Coenzyme q10 nanoliposome and preparation method and application thereof

Abstract

The application provides a coenzyme Q10 nanoliposome and a preparation method and application thereof, and belongs to the technical field of biological medicines. The coenzyme Q10 nanoliposome prepared by successively extruding a solution through 220nm, 100nm and 50nm polycarbonate filter membranes has a particle size range of 30-50nm, is not prone to fusion and collapse, can be endocytosed by neurons, and has a simple and efficient preparation method. The application further prepares nanoliposomes simultaneously coated with 5nm gold and coenzyme Q10, the distribution of the nanoliposomes can be tracked through a transmission electron microscope, and it is verified whether the drug coenzyme Q10 enters the brain extracellular space and is endocytosed into neurons. The coenzyme Q10 nanoliposome provided by the application can penetrate the blood-brain barrier, enter the extracellular space, and be endocytosed by neurons, thereby eliminating accumulated formaldehyde in the brain, and providing a basis for the research and development of drugs for brain diseases such as Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a coenzyme Q10 nanoliposome, its preparation method, and its application. Background Technology

[0002] The incidence of central nervous system (CNS) diseases is rising worldwide, but CNS drug development remains extremely challenging, characterized by high costs, long development cycles, and high failure rates. The CNS is highly protected by physiological barriers, particularly the blood-brain barrier (BBB) ​​and the extracellular space (ECS) of neurons, which is only 38-64 nm in diameter. These barriers prevent drugs from reaching their target sites in the brain from their entry points. The diameter of drugs also limits their internalization by neurons; drugs with excessively large diameters struggle to penetrate the neuronal cytoplasm to salvage neuronal activity. Therefore, the challenge of delivering drugs to the brain is often attributed to complex and highly regulated barriers, requiring drug delivery systems to penetrate the brain parenchyma to reach the target disease site. For example, a typical pathological feature of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aβ) in the 38-64 nm ECS, forming senile plaques (SPs). Age-related accumulation of endogenous formaldehyde promotes Aβ aggregation, which can clog the extracellular space and slow or halt the drainage of interstitial fluid (ISF). This leads to disruptions in neurotransmitter transmission and metabolic waste excretion, potentially inducing neuronal death and memory impairment. Consequently, drugs dissolved in the ISF cannot reach damaged neurons and thus fail to exert a rescue effect. Age plaques blocking the ECS are a key reason why drugs cannot reach target cells. Therefore, the development of nanomedicines that can cross the ECS and BBB is crucial for treating CNS diseases such as Alzheimer's disease (AD).

[0003] Liposomes are recognized worldwide as one of the safest nanomaterials and can serve as highly efficient carriers for drugs or other nanoparticles. Numerous studies have demonstrated that drugs with a particle size smaller than 100 nanometers can penetrate the blood-brain barrier (BBB). Currently, although many methods for preparing nanoliposomes have been reported, such as thin-film hydration and reverse evaporation, these methods are generally complex, and the resulting nanoliposomes are prone to melt-collapse, exhibiting poor stability and efficacy. Furthermore, existing methods struggle to produce nanoliposomes with a particle size smaller than 38-64 nm that can penetrate the ECS (extracorporeal membrane oxygenation) barrier, particularly for coenzyme Q10. Currently, there are no reports, either domestically or internationally, of preparing nanoliposomes with a particle size smaller than 50 nanometers that can be stably delivered to neurons in the brain. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a coenzyme Q10 nanoliposome that can be internalized by neurons and its preparation method. The prepared coenzyme Q10 nanoliposome has a particle size range of 30-50 nm and an average diameter of about 35 nm. It is not prone to melting and collapse, and can be internalized by neurons to exert an effective therapeutic effect.

[0005] The present invention also aims to provide a nanoliposome that simultaneously encapsulates gold nanoparticles and coenzyme Q10, and a method for preparing the same. By introducing gold nanoparticles into the nanoliposome, it is possible to directly observe the entry of the nanoliposome into the extracellular space of brain cells and its endocytosis into neurons.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing coenzyme Q10 nanoliposomes, comprising the following steps:

[0008] 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 8-12 times each through polycarbonate filter membranes with diameters of 220nm, 100nm and 50nm to obtain a coenzyme Q10 nanoliposome solution.

[0009] Preferably, 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).

[0010] Preferably, the mixing step of the homogeneous solution includes: taking egg yolk lecithin into a container, adding 95% ethanol, soybean oil and polyethanol 12-hydroxystearate in sequence, heating and stirring in a water bath until homogeneous, then adding coenzyme Q10, sealing, heating and stirring in a water bath to obtain a homogeneous solution.

[0011] Preferably, the homogeneous solution and the glucose injection are mixed at a volume ratio of 1:(90~110).

[0012] The present invention also provides coenzyme Q10 nanoliposomes prepared by the above preparation method.

[0013] Preferably, the particle size of the coenzyme Q10 nanoliposomes is 30-50 nm.

[0014] 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 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 nanoliposome solution simultaneously encapsulating gold nanoparticles and coenzyme Q10.

[0015] Preferably, 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).

[0016] The present invention also provides nanoliposomes that simultaneously encapsulate gold nanoparticles and coenzyme Q10, prepared by the above preparation method.

[0017] 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.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0019] The method for preparing coenzyme Q10 nanoliposomes provided by this invention is simple, efficient, and low-cost. It only requires sequentially pressing the solution with polycarbonate filter membranes of 220nm, 100nm, and 50nm to obtain coenzyme Q10 nanoliposomes with a particle size range of 30-50nm and an average diameter of about 35nm. Moreover, the prepared coenzyme Q10 nanoliposomes are not prone to melting and collapse and can be internalized by neurons to exert an effective therapeutic effect.

[0020] This invention encapsulates 5nm gold and coenzyme Q10 simultaneously to prepare nanoliposomes. Transmission electron microscopy can be used to track whether the nanoliposomes penetrate the blood-brain barrier and their distribution in the brain ECS, verifying whether the drug enters the extracellular space of brain cells and whether it is internalized into neurons, providing a foundation for the development of coenzyme Q10 nanomedicines. Attached Figure Description

[0021] Figure 1 TEM images of each group of coenzyme Q10 nanoliposomes;

[0022] Figure 2 Hydrodynamic diameter distribution of coenzyme Q10 nanoliposomes 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 120 nm 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 h.

[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, 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℃, preferably 60℃.

[0045] The homogeneous solution and glucose injection solution of the present invention are mixed at a volume ratio of 1:(90~110), and the preferred volume ratio is 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. It 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 after squeezing. Use a 50 nm polycarbonate filter membrane to squeeze the solution 8 times after squeezing 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 -13.19 mV, while that of the 120nm Q10@NPs group is -12.17 mV. 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 the in vitro release rate of the drug was determined using dialysis bags at different times (0, 5, 10, 20 h).

[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. It 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 60 r / min. After film formation, the film was vacuum dried for 50 min 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 1 h 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 AuNPs liposomes (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 after injecting AD mice with 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 brain tissue 3 hours later. Five minutes later, the mice were anesthetized with 1.25% tribromoethanol solution via intraperitoneal injection. 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 fixation solution. A 1 mm section was surgically removed. 1mm Tissue blocks of 1 mm in size were placed in EP tubes containing electron microscopy fixative and fixed at room temperature in the dark for 2 hours, then transferred to 4°C for storage. The tissue was then rinsed three times with 0.1 M phosphate buffer PB (pH 7.4), 15 min 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 solution was 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, and then twice in 100% acetone, 15 minutes each time.

[0114] 4. Infiltration and embedding: Acetone: 812 embedding agent = 1:1, 37℃ for 2~4h; Acetone: 812 embedding agent = 1:2, infiltrate overnight at 37℃; Pure 812 embedding agent, 37℃ for 5~8h. 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 60~80nm using an ultrathin microtome, and the sections are retrieved using a 150-mesh anti-slip copper screen.

[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 in 2.6% lead citrate solution for 8 min in the dark; wash 3 times with ultrapure water, and blot dry with filter paper. Place copper mesh sections in a copper mesh box and dry at room temperature overnight.

[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 Q10 Au@NPs (120nm Q10-Au@Lip) injected into the hippocampus via lateral ventricle and intraperitoneal injection (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 endocrine system (ECS) with a diameter of 38-64nm.

[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 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 The total fluorescence intensity of coenzyme Q10 in SH-SY5Y cells cultured with different coenzyme Q10 levels from 0 to 6 hours was statistically analyzed (n=4). (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 1000 μM standard coenzyme Q10, 120nm Q10 represents the cell culture medium group supplemented with 1000 μM 120nm Q10-Au@Lip, and 35nm Q10 represents the cell culture medium group supplemented with 1000 μM 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 show that the degradation rate of 35 nm Q10-Au@Lip is 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 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 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. 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); The mixing steps of the homogeneous solution include: taking egg yolk lecithin into a container, adding 95% ethanol, soybean oil and polyethanol 12-hydroxystearate in sequence, heating and stirring in a water bath until uniform, then adding coenzyme Q10, sealing, heating and stirring in a water bath to obtain a homogeneous solution. The homogeneous solution and glucose injection solution are mixed at a volume ratio of 1:(90-110).

2. Coenzyme Q10 nanoliposomes prepared by the preparation method according to claim 1.

3. The coenzyme Q10 nanoliposomes according to claim 2, characterized in that, The particle size of the coenzyme Q10 nanoliposomes is 30-50 nm.

4. 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. 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).

5. The nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 prepared by the preparation method of claim 4.

6. The use of the coenzyme Q10 nanoliposomes according to any one of claims 2 to 3 or the nanoliposomes simultaneously encapsulating gold nanoparticles and coenzyme Q10 according to claim 5 in the preparation of coenzyme Q10 nanomedicines.

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