nano-liposome simultaneously wrapping nano-gold and coenzyme Q10 in preparation or research and development of coenzyme Q10 nano-drugs.

[0018] Compared with the prior art, the technical scheme of the application has the following beneficial effects:

[0019] The preparation method of the coenzyme Q10 nano-liposome provided by the application is simple, efficient and low in cost, and only needs to extrude the solution using polycarbonate filter membranes with diameters of 220 nm, 100 nm and 50 nm in sequence to obtain coenzyme Q10 nano-liposomes with a particle size range of 30-50 nm and an average diameter of about 35 nm, and the prepared coenzyme Q10 nano-liposomes are not prone to fusion and collapse and can be endocytosed by neurons to play an effective therapeutic role.

[0020] The 5 nm gold and coenzyme Q10 are simultaneously wrapped to prepare nano-liposomes, and whether the nano-liposomes penetrate the blood-brain barrier and distribute in the brain ECS can be traced by transmission electron microscopy, and whether the drug enters the brain extracellular space and is endocytosed into neurons is verified, thereby providing a basis for research and development of coenzyme Q10 nano-drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : TEM micrographs of coenzyme Q10 nano-liposomes in each group;

[0022] Figure 2 : Hydrodynamic diameter distribution of coenzyme Q10 nano-liposomes in each group;

[0023] Figure 3Zeta potential distribution of coenzyme Q10 nanoliposomes in each group;

[0024] Figure 4 Images of the solutions of each group of coenzyme Q10 nanoliposomes;

[0025] Figure 5 Comparison of Coenzyme Q10 nanoliposome melting and collapse experiment results;

[0026] Figure 6 TEM images of nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 in each group;

[0027] Figure 7 Hydrodynamic diameter distribution of nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 in each group;

[0028] Figure 8 Zeta potential distribution of nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 in each group;

[0029] Figure 9 Transmission electron micrographs of the CA1 region of the hippocampus in WT and AD mice;

[0030] Figure 10 Statistical graph of AuNPs entering the extracellular space of the CA1 region of the hippocampus;

[0031] Figure 11 Transmission electron micrographs of the prefrontal cortex of C57 and AD mice;

[0032] Figure 12 Statistical graph of AuNPs entering the extracellular space of the prefrontal cortex;

[0033] Figure 13 Statistics on the number of 120nm Q10Au@NPs injected into the hippocampus via lateral ventricle injection and intraperitoneal injection;

[0034] Figure 14 : Intracellular coenzyme Q10 fluorescence imaging in SH-SY5Y cells;

[0035] Figure 15 Changes in the relative fluorescence intensity of coenzyme Q10 in SH-SY5Y cells over time;

[0036] Figure 16 Statistical analysis of total fluorescence intensity of coenzyme Q10 in SH-SY5Y cells after culturing with different coenzyme Q10 groups from 0 to 6 hours;

[0037] Figure 17 Changes in intracellular coenzyme Q10 content after SH-SY5Y cells were cultured with standard coenzyme Q10 at a concentration of 100 μM, coenzyme Q10 at a concentration of 120 nm, and coenzyme Q10 at a concentration of 35 nm, respectively.

[0038] Figure 18 Changes in extracellular coenzyme Q10 content after culturing SH-SY5Y cells with standard coenzyme Q10 at concentrations of 100 μM, 120 nm, and 35 nm.

[0039] Figure 19 Changes in intracellular coenzyme Q10 content after SH-SY5Y cells were cultured with standard coenzyme Q10 at a concentration of 1000 μM, coenzyme Q10 at a concentration of 120 nm, and coenzyme Q10 at a concentration of 35 nm, respectively.

[0040] Figure 20 Changes in extracellular coenzyme Q10 content after SH-SY5Y cells were cultured with standard coenzyme Q10 at a concentration of 1000 μM, coenzyme Q10 at a concentration of 120 nm, and coenzyme Q10 at a concentration of 35 nm, respectively.

[0041] Figure 21 Cytotoxicity of 120nm and 35nm coenzyme Q10. Detailed Implementation

[0042] This invention provides a method for preparing coenzyme Q10 nanoliposomes, comprising the following steps: mixing coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate and 95% ethanol to obtain a homogeneous solution; adding the homogeneous solution to glucose injection and mixing to obtain a mixed solution; extruding the mixed solution sequentially through polycarbonate filter membranes with diameters of 220 nm, 100 nm and 50 nm 8 to 12 times each to obtain a coenzyme Q10 nanoliposome solution.

[0043] The coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate, and 95% ethanol of this invention are mixed in a mass ratio of (9-11):(9-11):(9-11):(13-17):(50-60); preferably, they are mixed in a mass ratio of 10:10:10:15:55. This invention does not limit the specific sources of coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate, and 95% ethanol.

[0044] The mixing steps of the homogeneous solution described in this invention include: placing egg yolk lecithin in a container, sequentially adding 95% ethanol, soybean oil, and polyethylene glycol 12-hydroxystearate, heating and stirring in a water bath until homogeneous, then adding coenzyme Q10, sealing, and heating and stirring in a water bath to obtain a homogeneous solution. The water bath heating temperature is 55–65°C, preferably 60°C.

[0045] The homogeneous solution and glucose injection solution of the present invention are mixed at a volume ratio of 1:(90-110), preferably 1:100.

[0046] This invention employs polycarbonate filter membranes with diameters of 220 nm, 100 nm, and 50 nm, extruding them 8-12 times each. Specifically, a 220 nm diameter polycarbonate filter membrane is extruded 8-12 times, followed by a 100 nm diameter polycarbonate filter membrane, and finally a 50 nm diameter polycarbonate filter membrane. The preferred number of extrusions is 10. This invention, by sequentially extruding with 220 nm, 100 nm, and 50 nm polycarbonate filter membranes, yields coenzyme Q10 nanoliposomes that not only meet the required particle size but, most importantly, are less prone to melting and collapse. While extruding with 220 nm and 50 nm polycarbonate filter membranes, or with other sizes or particle size gradients, can also produce liposomes smaller than 50 nm, these liposomes tend to melt and collapse upon entering the body or cells, failing to achieve the desired therapeutic effect.

[0047] The present invention also provides coenzyme Q10 nanoliposomes prepared by the above preparation method, with a particle size range of 30-50 nm, an average diameter of 35 nm, and not prone to melting and collapse, and can be internalized by neurons to exert an effective therapeutic effect.

[0048] This invention also provides a method for preparing nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10, comprising the following steps: mixing coenzyme Q10, gold nanoparticles, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate, and 95% ethanol to obtain a homogeneous solution; adding the homogeneous solution to glucose injection solution and mixing thoroughly to obtain a mixed solution; sequentially pressing the mixed solution through polycarbonate filter membranes with diameters of 220 nm, 100 nm, and 50 nm 8-12 times each to obtain a nanoliposome solution simultaneously encapsulating gold nanoparticles and coenzyme Q10. The gold nanoparticles are preferably 5 nm gold particles.

[0049] The gold nanoparticles, coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate and 95% ethanol of the present invention are mixed in a mass ratio of (5-7):(9-11):(9-11):(9-11):(13-17):(50-60), preferably in a mass ratio of 6:10:10:10:15:55.

[0050] This invention also provides nanoliposomes prepared by the above method, simultaneously encapsulating gold nanoparticles and coenzyme Q10, with a particle size range of 30–50 nm, and which are not prone to melting and collapse. By introducing gold nanoparticles into the nanoliposomes, this invention allows direct observation of the nanoliposomes entering the extracellular space of brain cells and their endocytosis into neurons.

[0051] The present invention also provides the application of the above-mentioned coenzyme Q10 nanoliposomes or the above-mentioned nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 in the preparation or development of coenzyme Q10 nanomedicines.

[0052] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0053] In a specific embodiment of the present invention, coenzyme Q10 was purchased from Taizhou Hongyao Chemical Co., Ltd., batch number 20190601; soybean oil (LCT) was purchased from Zhonghang (Tieling) Pharmaceutical Co., Ltd., batch number Y13060103-2-01; polyethylene glycol 12-hydroxystearate (HS15) was purchased from BASF SE, batch number 28484147GO; egg yolk lecithin (E80) was purchased from LTpoTd, batch number 510300-21738951921; and 95% ethanol was purchased from Nanjing Reagent Co., Ltd., batch number 1908202061B. The DF-101S thermostatic magnetic stirrer was purchased from Zhengzhou Hengyan, equipment code DLO1A01029; the AL-204 electronic balance was purchased from Mettler, equipment code DLO1A02012; and the Nicomp2300 nanoparticle size analyzer was purchased from PSS, equipment code DL0ZA02029.

[0054] Unless otherwise specified, the following embodiments are all conventional methods.

[0055] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0056] Example 1

[0057] The formula for Coenzyme Q10 nanoliposomes is as follows: Coenzyme Q10 1g, soybean oil 1g, egg yolk lecithin (E80) 1g, polyethanol 12-hydroxystearate 1.5g, and 95% ethanol 5.5g.

[0058] Preparation of Coenzyme Q10 nanoliposomes:

[0059] (1) Weigh out the prescribed amount of coenzyme Q10 for later use; weigh the prescribed amount of E80 into a 10mL vial, add the prescribed amount of 95% ethanol, soybean oil and polyethanol 12-hydroxystearate in sequence, stir in a 60℃ water bath until uniform; then add the weighed coenzyme Q10, seal, and continue to stir in a 60℃ water bath to form a homogeneous solution.

[0060] (2) Take 1 mL of the homogeneous solution and add it to 100 mL of glucose injection solution. Shake well and squeeze the solution 10 times using a 220 nm polycarbonate filter membrane. Squeeze the squeezed solution 10 times using a 100 nm polycarbonate filter membrane. Squeeze the squeezed solution 10 times using a 50 nm polycarbonate filter membrane to obtain a coenzyme Q10 nanoliposome solution. This solution is denoted as 35 nm Q10@NPs.

[0061] Example 2

[0062] The formula for Coenzyme Q10 nanoliposomes is as follows: Coenzyme Q10 0.9g, soybean oil 0.9g, egg yolk lecithin (E80) 0.9g, polyethanol 12-hydroxystearate 1.3g, and 95% ethanol 6g.

[0063] Preparation of Coenzyme Q10 nanoliposomes:

[0064] (1) Weigh out the prescribed amount of coenzyme Q10 for later use; weigh the prescribed amount of E80 into a 10mL vial, add the prescribed amount of 95% ethanol, soybean oil and polyethanol 12-hydroxystearate in sequence, stir evenly in a 60℃ water bath; then add the weighed coenzyme Q10, seal, and continue to stir in a 60℃ water bath to form a homogeneous solution.

[0065] (2) Take 1 mL of homogeneous solution, add it to 90 mL of glucose injection solution and shake well. Use a 220 nm polycarbonate filter membrane to squeeze the solution 8 times. Use a 100 nm polycarbonate filter membrane to squeeze the solution 8 times. Use a 50 nm polycarbonate filter membrane to squeeze the solution 8 times to obtain a coenzyme Q10 nanoliposome solution.

[0066] Example 3

[0067] The formula for Coenzyme Q10 nanoliposomes is as follows: Coenzyme Q10 1.1g, soybean oil 1.1g, egg yolk lecithin (E80) 1.1g, polyethanol 12-hydroxystearate 1.7g, and 95% ethanol 5g.

[0068] Preparation of Coenzyme Q10 nanoliposomes:

[0069] (1) Weigh out the prescribed amount of coenzyme Q10 for later use; weigh the prescribed amount of E80 into a 10mL vial, add the prescribed amount of 95% ethanol, soybean oil and polyethanol 12-hydroxystearate in sequence, stir evenly in a 60℃ water bath; then add the weighed coenzyme Q10, seal, and continue to stir in a 60℃ water bath to form a homogeneous solution.

[0070] (2) Take 1 mL of homogeneous solution, add it to 110 mL of glucose injection solution and shake well. Use a 220 nm polycarbonate filter membrane to squeeze the solution 12 times. Use a 100 nm polycarbonate filter membrane to squeeze the solution 12 times. Use a 50 nm polycarbonate filter membrane to squeeze the solution 12 times to obtain a coenzyme Q10 nanoliposome solution.

[0071] Comparative Example 1

[0072] The formulation of coenzyme Q10 nanoliposomes is the same as in Example 1.

[0073] Preparation of Coenzyme Q10 nanoliposomes:

[0074] (1) Same as Example 1.

[0075] (2) Take 1 mL of the homogeneous solution and add it to 100 mL of glucose injection solution. Shake well and squeeze the solution 10 times using a 220 nm polycarbonate filter membrane. After squeezing, squeeze the solution 10 times using a 100 nm polycarbonate filter membrane to obtain a coenzyme Q10 nanoliposome solution. It is denoted as 120 nm Q10@NPs.

[0076] Comparative Example 2

[0077] The formulation of coenzyme Q10 nanoliposomes is the same as in Example 1.

[0078] Preparation of Coenzyme Q10 nanoliposomes:

[0079] (1) Same as Example 1.

[0080] (2) Take 1 mL of homogeneous solution, add it to 100 mL of glucose injection solution and shake well. Use a 220 nm polycarbonate filter membrane to squeeze the solution 10 times. After squeezing, use a 50 nm polycarbonate filter membrane to squeeze the solution 10 times to obtain a coenzyme Q10 nanoliposome solution.

[0081] Experimental Example 1

[0082] The coenzyme Q10 nanoliposome solutions prepared in Example 1 and Comparative Example 1 were characterized as follows:

[0083] 1. The coenzyme Q10 nanoliposome solutions prepared in Example 1 and Comparative Example 1 were scanned using TEM. The TEM micrographs are shown below. Figure 1 As shown in the figure, the scale bar is 100 nm. The results show that the 120 nm Q10@NPs and 35 nm Q10@NPs are white spheres with particle sizes of approximately 120 nm and 35 nm, respectively, by TEM scanning, and their hydrodynamic diameters are 125.79 nm and 34.67 nm, respectively.

[0084] 2. The coenzyme Q10 nanoliposome solutions prepared in each group were analyzed by laser scattering using an Anton Paar particle size analyzer. The results are as follows: Figure 2 As shown in the figure. The results showed that 73% of the liposomes in the 120nm Q10@NPs group had a particle size of 100-130nm, with an average particle size of 125.79nm; while 69% of the liposomes in the 35nm Q10@NPs group had a particle size of 31-38nm, with an average particle size of 34.67nm.

[0085] 3. The zeta potential distribution of the coenzyme Q10 nanoliposome solutions prepared in each group was detected using a nanoparticle size analyzer. The results are as follows: Figure 3 As shown in the figure. The results show that the average Zeta potential of the 35nm Q10@NPs group is -13.19mV, while that of the 120nm Q10@NPs group is -12.17mV. The absolute value of the Zeta potential of the 35nm Q10@NPs group is greater than that of the 120nm Q10@NPs group, indicating that the 35nm Q10@NPs group has higher stability.

[0086] 4. The coenzyme Q10 nanoliposome solutions prepared in each group were diluted 5 times with a 5% glucose solution. A glucose solution of the same concentration and a coenzyme Q10 solution (0.9% coenzyme Q10 solution dissolved in a 5% glucose solution) were used as controls. The solutions in each group were as follows: Figure 4 As shown in the figure, empty liposomes represent the glucose solution group, CoQ10 liposomes represent the coenzyme Q10 solution group, 120nm Q10@NPs represent the 120nm Q10@NPs group, and 35nm Q10@NPs represent the 35nm Q10@NPs group. The results show that the formulation is an orange-red oily solution, with uniform mixing of raw materials and excipients. The formulation mixes relatively quickly, and after preparation, it exhibits good flowability and greater clarity. The 35nm Q10@NPs, after dilution, appears as an orange-yellow emulsion, uniformly dispersed in glucose, and shows no precipitation after prolonged standing at room temperature, indicating good stability.

[0087] 5. The encapsulation efficiency of the coenzyme Q10 nanoliposomes prepared in Example 1 was tested and found to be 69%.

[0088] 6. Melting-disintegration experiments were conducted on the coenzyme Q10 nanoliposomes prepared in Example 1 and Comparative Example 2.

[0089] The experimental procedure was as follows: Coenzyme Q10 nanoliposomes with different nanodiameters were extruded and released at different times (0, 5, 10, 20 h) using dialysis bags to determine the in vitro release rate of the drug.

[0090] The results are as follows Figure 5As shown in the figure, 200nm-100nm-50nm-Q10 represents 35nm coenzyme Q10 obtained by extrusion through three filter membranes (35nm Q10@NPs obtained in Example 1), 200nm-50nm-Q10 represents 35nm coenzyme Q10 obtained by extrusion through two filter membranes (35nm Q10@NPs obtained in Comparative Example 2), and standard Q10 represents coenzyme Q10 solution (coenzyme Q10 solution concentration of 0.9%, obtained by dissolving in 5% glucose solution). The results show that the 35nm coenzyme Q10 obtained by extrusion through the three filter membranes is released uniformly; while the coenzyme Q10 that has not been extruded through the 100nm filter membrane shows early disintegration, and in the later stage, like the control, there is no sustained release effect.

[0091] Example 4

[0092] The formulation of nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 is as follows: 0.5 mg gold nanoparticles, 1.1 g coenzyme Q10, 1.1 g soybean oil, 1.1 g egg yolk lecithin (E80), 1.7 g polyethanol 12-hydroxystearate, and 5 g 95% ethanol.

[0093] Preparation of Coenzyme Q10 nanoliposomes:

[0094] (1) Weigh out the prescribed amount of coenzyme Q10 for later use; weigh the prescribed amount of E80 into a 10mL vial, add the prescribed amount of nano gold particles, 95% ethanol, soybean oil and polyethanol 12-hydroxystearate in sequence, stir evenly in a 60℃ water bath; then add the weighed coenzyme Q10, seal, and continue to stir in a 60℃ water bath to form a homogeneous solution.

[0095] (2) Take 1 mL of the homogeneous solution and add it to 100 mL of glucose injection solution. Shake well and squeeze the solution 10 times using a 220 nm polycarbonate filter membrane. Squeeze the squeezed solution 10 times using a 100 nm polycarbonate filter membrane. Squeeze the squeezed solution 10 times using a 50 nm polycarbonate filter membrane to obtain a coenzyme Q10 nanoliposome solution. This solution is denoted as 35 nm Q10-Au@Lip.

[0096] Comparative Example 3

[0097] The formulation of nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 is the same as in Example 4.

[0098] Preparation of Coenzyme Q10 nanoliposomes:

[0099] (1) Same as Example 4.

[0100] (2) Take 1 mL of the homogeneous solution and add it to 100 mL of glucose injection solution. Shake well and squeeze the solution 10 times using a 220 nm polycarbonate filter membrane. After squeezing, squeeze the solution 10 times using a 100 nm polycarbonate filter membrane to obtain a coenzyme Q10 nanoliposome solution. It is denoted as 120 nm Q10-Au@Lip.

[0101] Experimental Example 2

[0102] The nanoliposome solutions simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared in Example 4 and Comparative Example 3 were characterized:

[0103] 1. The nanoliposome solutions simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared in each group were scanned using TEM. The TEM micrographs are shown below. Figure 6 As shown in the figure, the scale bar is 100 nm, the circled areas represent liposome particles, and the black area on the right represents 5 nm gold. The results show that the liposome complex of 120 nm Q10-Au@Lip has a particle size between 100 and 130 nm and a hydrodynamic diameter of 122.26 nm; the liposome complex of 35 nm Q10-Au@Lip has a particle size between 30 and 50 nm and a hydrodynamic diameter of 35.03 nm.

[0104] 2. The nanoliposome solutions simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared in each group were analyzed by laser scattering using an Anton Paar particle size analyzer. The results are as follows: Figure 7 As shown in the figure. The results showed that 81% of the liposomes in the 120nm Q10-Au@Lip group had a particle size of 100-130nm, with an average particle size of 122.26nm; while 74% of the liposomes in the 35nm Q10-Au@Lip group had a particle size of 30-35nm, with an average particle size of 35.03nm.

[0105] 3. The zeta potential distribution of the nanoliposome solutions simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared in each group was detected using a nanoparticle size analyzer. The results are as follows: Figure 8 As shown in the figure. The results show that the average Zeta potential of the 35nm Q10-Au@Lip group is -15.6mV, while that of the 120nm Q10-Au@Lip group is -13.4mV. The absolute value of the 35nm Q10-Au@Lip group is greater than that of the 120nm Q10-Au@Lip group, indicating that the 35nm Q10-Au@Lip has higher stability.

[0106] 4. The encapsulation efficiency of the nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared in Example 4 was tested and found to be 74%.

[0107] Experimental Example 3

[0108] Electron microscopy was used to demonstrate the extracellular distribution of the in vivo nanomedicines in the 120nm Q10-Au@Lip prepared in Comparative Example 3 and the 35nm Q10-Au@Lip prepared in Example 4.

[0109] Preparation of Au@Lip: 5g of lecithin and 1g of cholesterol were dissolved in 10mL of dichloromethane. The solution was transferred to a round-bottom flask and rotary evaporated to form a thin film. The rotary evaporation temperature was 50℃ and the rotation speed was 60r / min. After film formation, the film was vacuum dried for 50min to remove residual dichloromethane. 2g of AuNPs were added to 10mL of 200mM (NH4)2SO4 solution to obtain a solution of AuNPs. The solution of AuNPs was poured into a round-bottom flask and hydrated at 50℃ for 1h to obtain liposomes encapsulating AuNPs (Au@Lip).

[0110] Preparation of Q10@AuNPs: Take 1 mL of 0.9% coenzyme Q10 solution, add it to 100 mL of liposomes containing AuNPs (Au@Lip) and shake well. Squeeze the solution 10 times using a 220 nm polycarbonate filter membrane. After squeezing, squeeze the solution 10 times using 100 nm and 50 nm polycarbonate filter membranes respectively to obtain a coenzyme Q10 nanoliposome solution, which is 35 nm Q10-Au@Lip.

[0111] 1. Tissue Collection: Acute tissue samples were collected from AD mice after injection of 120nm Q10-Au@Lip, 35nm Q10-Au@Lip, Q10@AuNPs, and Au@Lip, respectively. Intraperitoneal injection was administered 4 weeks prior, followed by a stereotactic injection of 1 ml of the brain 3 hours later. Five minutes later, the mice were anesthetized with 1.25% tribromoethanol intraperitoneal solution. After the mice were unconscious, they were euthanized by cervical dislocation. Brain tissue was removed and placed in ice-cold culture dishes. The bilateral prefrontal cortex and the CA1 region of the hippocampus were immediately removed and placed in culture dishes containing electron microscopy fixative. 1mm*1mm*1mm tissue blocks were surgically cut and placed in EP tubes containing electron microscopy fixative. The tissue was fixed at room temperature in the dark for 2 hours, then transferred to 4℃ for storage. The mice were rinsed three times with 0.1M phosphate buffer PB (pH 7.4), 15 minutes each time.

[0112] 2. Post-fixation: 1% osmium tetroxide was fixed at room temperature for 2 hours in the dark using 0.1M phosphate buffer PB (pH 7.4). The sample was then rinsed three times with 0.1M phosphate buffer PB (pH 7.4), 15 minutes each time.

[0113] 3. Room temperature dehydration: The tissue was sequentially immersed in 30%-50%-70%-80%-95%-100%-100% alcohol for upward dehydration, 20 minutes each time, followed by 100% acetone twice, 15 minutes each time.

[0114] 4. Infiltration embedding: Acetone: 812 embedding agent = 1:1, 37℃ for 2-4 hours; Acetone: 812 embedding agent = 1:2, infiltrate overnight at 37℃; Pure 812 embedding agent, 37℃ for 5-8 hours. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate, and bake in an oven at 37℃ overnight.

[0115] 5. Polymerization: Place the embedding plate in a 60℃ oven for polymerization for 48 hours, then remove the resin block for later use.

[0116] 6. Ultrathin sectioning: The resin block is used to make ultrathin sections of 60-80 nm using an ultrathin microtome, and the sections are retrieved using a 150-mesh anti-slip copper mesh.

[0117] 7. Staining: Stain copper mesh in 2% uranium acetate saturated alcohol solution for 8 min in the dark; wash 3 times with 70% alcohol; wash 3 times with ultrapure water; stain 8 min in 2.6% lead citrate solution in the dark; wash 3 times with ultrapure water, and blot dry slightly with filter paper. Place copper mesh sections in a copper mesh box and dry overnight at room temperature.

[0118] 8. Transmission electron microscopy (TEM) imaging:

[0119] Manufacturer: JEOL Ltd.; Model: JEM-F200;

[0120] Imaging conditions: Voltage 200KV, magnification 50K×;

[0121] Imaging process: Place the copper mesh into the sample loading well, evacuate for 30 minutes, and then locate the target area.

[0122] Figure 9Transmission electron microscopy (TEM) images of the CA1 region of the hippocampus in WT and AD mice are shown. The scale bars from left to right are 2 μm, 100 nm, and 25 nm, respectively. From top to bottom, the images represent WT, WT+AuNPs, WT+120 nm Q10@AuNPs, WT+35 nm Q10@AuNPs, AD+AuNPs, AD+Q10@AuNPs, AD+120 nm Q10@AuNPs, and AD+35 nm Q10@AuNPs. Small triangles represent 5 nm gold in the intercellular space (ECS); large triangles represent 5 nm gold that has entered the cell (cytoplasm). The rectangular inset is magnified. AD+35nmQ10@AuNPs indicates AD mice injected with 35nm Q10-Au@Lip, AD+120nm Q10@AuNPs indicates AD mice injected with 120nm Q10-Au@Lip, AD+Q10@AuNPs indicates AD mice injected with Q10@AuNPs, AD+AuNPs indicates AD mice injected with Au@Lip (5nm gold liposomes), WT+35nm Q10@AuNPs indicates WT mice injected with 35nm Q10-Au@Lip, WT+120nm Q10@AuNPs indicates WT mice injected with 120nm Q10-Au@Lip, WT+AuNPs indicates WT mice injected with Au@Lip, and WT indicates WT mice that did not receive any injections. Figure 10 A statistical plot of AuNPs entering the extracellular space of the CA1 region of the hippocampus (n=5), F (4,20) =8.514, P=0.0004, one-way ANOVA.

[0123] Figure 11Transmission electron microscopy (TEM) images of the prefrontal cortex of C57 and AD mice are shown. The scale bars from left to right are 100 nm and 25 nm, respectively. From top to bottom, the images represent WT+AuNPs, WT+120 nm Q10+AuNPs (WT+120 nm Q10@AuNPs), WT+35 nm Q10+AuNPs (WT+35 nm Q10@AuNPs), AD+AuNPs, AD+120 nm Q10+AuNPs (AD+120 nm Q10@AuNPs), and AD+35 nm Q10+AuNPs (AD+35 nm Q10@AuNPs). Small triangles represent 5 nm gold in the intercellular space (ECS); large triangles represent 5 nm gold that has entered the cell (cytoplasm). The rectangular inset is magnified. AD+35nm Q10@AuNPs indicates AD mice injected with 35nm Q10-Au@Lip, AD+120nm Q10@AuNPs indicates AD mice injected with 120nm Q10-Au@Lip, AD+AuNPs indicates AD mice injected with Au@Lip (5nm gold liposomes), WT+35nm Q10@AuNPs indicates WT mice injected with 35nm Q10-Au@Lip, WT+120nm Q10@AuNPs indicates WT mice injected with 120nm Q10-Au@Lip, and WT+AuNPs indicates WT mice injected with Au@Lip. Figure 12 A statistical plot of AuNPs entering the extracellular space of the prefrontal cortex (n=5), F (4,20) =7.214, P=0.0009, one-way ANOVA.

[0124] The results showed that gold particles of varying degrees were found in the extracellular space (small triangles) and intraneurons (large triangles) of both WT and AD mice in the CA1 region of the hippocampus and the prefrontal cortex. In both WT and AD mice, the 35nm Q10-Au@Lip group had more gold particles in the ECS of the CA1 region of the hippocampus and the prefrontal cortex than the 120nm Q10-Au@Lip group.

[0125] Figure 13 The number of 120nm Q10Au@NPs (120nm Q10-Au@Lip) injected into the hippocampus via lateral ventricle and peritoneum (n=4). (3,9) =1.500, P=0.2797; F (3,9) =35.70, P<0.0001, two-way ANOVA. *: P<0.05, **: P<0.01, ***: P<0.001.

[0126] The results showed that 35nm Q10-Au@Lip could cross the blood-brain barrier (BBB), but the number of gold particles injected intraperitoneally was less than that injected intraventricularly, indicating that 35nm Q10-Au@Lip experienced some loss during blood circulation and BBB crossing. While 120nm Q10-Au@Lip could cross the BBB, it struggled to penetrate the extracorporeal membrane oxygenation (ECS) with a diameter of 38–64 nm.

[0127] Test Example 4

[0128] Evidence of nanomedicines being endocytotic by neurons:

[0129] 1. SH-SY5Y cells were cultured from the Institute of Geriatrics, Wenzhou Medical University, China. SH-SY5Y cells were seeded in complete culture medium (DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) and cultured at 37°C and 5% CO2.

[0130] 2. CoQ10 Fluorescence Imaging: Standard coenzyme Q10, 120nm Q10-Au@Lip, and 35nm Q10-Au@Lip cell culture media were prepared using DMEM / F-12 cell culture medium, with concentrations of 100μM and 1000μM, respectively. After SH-SY5Y cells reached 70% confluence, the medium was replaced with cell culture media containing standard coenzyme Q10, 120nm Q10-Au@Lip, and 35nm Q10-Au@Lip, and cultured for 0h, 0.5h, 1h, 3h, and 6h, respectively. The control group was replaced with normal cell culture medium. 1mL of coenzyme Q10 staining solution was added to the culture dish, and staining was performed at room temperature in the dark for 15min. Images were scanned and captured using a laser confocal microscope, with the excitation wavelength set to 450nm and the emission wavelength to 515nm. The relative fluorescence intensity of intracellular coenzyme Q10 was statistically analyzed using ZEISS microscopy image analysis software (ZEN 3.5).

[0131] Figure 14 Fluorescence imaging of coenzyme Q10 in SH-SY5Y cells, scale bar is 20 μm; Figure 15 The relative fluorescence intensity of coenzyme Q10 in SH-SY5Y cells changes over time (n=4), F (12,60) =3.633, P=0.0004; F (4,60) =3.669, P=0.0097; F (3,60) =4.129, P=0.0100; Two-way ANOVA; Figure 16 Statistical analysis of total fluorescence intensity of coenzyme Q10 in SH-SY5Y cells cultured with different coenzyme Q10 groups from 0 to 6 hours (n=4), F (3,12)=14.44, P=0.0003, one-way ANOVA. In the figure, Con represents the control group, Q10 represents the cell culture medium group supplemented with 1000μM standard coenzyme Q10, 120nm Q10 represents the cell culture medium group supplemented with 1000μM 120nm Q10-Au@Lip, and 35nm Q10 represents the cell culture medium group supplemented with 1000μM 35nm Q10-Au@Lip. The results showed that standard coenzyme Q10, 120nm Q10-Au@Lip, and 35nm Q10-Au@Lip all had a certain degree of neuronal cell membrane permeability. Among them, 35nm Q10-Au@Lip had the strongest cell membrane permeability, and the cell membrane permeability of 35nm Q10-Au@Lip reached its peak at 1 hour, showing high efficiency in cell membrane permeability.

[0132] Figure 17 After culturing SH-SY5Y cells with standard coenzyme Q10 at concentrations of 100 μM, 120 nm Q10-Au@Lip, and 35 nm Q10-Au@Lip, the changes in intracellular coenzyme Q10 content (n=3) were observed. (12,40) =7.516, P<0.0001; F (4,40) =45.90, P<0.0001; F (3,40) =22.55, P<0.0001; two-way ANOVA. Figure 18 The changes in extracellular (in culture medium) coenzyme Q10 content (n=3) after culturing SH-SY5Y cells with standard coenzyme Q10 at concentrations of 100 μM, 120 nm Q10-Au@Lip, and 35 nm Q10-Au@Lip, respectively, were observed. (12,60) =10.32, P<0.0001; F (4,60) =8.969, P<0.0001; F (3,60) =1.231, P=0.3065; two-way ANOVA. Figure 19 After culturing SH-SY5Y cells with standard coenzyme Q10 at concentrations of 1000 μM, 120 nm Q10-Au@Lip, and 35 nm Q10-Au@Lip, the changes in intracellular coenzyme Q10 content (n=3) were observed. (12,20) =2.489, P=0.0345; F (4,20) =18.12, P<0.0001; F (3,20) =0.7020, P=0.5619, two-way ANOVA. Figure 20 The changes in extracellular (in culture medium) coenzyme Q10 content (n=3) after culturing SH-SY5Y cells with standard coenzyme Q10 at a concentration of 1000 μM, 120 nm Q10-Au@Lip, and 35 nm Q10-Au@Lip, respectively, were observed.(12,60) =2.944, P=0.0028; F (4,60) =16.06, P<0.0001; F (3,60) = 85.22, P < 0.0001; two-way ANOVA. In the figure, Con represents the control group, Q10 represents the cell culture medium group supplemented with 1000 μM standard coenzyme Q10, 120 nm Q10 represents the cell culture medium group containing 120 nm Q10-Au@Lip, and 35 nm Q10 represents the cell culture medium group containing 35 nm Q10-Au@Lip. The results showed that the degradation rate of 35 nm Q10-Au@Lip was slower, indicating that the nano-coating has a protective effect on the structure of 35 nm Q10-Au@Lip and can play a role in sustained release.

[0133] Experimental Example 5

[0134] Verification of the cytotoxicity of nanomedicines on neuronal cells:

[0135] SH-SY5Y cells were cultured from the Institute of Geriatrics, Wenzhou Medical University, China. SH-SY5Y cells were seeded in complete culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) and cultured at 37°C and 5% CO2. 120nm Q10-Au@Lip and 35nm Q10-Au@Lip cell culture media were prepared at concentrations of 20, 40, 60, 80, and 100 μM using DMEM / F-12 cell culture medium. After the SH-SY5Y cells reached 70% confluence, the medium was replaced with 120nm Q10-Au@Lip and 35nm Q10-Au@Lip cell culture media, and the cells were cultured for 12 hours. Cell viability was then assessed.

[0136] Figure 21 Cytotoxicity of 120nm Q10 and 35nm Q10 (n=5), F (4,40) =11.62, P<0.0001; F (4,40) =28.29, P<0.0001; F (1,40) = 32.19, P < 0.0001; Two-way ANOVA. In the figure, 120nm Q10 represents the cell culture medium containing 120nm Q10-Au@Lip, and 35nm Q10 represents the cell culture medium containing 35nm Q10-Au@Lip. The results show that 35nm Q10-Au@Lip has lower cytotoxicity.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing coenzyme Q10 nanoliposomes, characterized in that, Includes the following steps: Coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate and 95% ethanol were mixed to obtain a homogeneous solution; the homogeneous solution was added to glucose injection solution and mixed to obtain a mixed solution; the mixed solution was extruded 8 to 12 times each through polycarbonate filter membranes with diameters of 220 nm, 100 nm and 50 nm to obtain a coenzyme Q10 nanoliposome solution.

2. The preparation method according to claim 1, characterized in that, The coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate and 95% ethanol are mixed in a mass ratio of (9-11):(9-11):(9-11):(13-17):(50-60).

3. The preparation method according to claim 1, characterized in that, The mixing step of the homogeneous solution includes: Take egg yolk lecithin into a container, add 95% ethanol, soybean oil and polyethylene glycol 12-hydroxystearate in sequence, heat in a water bath and stir until uniform, then add coenzyme Q10, seal, heat in a water bath and stir to obtain a homogeneous solution.

4. The preparation method according to claim 3, characterized in that, The homogeneous solution and glucose injection solution are mixed at a volume ratio of 1:(90-110).

5. Coenzyme Q10 nanoliposomes prepared by the preparation method according to any one of claims 1 to 4.

6. The coenzyme Q10 nanoliposomes according to claim 5, characterized in that, The particle size of the coenzyme Q10 nanoliposomes is 30-50 nm.

7. A method for preparing nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10, characterized in that, Includes the following steps: Coenzyme Q10, gold nanoparticles, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate, and 95% ethanol were mixed to obtain a homogeneous solution. The homogeneous solution was added to glucose injection solution and mixed to obtain a mixed solution. The mixed solution was extruded 8-12 times each through polycarbonate filter membranes with diameters of 220 nm, 100 nm, and 50 nm to obtain a nanoliposome solution that simultaneously encapsulates gold nanoparticles and coenzyme Q10.

8. The preparation method according to claim 7, characterized in that, The gold nanoparticles, coenzyme Q10, soybean oil, egg yolk lecithin, polyethanol 12-hydroxystearate and 95% ethanol are mixed in a mass ratio of (5-7):(9-11):(9-11):(9-11):(13-17):(50-60).

9. The nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared by the preparation method according to claim 7 or 8.

10. The use of the coenzyme Q10 nanoliposomes according to any one of claims 5 to 6 or the nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 according to claim 9 in the preparation or development of coenzyme Q10 nanomedicines.

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