A nano drug delivery system and its preparation method and application

Abstract

The present invention discloses a nano drug delivery system and a preparation method and application thereof, wherein the nano drug delivery system comprises dihydrochlorin e6 and chloroquine. The nano drug delivery system prepared by the scheme of the present invention has a suitable size, can be selectively enriched at the tumor site, has a good tumor cell killing effect, has no toxic side effects on the body, has a simple preparation method, low cost, and significant effect, and provides a new idea for the preparation of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomaterials, and in particular relates to a nano-drug delivery system and a preparation method and application thereof. Background Art

[0002] Due to the intensification of industrialization and population aging, malignant tumors have gradually become a public health issue that cannot be ignored. Malignant tumors are often extremely difficult to cure, which brings great economic burden and mental pressure to society, families and individuals. Although medical workers have made great efforts to treat malignant tumors in the past few decades, and the research on new anti-tumor drugs and tumor treatment methods has also made certain progress, the incidence and mortality of tumors are still high worldwide, seriously threatening human health and life. In the treatment of tumors, surgery and radiotherapy are usually used to treat local malignant tumors, while chemotherapy, immunotherapy and targeted therapy are often used alone or in combination for systemic treatment of tumors. However, these methods all have their own shortcomings. Therefore, there is an urgent need for new and reliable tumor treatment methods in clinical practice.

[0003] In recent years, photodynamic therapy has become an increasingly important focus in tumor treatment research due to its advantages such as spatiotemporal controllability and low side effects. Photodynamic therapy mainly involves injecting photosensitizers into patients and then irradiating the tumor site with lasers of a specific wavelength. After being excited by light of the corresponding wavelength, the photosensitizer is converted from the ground state to the excited state, and then to the electronically excited triplet state, ultimately triggering a type I photochemical reaction or a type II photochemical reaction. In the former reaction, the photosensitizer in the electronically excited triplet state directly reacts with substrates such as biomacromolecules through electron transfer to form free radicals and kill cells. In the latter reaction, the photosensitizer in the electronically excited triplet state directly transfers energy to oxygen molecules in the surrounding environment, producing singlet oxygen to kill the target. Therefore, type II photochemical reactions often dominate in an environment with sufficient oxygen, while type I photochemical reactions can also occur under hypoxic conditions. However, like other drugs, photosensitizers have low drug concentrations at the tumor site due to the physiological barriers and active drug metabolism capabilities in the human body, and cannot completely cure the tumor.

[0004] In the treatment of tumors, the inherent defects of various drugs have promoted the application and development of nanotechnology and various nanomaterials in the biomedical field, forming an emerging medical research field, namely, nano drug delivery system. Nano drug carriers have very obvious advantages over free drugs. They can not only increase drug stability, but also increase drug accumulation and delay release in tumor tissues, which can significantly improve the bioavailability of drugs and reduce their systemic toxicity. The discovery of the enhanced permeability and retention effect (EPR) of nanomaterials in solid tumors is the driving force for the rapid development of nano drug carriers. With the discovery of the EPR phenomenon, more and more nano materials for anti-tumor research have been developed, such as liposomes. In order to further obtain the targeting function, the design of nano carriers can also be combined with specific targeting molecules to increase the recognition of target cells by the carrier, so that the nano material is enriched at the tumor site, thereby increasing the therapeutic effect. At present, many nano drugs have been used clinically, such as doxorubicin liposomes, irinotecan liposomes, paclitaxel albumin, etc. However, the long-term use of a single drug delivery will still cause drug resistance. Based on this, many research teams have developed various new nanocarriers to simultaneously deliver two drugs with different mechanisms of action, in order to better exert the synergistic effect of the drugs and increase the anti-tumor effect. However, this strategy of using exogenous nanocarriers to load two drugs at the same time, on the one hand, cannot accurately control the drug loading between different batches, and on the other hand, due to the complexity of the structure, the metabolism and excretion process in the body, and potential toxic side effects of exogenous nanocarriers, further limit their clinical transformation. In addition, there are also problems such as high price, complex preparation scheme, high toxicity, and unsatisfactory therapeutic effect, which urgently need to be further improved by medical researchers. Self-assembled nanomaterials are structural units that spontaneously form a stable aggregation system with specific structure and function, mainly bound by non-covalent bonds, with the help of weak intermolecular interactions. In recent years, the widespread application of prodrugs and nanotechnology in the field of drug delivery has greatly enriched the delivery strategies of anti-tumor drugs. The self-assembled nanodrug delivery system based on small molecule drugs combines the advantages of prodrugs and nanotechnology. With its advantages such as high drug loading capacity, good stability, low toxicity and side effects, and strong tumor targeting, it has become a hot spot in photodynamic drug delivery research.

[0005] In addition, photodynamic therapy also has the problem of tumor resistance. A large amount of reactive oxygen can easily cause tumor cell stress, activate tumor autophagy, and alleviate or offset the therapeutic effect of photodynamic therapy on tumors. Autophagy is a highly conservative and ubiquitous physiological phenomenon in cells. It wraps damaged organelles or oxidized biomacromolecules into autophagosomes through a double membrane structure, and then transports them to lysosomes for degradation, and then decomposes them into nutrients required for cell growth. Autophagy not only promotes cells to maintain their own internal environment stability for normal proliferation and differentiation, but also helps cells survive stress. Therefore, how to inhibit tumor autophagy while performing photodynamic therapy has become the key to photodynamic therapy for tumors. Therefore, in order to solve the above problems, combined therapy or seeking more efficient drug delivery methods are the current key directions in the field of tumor treatment. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a nano drug delivery system.

[0007] The present invention also provides a method for preparing the nano drug delivery system.

[0008] The present invention also proposes the application of the nano drug delivery system.

[0009] According to one aspect of the present invention, a nano drug delivery system is provided, wherein the nano drug delivery system comprises chlorin e6 and chloroquine.

[0010] In some embodiments of the present invention, the particle size of the nano drug delivery system is 10-120 nm.

[0011] In some embodiments of the present invention, the particle size of the nano drug delivery system is 30-50 nm.

[0012] In some embodiments of the present invention, the potential of the nano drug delivery system is -15.26±0.1320 mV.

[0013] In some embodiments of the present invention, the mass addition ratio of the dihydrochlorin e6 and chloroquine is (1-3): (1-5).

[0014] According to a second aspect of the present invention, a method for preparing the above-mentioned nano drug delivery system is proposed, and the method comprises the following steps: subjecting dihydrochlorin e6 and chloroquine to ultrasound to obtain the nano drug delivery system.

[0015] In some embodiments of the present invention, the concentration of the dihydrochlorin e6 is 8-12 mg / ml.

[0016] In some embodiments of the present invention, DMSO is selected as the solvent for dissolving chlorin e6.

[0017] In some embodiments of the invention, the concentration of chloroquine is 8-12 mg / ml.

[0018] In some embodiments of the present invention, DMSO is selected as the solvent for dissolving chloroquine.

[0019] In some embodiments of the present invention, the ultrasonic time is 20-40 min, and the ultrasonic temperature is 15-25°C.

[0020] In some embodiments of the present invention, after the ultrasound, a step of purifying the product obtained by ultrasound is also included.

[0021] In some embodiments of the present invention, the purification is performed by centrifugation at a speed of 4200 rpm for 10 min.

[0022] In some embodiments of the present invention, the centrifugation is performed using an ultrafiltration tube.

[0023] In some embodiments of the present invention, the molecular cutoff of the ultrafiltration membrane of the ultrafiltration tube is 8-12 kD.

[0024] In some embodiments of the present invention, the molecular cutoff of the ultrafiltration membrane of the ultrafiltration tube is 10 kD.

[0025] In the third aspect of the present invention, an application of the nano drug delivery system is proposed, wherein the application is application in the preparation of tumor-killing drugs.

[0026] In some embodiments of the invention, the tumor comprises a superficial tumor.

[0027] In some embodiments of the invention, the superficial tumor comprises breast cancer or melanoma.

[0028] In some embodiments of the present invention, the application is application in the preparation of a tumor autophagy promoter.

[0029] In some embodiments of the present invention, the application is application in the preparation of tumor autophagy repair inhibitors.

[0030] In some embodiments of the present invention, the method for using the nano drug delivery system is as follows: the nano drug delivery system is introduced into the body, and when the enrichment amount of the nano drug delivery system at the tumor site reaches the maximum, the tumor site is irradiated with laser.

[0031] In some embodiments of the present invention, the wavelength of the laser is 630-680 nm, and the irradiation time is 0-1 h.

[0032] In some embodiments of the present invention, the laser light is infrared light.

[0033] In some embodiments of the present invention, the introduction method is intravenous injection.

[0034] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the nano drug delivery system prepared by the scheme of the present invention has a suitable size, can be selectively enriched in the tumor site, has a good tumor cell killing effect, and has no toxic side effects on the body.

[0035] Chlorin e6 (i.e., Ce6) can produce highly cytotoxic singlet oxygen and hydrogen peroxide under illumination conditions. Since Ce6 requires oxygen to participate in the process of producing singlet oxygen, the oxygen produced by the Fenton reaction can be used as an oxygen source, and the produced hydrogen peroxide can be used in the Fenton reaction to produce hydroxyl radicals and oxygen, thereby realizing an enzyme-free cascade reaction, and having the performance of synergistic chemical kinetics and photodynamic combined therapy. The enzyme-free cascade reaction is used for tumor treatment to achieve better therapeutic effects, and the chloroquine in the nanomedicine can neutralize the lysosome, inhibit the tumor autophagy channel, and cause the oxidized biomacromolecules and damaged organelles to be unable to be degraded through the autophagy pathway, and the continuous accumulation further causes metabolic disorders and toxic protein generation, resulting in a cascade amplified toxic effect, thereby achieving an efficient tumor killing effect. Therefore, the nano drug delivery system prepared by the present invention has good stability, and exhibits good water solubility, biocompatibility, and strong near-infrared absorption. It can achieve the effect of chemical photodynamic synergistic tumor killing by generating reactive oxygen and inhibiting tumor autophagy repair under near-infrared light irradiation. The present invention prepares a new type of self-assembled nano drug delivery system for the first time, which has spatiotemporal controllability, can be enriched in tumor cells, effectively reduce the concentration of photosensitizer, improve drug solubility, and reduce adverse reactions of the body, thereby achieving a system for efficient tumor treatment, providing new ideas and theoretical basis for the development of clinical anti-tumor drugs. In addition, exploring new strategies for enhanced photodynamic therapy has very huge potential application value and economic and social benefits.

[0036] The nano drug delivery system prepared by the scheme of the present invention is prepared by self-assembly of dihydrochlorin e6 and chloroquine, has a simple preparation method, low cost and significant effect, and provides a new idea for the preparation of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 This is a graph showing the particle size distribution results of the self-assembled nano drug delivery system with different concentrations in the test examples of the present invention;

[0039] Figure 2 The UV spectra of the self-assembled nano drug delivery system with different concentrations in the test examples of the present invention;

[0040] Figure 3 It is a transmission electron microscopy image of the optimal ratio self-assembled nano drug delivery system in the test example of the present invention;

[0041] Figure 4 The graph is a test result of the particle size distribution and potential of the self-assembled nano-drug with the optimum ratio in the test example of the present invention;

[0042] Figure 5 This is a graph showing the cell viability test results in a test example of the present invention;

[0043] Figure 6 This is the MDC staining diagram of cell autophagy of 4T1 cells under different treatments in the test example of the present invention;

[0044] Figure 7 The distribution of the same concentration of dihydrochlorin e6 and the self-assembled nanomedicine in the main organs of 4T1 tumor-bearing mice and the relative fluorescence intensity statistics in the test example of the present invention;

[0045] Figure 8 This is a record of tumor volume of tumor-bearing mice after being treated with different drugs in the test example of the present invention;

[0046] Fig. 9 This is a graph recording the weight of the tumor on the 16th day after the tumor-bearing mice were treated with different drugs in the test example of the present invention. DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0048] Example 1 A Nano-drug Delivery System

[0049] This example prepares a nano drug delivery system, and the specific preparation process is as follows:

[0050] (1) Solution preparation: Chlorin e6 (Ce6) was dissolved in DMSO to prepare a stock solution with a concentration of 10 mg / ml; chloroquine (CQ) was dissolved in DMSO to prepare a stock solution with a concentration of 10 mg / ml.

[0051] (2) Synthesis of nanomedicines: Self-assembled nanomedicines were synthesized by ultrasonic precipitation. The prepared dihydrochlorin e6 and chloroquine were added into a 5 mL glass bottle in different proportions. The ratio of dihydrochlorin e6 to chloroquine was 1:2 (i.e., the added amount was 50 μL:100 μL). Ultrasonication was performed for 30 min, and the temperature was set at 20°C. After the reaction was completed, 2 ml of deionized water was added and ultrasonication was continued for 30 minutes.

[0052] (3) After the reaction is completed, the obtained product is transferred to a 10 kd ultrafiltration tube and centrifuged at 4200 rpm for 10 min. The liquid in the tube is collected to obtain the nano drug delivery system and stored at 4°C for future use.

[0053] Example 2

[0054] This example prepares a nano drug delivery system, which is different from Example 1 only in that the ratio of dihydrochlorin e6 to chloroquine is 3:1 (ie, the added amount is 150 μL:50 μL).

[0055] Example 3

[0056] This embodiment prepares a nano drug delivery system, which is different from the embodiment 1 only in that the ratio of dihydrochlorin e6 to chloroquine is 2:1 (ie, the added amount is 100 μL:50 μL).

[0057] Example 4

[0058] This example prepares a nano drug delivery system, which is different from Example 1 only in that the ratio of dihydrochlorin e6 to chloroquine is 1:1 (ie, the added amount is 50 μL:50 μL).

[0059] Example 5

[0060] This example prepares a nano drug delivery system, which is different from Example 1 only in that the ratio of dihydrochlorin e6 to chloroquine is 1:5 (ie, the added amount is 50 μL:250 μL).

[0061] Test example

[0062] This test example tests the performance of the nano drug delivery system prepared in Examples 1-5.

[0063] 1. Physical and chemical determination

[0064] (1) Particle size detection

[0065] The particle sizes of the nano drug delivery systems prepared in Examples 1-5 were detected using a transmission electron microscope.

[0066] (2) Spectral detection

[0067] The UV-visible spectra of the nano drug delivery systems prepared in Examples 1-5 were detected using a multifunctional ELISA instrument.

[0068] (3) Potential detection

[0069] The potential of the nano drug delivery system prepared in Example 1 was detected using a dynamic light scattering instrument.

[0070] Test results such as Figure 1-4 As shown in the figure, it can be seen that among the different ratios of dihydrochlorin e6 and chloroquine, only when the ratio is 1:2, the nanoparticle size is the smallest and has the ultraviolet absorption peaks of both drugs. Therefore, 1:2 is the optimal ratio. The prepared nanomedicine is spherical particles of 30-50nm, and the potential is -15.26±0.1320mV.

[0071] 2. Determination of anti-tumor effect

[0072] Preparation of solution: Chlorin e6, chloroquine and the nanocarrier prepared in Example 1 were dissolved in DMEM complete culture medium respectively.

[0073] Different groups were used for anti-tumor determination, specifically grouped as: dihydrochlorin e6 group, chloroquine group, drug mixture group (the added volume ratio of dihydrochlorin e6 and chloroquine was 1:2), nanodrug group (nano drug delivery system prepared in Example 1), dihydrochlorin e6 + light group, drug mixture (the added volume ratio of dihydrochlorin e6 and chloroquine was 1:2) + light group, nanodrug (nano drug delivery system prepared in Example 1) + light group.

[0074] 4T1 breast cancer cells were cultured in DMEM + 10% FBS medium and seeded in a 96-well plate. When the density reached 30%, dihydrochlorin e6 (dihydrochlorin e6 group), chloroquine (chloroquine group), the volume ratio of dihydrochlorin e6 and chloroquine was 1:2 (drug mixture group), the nano drug delivery system prepared in Example 1 (nano drug group), dihydrochlorin e6 + light (dihydrochlorin e6 + light group), the mixed drug with the volume ratio of dihydrochlorin e6 and chloroquine being 1:2 + light (drug mixture + light group), and the nano drug delivery system prepared in Example 1 + light treatment (nano drug + light group) were added to the cells at final concentrations of (0, 0.15625, 0.3125, 0.625, 1.25, 2.5 μg / ml). After incubation for 6 hours, all the light groups were irradiated with 650nm near-infrared light. The cells were cultured for 48 hours, and then the cell survival rate was detected using the CCK-8 method.

[0075] The results are as follows Figure 5 As shown in the figure, it can be seen that the survival rate of tumor cells gradually decreases with the increase of the treatment concentration of nanodrug. At the same time, the nanodrug + light group shows better tumor cell killing effect, indicating that nanodrugs can achieve better tumor cell killing effect under light.

[0076] 3. Application of nanomedicine as an autophagy promoter in tumor cells

[0077] Different groups were used to detect nanomedicine as a promoter of tumor cell autophagy, and the specific groups were: Different groups were used for anti-tumor determination, and the specific groups were: dihydrochlorin e6 group, chloroquine group, nanomedicine group (nanodrug delivery system prepared in Example 1), dihydrochlorin e6 + light group, drug mixture (the added mass ratio of dihydrochlorin e6 and chloroquine is 1:2) + light group, nanomedicine (nanodrug delivery system prepared in Example 1) + light group and phosphate buffer control group.

[0078] 4T1 breast cancer cells were cultured with DMEM + 10% FBS medium and seeded in a confocal microscope. When the density reached 30%, 1.25 μg / ml dihydrochlorin e6 (dihydrochlorin e6 group), 2.5 μg / ml chloroquine (chloroquine group), 1.25 μg / ml nano drug delivery system prepared in Example 1 (nano drug group), 1.25 μg / ml dihydrochlorin e6 + light (dihydrochlorin e6 + light group), a mixed drug of 1.25 μg / ml dihydrochlorin e6 and 2.5 μg / ml chloroquine + light (drug mixture + light group), and 1.25 μg / ml nano drug delivery system prepared in Example 1 + light treatment (nano drug + light group) were added to the control group. The same volume of phosphate buffer as that of the experimental group was added. After 6 hours of incubation, all illumination groups were irradiated with 650 nm near-infrared light. After 24 hours of incubation, the cells were stained with MDC staining kit and the autophagy was observed by laser confocal microscopy.

[0079] The experimental results are as follows Figure 6 As shown in the figure, it can be seen that nanomedicine + light can significantly increase the level of autophagy in tumor cells. At the same time, due to the inhibition of autophagy repair of tumor cells, a large number of autophagic bodies accumulate in tumor cells in the nanomedicine + light group compared with the chloroquine group.

[0080] 4. Animal experiments

[0081] (1) Enrichment of Nano-drug Delivery Systems

[0082] 4T1 breast cancer cells (purchased from ATCC) were cultured in DMEM + 10% FBS medium. Each mouse was injected with 10 6 4T1 breast cancer cells were used to establish a subcutaneous tumor-bearing model in BALB / c mice. 3 The tumor-bearing mice were randomly divided into two groups: the dihydrochlorin e6 group and the nanodrug group.

[0083] The two groups were given equal concentrations (333.3 μg / mL) of dihydrochlorin e6 and 200 μL of nanodrug (nano drug delivery system prepared in Example 1) via tail vein administration, and in vitro live imaging of the main organs (heart, liver, spleen, lung, kidney) and tumors of the mice was performed 6 hours after administration, and statistical analysis was performed.

[0084] The experimental results are as follows Figure 7 As shown in the figure, it can be seen that the nanomedicine is more obviously enriched in tumor tissue than simple dihydrochlorin e6.

[0085] (2) The inhibitory effect of nano-drug delivery system on tumors

[0086] 4T1 breast cancer cells (purchased from ATCC) were cultured in DMEM + 10% FBS medium. Each mouse was injected with 10 6 4T1 breast cancer cells were used to establish a subcutaneous tumor-bearing model in BALB / c mice. When the tumor volume grew to 100 mm 3 At the same time, all tumor-bearing mice were randomly divided into 7 groups, namely: phosphate buffer group (administered phosphate buffer), dihydrochlorin e6 group (administered dihydrochlorin e6), chloroquine group (administered chloroquine), nano drug group (administered nano drug delivery system prepared in Example 1), dihydrochlorin e6 + light irradiation group (administered dihydrochlorin e6), drug mixture + light irradiation group (administered dihydrochlorin e6 and chloroquine added volume ratio of 1:2), nano drug + light irradiation group (administered nano drug delivery system prepared in Example 1). Among them, the concentration of dihydrochlorin e6 in each group was 333.3 μg / mL, the concentration of chloroquine was 666.6 μg / mL, and the concentration of nano drug was 333.3 μg / mL. All drugs were administered by tail vein injection, and 200 μL was injected into the tail vein of each mouse. After 6 hours of drug administration, the tumor sites of mice in the dihydrochlorin e6 + light group, drug mixture + light group, and nanodrug + light group were irradiated with 650nm near-infrared light for 5 minutes. Mice were treated once every two days for a total of 4 times (the first treatment day was day 0). The size of mouse tumors was recorded every two days until day 16. After day 16, the tumors were removed and weighed.

[0087] The experimental results are as follows Figure 8-9 As shown, from Figure 8 It can be seen that after treatment, the growth rate of tumor volume in the light-exposed group was significantly lower than that in the non-light-exposed group. At the same time, the tumor volume in the other groups gradually increased. On the 16th day, there was a clear difference between the nanomedicine + light-exposed group and the other groups, proving that the nanomedicine + light-exposed group can effectively inhibit tumor growth. Fig. 9It can be seen that on the 16th day, all mice were anesthetized and killed by cervical dislocation, and the ex vivo tumor tissue was taken out for weighing. The tumor mass of the nanomedicine + light group was significantly smaller than that of the other groups, with a significant difference, indicating that it can effectively inhibit tumor growth.

[0088] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A nano drug delivery system, characterized in that: The nano drug delivery system comprises dihydrochlorin e6 and chloroquine; the mass addition ratio of the dihydrochlorin e6 to chloroquine is 3:1, 1:2 or 1:5; The nano drug delivery system is prepared by using ultrasound to react dihydrochlorin e6 and chloroquine.

2. The nano drug delivery system according to claim 1, characterized in that: The particle size of the nano drug delivery system is 10-120 nm.

3. The nano drug delivery system according to claim 1, characterized in that: The potential of the nano drug delivery system is -15.26±0.1320 mV.

4. A method for preparing the nano drug delivery system according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: subjecting dihydrochlorin e6 and chloroquine to ultrasonic treatment; the mass addition ratio of the dihydrochlorin e6 and chloroquine is 3:1, 1:2 or 1:

5.

5. The method according to claim 4, characterized in that The ultrasonic time is 20-40 minutes, and the temperature is 15-25°C.

6. Use of the nano drug delivery system according to any one of claims 1 to 3 in the preparation of tumor-killing drugs, characterized in that: The tumor is breast cancer.

7. The use according to claim 6, characterized in that: The tumor-killing drug can be used as a tumor autophagy promoter.

8. The use according to claim 6, characterized in that: The tumor-killing drug can be used as a tumor autophagy repair inhibitor.

9. The use according to any one of claims 6 to 8, characterized in that: The method for using the nano drug delivery system is as follows: the nano drug delivery system is introduced into the body, and when the enrichment amount of the nano drug delivery system at the tumor site reaches the maximum, the tumor site is irradiated with laser.

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