Method for loading drug by using gram-negative bacteria vacuum method and drug-loaded compound thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for loading a medicine by using a gram-negative bacterium vacuum method and a medicine-loaded compound thereof. The drug-loaded compound is composed of gram-negative bacteria and an internally-loaded drug, wherein the gram-negative bacteria is a carrier of the internally-loaded drug; the medicine comprises any one or more of salidroside and epigallocatechin gallate, and the gram-negative bacteria comprise any one or more of 1917 type Escherichia coli and Ackermann mucin. According to the invention, gram-negative bacteria are taken as a carrier, and a vacuum method is adopted to load the medicine, so that the problems of short half-life period, low bioavailability, poor stability, easiness in oxygenolysis and the like of common medicines such as salidroside and EGCG can be effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for loading drugs using a Gram-negative bacteria vacuum method and a drug-loading complex thereof. Background Art

[0002] Salidroside (SAL) is a natural glycoside compound extracted from the Crassulaceae plant Rhodiola rosea. Its chemical structure is composed of salidroside (i.e., the non-sugar part of salidroside) and sugar molecules (such as glucose). Salidroside itself has strong biological activities such as antioxidant, anti-fatigue, and anti-stress. Existing studies have confirmed that SAL has multiple pharmacological effects. For example: 1) SAL helps improve the body's ability to adapt to environmental stress by regulating the functions of the nervous system and endocrine system. 2) SAL can reduce physical and mental fatigue and improve physical endurance by promoting energy metabolism in the body and reducing the level of stress hormones (such as cortisol). 3) SAL has strong antioxidant activity and can scavenge free radicals in the body, thereby reducing the damage caused by oxidative stress to the body. However, SAL still has defects in practical applications, such as short half-life, low bioavailability, and easy to cause the efflux effect of P-glycoprotein and degradation of intestinal flora. Therefore, it is worth exploring the idea of ​​modifying its appropriate oral delivery system.

[0003] Epigallocatechin gallate (EGCG) is a polyphenol compound extracted from tea leaves and belongs to the catechin class. Its chemical structure is composed of a catechin molecule and a gallic acid molecule connected by an ester bond. EGCG has a strong antioxidant effect and can reduce the damage of oxidative stress to cells and tissues by removing free radicals and reactive oxygen species (ROS) in the body. Many studies have shown that EGCG can inhibit the proliferation of cancer cells, induce cancer cell apoptosis, inhibit tumor angiogenesis, and block the metastasis of cancer cells through various pathways. EGCG also exerts anti-inflammatory effects by regulating inflammatory signaling pathways in the body. EGCG can reduce chronic inflammatory responses by inhibiting the activity of key inflammatory factors such as NF-κB and COX-2. In addition, EGCG also has a certain protective effect on the nervous system. Although EGCG has many pharmacological activities and potential health benefits, there are also some disadvantages and limitations in its application, such as short half-life, poor bioavailability, instability and easy degradation, poor absorption in the blood, and hepatotoxicity at high doses. Therefore, loading EGCG into a suitable carrier to improve the bioavailability and stability of the drug is a technical problem that needs to be solved urgently.

[0004] There are many types of drug delivery methods currently available, each with its own unique advantages, but also with certain disadvantages and limitations. For example, 1) Liposome drug delivery: has disadvantages such as poor stability, easy drug leakage, complex preparation process, high cost, and difficulty in loading large molecule drugs; 2) Nanoparticle drug delivery: has advantages such as high drug loading capacity, adjustable drug release curve, and good targeting, but has potential biological toxicity, slow clearance, may cause long-term accumulation, and its metabolic behavior in the body is not yet fully understood; 3) Microsphere drug delivery: can load various types of drugs, but has disadvantages such as microsphere size affecting biodistribution, complex preparation, and may cause drug denaturation; 4) Hydrogel drug delivery: has high water absorption and biocompatibility, but has limited carrying capacity, is difficult to load hydrophobic drugs, and has weak mechanical strength and is easily degraded in complex in vivo environments; 5) Polymer micelle drug delivery: can improve the water solubility of hydrophobic drugs and has good blood circulation stability, but has limited capacity and is only suitable for small molecule hydrophobic drugs; 6) Virus drug delivery: has excellent targeting and can efficiently penetrate cells, but has potential immunogenicity and complex preparation and modification techniques, and may even cause the risk of viral genetic recombination.

[0005] Gram-negative bacteria are a type of bacteria with a special cell wall structure. Its cell wall consists of a thin peptidoglycan layer and an outer membrane, which contains lipopolysaccharide (LPS) and lipoproteins. This structure makes Gram-negative bacteria have strong stress resistance and drug tolerance. Using Gram-negative bacteria to load drugs has the advantages of high drug loading efficiency, good biocompatibility, and targeted drug delivery. The technology of using Gram-negative bacteria to load drugs has a wide range of potential applications, such as:

[0006] 1) Anti-cancer therapy: Gram-negative bacteria such as Escherichia coli Nissle 1917 (EcN) and Akkermansia muciniphila (AKK) naturally tend to accumulate in the hypoxic tumor microenvironment. Using the vacuum method to load anti-cancer drugs can achieve tumor-targeted delivery of drugs, improve efficacy and reduce toxicity to normal tissues.

[0007] 2) Vaccine carriers: Gram-negative bacteria have strong immune activation ability and can be used as vaccine carriers. By loading antigens or immunomodulatory molecules and utilizing their natural immune stimulation ability, the immunogenicity of the vaccine can be significantly improved.

[0008] 3) Antimicrobial infection treatment: Gram-negative bacteria loaded with antibiotics can target infections caused by drug-resistant bacteria, especially in biofilm-related infections. Gram-negative bacteria loaded with antibiotics can penetrate the biofilm and act directly on the site of infection.

[0009] 5) Inflammatory diseases: Gram-negative bacteria can naturally accumulate at sites of inflammation and can be used to treat diseases such as inflammatory bowel disease (IBD) and rheumatoid arthritis by loading them with anti-inflammatory drugs (such as steroids or non-steroidal anti-inflammatory drugs).

[0010] 6) Treatment of neurological diseases: Using modified Gram-negative bacteria to deliver neuroprotectants or neurotransmitter repair agents through drug delivery technology can break through the blood-brain barrier and treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0011] 7) Intestinal microecological restoration: Using Gram-negative bacteria as carriers to load prebiotics or specific drugs can repair the imbalance of intestinal flora and treat diseases related to microecological imbalance, such as metabolic syndrome and colitis.

[0012] 8) Personalized medicine: Gram-negative bacteria can be used to load patient-specific drugs and release them in a targeted manner to achieve personalized treatment, improve drug efficacy and reduce side effects.

[0013] Some prior art discloses the technology of using bacteria as carriers to load drugs. For example, patent CN108324956A discloses a preparation method of using Escherichia coli ghosts as carriers to load paclitaxel drugs. The invention uses inactive Escherichia coli ghosts as carriers of paclitaxel drugs to improve the targeting of drugs to tumor cells and increase the anti-tumor effect of paclitaxel. For another example, patent CN111001014B discloses an anti-tumor drug based on fixed bacteria as carriers and its application. The invention uses fixed bacteria as carriers of chemotherapy drugs. The fixed bacteria include Gram-positive bacteria and Gram-negative bacteria, preferably Salmonella. Summary of the invention

[0014] In view of this, in order to overcome the defects of short half-life, poor bioavailability, poor stability, easy oxidation and decomposition of drugs such as salidroside and EGCG, the present invention proposes a method for loading drugs using a Gram-negative bacteria vacuum method and a drug-loaded complex thereof. The present invention uses Gram-negative bacteria as a carrier and adopts a vacuum method to load drugs such as salidroside and epigallocatechin gallate, which has the characteristics of good targeting, high drug loading rate, and long-term sustained release of drugs.

[0015] One of the objectives of the present invention is to provide a drug-loaded complex.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

[0017] A drug-loaded complex, which consists of Gram-negative bacteria and loaded drugs, wherein the Gram-negative bacteria are carriers of the drugs; the drugs include any one or more of salidroside and epigallocatechin gallate.

[0018] Preferably, the Gram-negative bacteria include any one or more of Escherichia coli type 1917 and Akkermansia mucinosa.

[0019] A second object of the present invention is to provide a drug-loaded bacterium.

[0020] To achieve the above object, the present invention adopts the following technical solutions:

[0021] A drug-loaded bacterium, wherein the bacterium is Akkermansia mucinosa loaded with salidroside and / or 1917-type Escherichia coli loaded with epigallocatechin gallate.

[0022] The third object of the present invention is to provide a method for loading drugs using the Gram-negative bacteria vacuum method.

[0023] To achieve the above object, the present invention adopts the following technical solutions:

[0024] The Gram-negative bacteria vacuum drug loading method is used to co-incubate the Gram-negative bacteria with the loaded drug under vacuum conditions.

[0025] Preferably, the Gram-negative bacteria include any one or more of Escherichia coli type 1917 and Akkermansia mucinosa; the loaded drug includes any one or more of salidroside and epigallocatechin gallate.

[0026] Preferably, the vacuum treatment is performed for 0.5-6 hours.

[0027] Preferably, the drug mass ratio of the loaded drug to the Gram-negative bacteria is 0.5-5.

[0028] Preferably, the final concentration of the loaded drug is 3-7 mg / mL, more preferably 5 mg / mL.

[0029] A fourth object of the present invention is to provide a method for preparing the aforementioned drug-loaded bacteria.

[0030] To achieve the above object, the present invention adopts the following technical solutions:

[0031] The method for preparing the aforementioned drug-loaded bacteria is prepared by utilizing the aforementioned method for loading drugs using Gram-negative bacteria by vacuum method.

[0032] Preferably, the mucin Akkermansia and the salidroside are co-incubated under vacuum conditions for 0.5-4 hours to obtain the mucin Akkermansia loaded with salidroside, and the drug-to-drug ratio of the salidroside to the mucin Akkermansia is 0.5-5.

[0033] And / or the 1917 type Escherichia coli and the epigallocatechin gallate are co-incubated under vacuum conditions for 1-4 hours to obtain the 1917 type Escherichia coli loaded with epigallocatechin gallate, and the drug-to-drug ratio of the epigallocatechin gallate to the 1917 type Escherichia coli is 0.5-2.5.

[0034] More preferably, the drug-to-drug ratio of the salidroside to the Akkermansia mucinosa is 5.

[0035] More preferably, the drug-to-drug ratio of the epigallocatechin gallate to the 1917 Escherichia coli is 2.5.

[0036] More preferably, the Akkermansia muciniphila and the salidroside are co-incubated under vacuum conditions for 4 hours.

[0037] More preferably, the 1917 Escherichia coli and the epigallocatechin gallate are co-incubated under vacuum conditions for 2 hours.

[0038] Preferably, the final concentration of salidroside is 3-7 mg / mL, more preferably 5 mg / mL.

[0039] Preferably, the final concentration of epigallocatechin gallate is 3-7 mg / mL, more preferably 5 mg / mL.

[0040] Preferably, the final concentration of Akkermansia muciniphila is OD value = 1.0.

[0041] Preferably, the final concentration of the 1917 type Escherichia coli is OD value = 0.5.

[0042] The beneficial effects of the present invention are:

[0043] 1. The present invention provides a method for loading drugs using the vacuum method of Gram-negative bacteria. Existing drug loading methods all have some disadvantages, such as low bioavailability, small drug loading amount, no targeting after drug loading, and inability to release quantitatively. The present invention uses Gram-negative bacteria as a carrier and adopts the vacuum method to load drugs, which has many advantages over the existing technology and can solve the problems of short half-life, low bioavailability, poor stability and easy oxidation and decomposition of some commonly used drugs in practical applications. And based on the health benefits of probiotics themselves in recent years, the present invention uses the vacuum method to load drugs to not only solve the above problems, but also can colonize in the intestine, with targeting to achieve the function of 1+1>2.

[0044] 2. The SAL@AKK and EGCG@EcN prepared by the present invention have good drug loading rate and encapsulation rate. Under the optimal conditions, the drug loading rate of the prepared SAL@AKK is 34.88%, and the encapsulation rate is 10.71%; the drug loading rate of EGCG@EcN is 35.0%, and the encapsulation rate is 21.5%.

[0045] 3. The SAL@AKK of the present invention can slowly release SAL in the intestinal fluid and continue to release SAL after 5 hours with a release rate of 8.89%, which successfully solves the problem of short drug half-life.

[0046] 4. The present invention adopts a vacuum method to load drugs. Under vacuum conditions, the drugs will not be oxidized and decomposed, which effectively improves the stability of the drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The SEM and TEM test results of AKK are shown in Figure 1. Figure 1 -A is the SEM image of AKK, Figure 1 -B is the TEM image of AKK.

[0048] Figure 2 This is the DLS particle size detection result of AKK.

[0049] Figure 3 This is the Zeta potential detection result of AKK.

[0050] Figure 4 This is the result of the flat plate coating test after AKK vacuum treatment, where: Figure 4 -A is the control plate coating test result diagram, Figure 4 -B is the flat plate coating test result after 2 hours of AKK vacuum treatment, Figure 4 -C is the flat plate coating test result after AKK vacuum treatment for 4 hours, Figure 4 -D is the flat plate coating test result after AKK vacuum treatment for 6 hours, Figure 4 -F is the flat plate coating test result after AKK vacuum treatment for 8 hours, Figure 4 -G is the flat plate coating test result after AKK vacuum treatment for 10 hours.

[0051] Figure 5 The SEM and TEM test results of AKK after vacuum treatment are shown in Figure 1. Figure 5 -A is the SEM image of AKK after vacuum treatment, Figure 5 -B is the TEM image after AKK vacuum treatment.

[0052] Figure 6 This is the DLS particle size test result after AKK vacuum treatment.

[0053] Figure 7 This is the Zeta potential detection result of AKK after vacuum treatment.

[0054] Figure 8 This is the SAL standard curve.

[0055] Fig. 9 Representative HPLC images of 0.5 mg / mL SAL solution in PBS.

[0056] Fig.10 The SEM, TEM and flat coating test results of SAL@AKK are shown below. Fig.10 -A is the SEM image of SAL@AKK, Fig.10 -B is the TEM image of SAL@AKK, Fig.10 -C is a graph showing the plate coating activity of SAL@AKK prepared by vacuum treatment for 4 h.

[0057] Fig.11 This is the DLS particle size detection result of SAL@AKK.

[0058] Fig.12 This is the DLS particle size detection result of SAL.

[0059] Fig.13 This is the Zeta potential detection result of SAL@AKK.

[0060] Fig.14 This is the Zeta potential detection result of SAL@AKK.

[0061] Fig.15 is the structural characterization result diagram of EcN, where: Fig.15 -A is the SEM image of EcN, Fig.15 -B is the TEM image of EcN.

[0062] Fig.16 This is the DLS particle size detection result of EcN.

[0063] Fig.17 This is the Zeta potential detection result of EcN.

[0064] Fig.18 This is the result of the flat plate coating test after EcN vacuum treatment, where: Fig.18 -A is the control plate coating test result diagram, Fig.18 -B is the flat plate coating test result after EcN vacuum treatment for 2 hours, Fig.18 -C is the flat plate coating test result after EcN vacuum treatment for 4 hours, Fig.18 -D is the flat plate coating test result after EcN vacuum treatment for 6 hours, Fig.18-E is the flat plate coating test result after EcN vacuum treatment for 8 hours, Fig.18 -F is the flat plate coating test result after EcN vacuum treatment for 10 hours.

[0065] Fig.19 The TEM and SEM test results of EcN after vacuum treatment are shown in Figure 1. Fig.19 -A is the TEM image of EcN after vacuum treatment, Fig.19 -B is the SEM image of EcN after vacuum treatment.

[0066] Fig. 20 This is the DLS particle size test result of EcN after vacuum treatment.

[0067] Fig.21 The results of plate coating after mixing different EGCG concentrations with bacterial solution are shown in Figure 1. Fig.21 -A is the plate coating result diagram of the normal group; Fig.21 -B is the plate coating result after EGCG with a concentration of 0.1 mg / mL was mixed with bacterial solution; Fig.21 -C is the plate coating result after mixing EGCG with bacterial solution at a concentration of 0.5 mg / mL; Fig.21 -D is the plate coating result after mixing EGCG with bacterial solution at a concentration of 1 mg / mL; Fig.21 -E is the plate coating result after EGCG with a concentration of 2.5 mg / mL was mixed with bacterial solution; Fig.21 -F is the plate coating result after EGCG with a concentration of 5 mg / mL was mixed with bacterial solution; Fig.21 -G is the plate coating result after mixing EGCG with bacterial solution at a concentration of 10 mg / mL; Fig.21 -H is the plate coating result after EGCG with a concentration of 20 mg / mL was mixed with bacterial solution.

[0068] Fig. 22 The SEM and TEM test results of EGCG@EcN are shown in Figure 2. Fig. 22 -A is the SEM image of EGCG@EcN, Fig. 22 -B is the TEM image of EGCG@EcN.

[0069] Fig.23 This is the DLS particle size test result of EcN after vacuum treatment.

[0070] Fig.24 This is the Zeta potential detection result of EcN after vacuum treatment. DETAILED DESCRIPTION

[0071] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0072] In the embodiments of the present invention, Gram-negative bacteria such as Escherichia coli Nissle 1917 (EcN) and Akkermansia muciniphila (AKK) are involved, wherein EcN is purchased from Bioboway Biotechnology.

[0073] In an embodiment of the present invention, a method for loading drugs using a Gram-negative bacteria vacuum method mainly comprises the following steps:

[0074] (1) Bacterial culture

[0075] (2) Activity of bacteria after vacuum treatment

[0076] The concentration was 1×10 9 1 mL of the Gram-negative bacterial solution with a CFU / mL was placed in a 10 mL centrifuge tube and placed in a vacuum drying oven, set to 37°C and evacuated. The tube was taken out at 2, 4, 6, 8 and 10 h, diluted in parallel to the appropriate concentration, and 10 μL was taken out for plate coating in the incubator. After 24-48 h, the number of colonies was observed to evaluate the activity of Gram-negative bacteria after vacuum treatment, thereby determining the vacuum treatment conditions that retain the activity of Gram-negative bacteria.

[0077] (3) Determination of drug antibacterial concentration

[0078] The concentration was 0.5×10 9 1 mL of Gram-negative bacterial solution with a CFU / mL was placed in a 10 mL centrifuge tube and incubated with different concentrations of drugs (EGCG) for 24 hours. After parallel dilution to the appropriate concentration, 10 μL was taken for plate coating. After 24-48 hours, the number of colonies was observed to evaluate the minimum inhibitory concentration, so as to determine the optimal concentration of the drug.

[0079] (4) Preparation and characterization

[0080] First, a standard curve of drug concentration is obtained by high performance liquid chromatography or other methods.

[0081] 1 mL of Gram-negative bacteria with a final concentration of OD value = 0.5 or 1.0 was centrifuged (wet weight was about 1 mg) and mixed with a drug solution with the optimal drug loading concentration, and incubated under vacuum conditions of 0.5, 1, 2, 4, 6, 8, and 10 h. After the treatment, the supernatant was taken after centrifugation at 6000×rpm, and diluted in proportion to a final concentration of the drug stock solution of 0.5 mg / mL for easy comparison. The concentration obtained in the supernatant was measured by high performance liquid chromatography to obtain the vacuum treatment time with the optimal drug loading amount, and different amounts of Gram-negative bacteria drug solution were incubated at this time point to obtain the drug-to-mass ratio and vacuum treatment conditions with the maximum drug loading amount, and prepared under these conditions, and at the same time, a part of the prepared drug loading system was taken for scanning electron microscopy and transmission electron microscopy, and DLS and Zeta potential were measured to determine the bacterial morphology and the dispersion coefficient in PBS.

[0082] In the embodiment of the present invention, the preparation method of SAL@AKK is as follows:

[0083] 1 mL of AKK with a final concentration of OD value = 1.0 was centrifuged (wet weight was about 1 mg), and incubated with 1 mL of SAL with a final concentration of 5 mg / mL and a solvent of PBS under vacuum for 0.5-6 h (preferably 4 h) to obtain SAL@AKK.

[0084] In the embodiment of the present invention, the preparation method of EGCG@EcN is as follows:

[0085] After centrifuging EcN with a final concentration of OD value = 0.5 (wet weight is about 1 mg), it is mixed with 1 mL of EGCG solution with a final concentration of 5 mg / mL, and incubated under vacuum conditions for 0.5-6 h, preferably 2 h, to obtain EGCG@EcN.

[0086] Example 1: Loading SAL using the AKK vacuum method

[0087] (1) Cultivation, management and detection of AKK

[0088] Prepare liquid culture medium at a ratio of 29.25 g / L in a 500 mL anaerobic culture bottle. Continue to deoxygenate with nitrogen for 5 minutes and sterilize with high pressure steam at 121°C for 15 minutes. After natural cooling, place in an anaerobic incubator and inoculate AKK. Anaerobic culture for 36 hours until the logarithmic growth phase, collect bacteria by centrifugation at 9000×rpm, and wash three times with sterile PBS. After resuspending in PBS, measure the OD value of the bacterial solution at 600nm. Dilute the bacterial solution to a concentration of 1×10 9CFU / mL. At the same time, some bacteria were fixed with 4% paraformaldehyde for 2-4h, washed 3 times with sterile PBS, and then dehydrated with 30%, 50%, 70%, 90%, 95%, and 100% ethanol solutions. After appropriate dilution, drop them on the silicon wafer and observe them by scanning electron microscope. At the same time, ultrathin sections were prepared, pre-embedded with agar, and fixed with 1% osmium acid prepared in 0.1M phosphate buffer (pH=7.4) at room temperature in the dark for 2h for post-fixation. Then rinsed with 0.1M phosphate buffer PB (pH=7.4) 3 times, 15min each time. Then the tissue was dehydrated with 30%-50%-70%-80%-95%-100%-100% ethanol gradient, 20min each time, and 100% acetone twice, 15min each time. Then embed with acetone:812 embedding agent = 1:1 at 37℃ for 2-4h, acetone:812 embedding agent = 1:2 at 37℃ for overnight penetration, and pure 812 embedding agent at 37℃ for 5-8h. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate and place it in a drying oven at 37℃ overnight, place the embedding plate at 60℃ for polymerization for 48h, take out the resin block for use, slice the resin block into 60-80nm ultrathin slices on an ultrathin slicer, observe the appearance with field emission transmission electron microscopy, and then use a laser particle size analyzer to measure the particle size (DLS) and Zeta potential to determine the dispersion coefficient of AKK in PBS.

[0089] Results: SEM test results are as follows Figure 1 -A, TEM test results are as follows Figure 1 -B, the DLS particle size test results are as follows Figure 2 The Zeta potential test results are shown in Figure 3 As shown in Figure 2. SEM results show that AKK in this study is an oval fungus with some irregular structures on the surface, which is plump and complete, consistent with the structure observed by TEM. The average DLS particle size is 1654.9nm and the average Zeta potential is -15.6mV.

[0090] (2) Activity of AKK after vacuum treatment

[0091] The concentration was 1×10 9 1 mL of AKK bacterial solution with CFU / mL was placed in a 10 mL centrifuge tube and placed in a vacuum drying oven, set to 37°C and vacuumed. It was taken out at 2, 4, 6, 8 and 10 h, diluted in parallel to the appropriate concentration, and 30 μL was taken out for plate coating in an anaerobic incubator. After 48 h, the number of colonies was observed to evaluate the activity of AKK after vacuum treatment, so as to determine the vacuum treatment conditions that retain AKK activity.

[0092] Results: The coating results of the flat plate after AKK vacuum treatment are as follows Figure 4As shown in the results, the activity of AKK was almost unaffected before 12 hours after vacuum treatment, but its activity dropped significantly at 12 hours. Therefore, vacuum treatment within 6 hours was selected as the vacuum condition for subsequent experiments. The SEM and TEM test results are shown in Figure 2. Figure 5 -A and Figure 5 -B, the results show that the vacuum AKK morphology reflected by TEM is consistent with SEM, proving that after vacuum treatment, many wrinkles appeared on the surface of AKK. These wrinkles increased the specific surface area and the pore size of the membrane, which may be the main reason why bacteria can carry more drugs after vacuum treatment. The DLS particle size and Zeta potential test results of AKK after vacuum treatment are shown in Figure 1. Figure 6 and Figure 7 As shown, the results showed that after vacuum treatment, the DLS particle size of AKK bacteria decreased from an average of 1654.9 nm to 949.4 nm, which was consistent with the bacterial shrinkage phenomenon observed by SEM and TEM. The average Zeta potential was -16.5 mV, with little change.

[0093] (3) Preparation and characterization of SAL@AKK

[0094] The standard curve of SAL 50%:50% sterile PBS-methanol solution was obtained by high performance liquid chromatography. The conditions were as follows: the mobile phase was methanol-water (20:80), the detection wavelength was 275nm, the flow rate was 0.5mL / min, the column temperature was 40℃, the sample collection volume was 10μL, and the analysis time was 20min.

[0095] 1 mL of AKK with a final concentration of OD value = 1.0 was centrifuged (wet weight was about 1 mg) and co-incubated with 1 mL of SAL with a final concentration of 5 mg / mL and PBS as solvent under vacuum conditions for 0.5, 1, 2, 4, 6, 8, and 10 hours. After the treatment, the supernatant was centrifuged at 9000×rpm and diluted to a final concentration of 0.5 mg / mL of SAL stock solution for easy comparison. The concentration of SAL in the supernatant was measured by high-performance liquid chromatography to obtain the vacuum treatment time with the best drug loading. At this time point, AKK was co-incubated with different concentrations of SAL (10 mg / mL and 1 mg / mL) to obtain the drug-to-mass ratio and vacuum treatment conditions with the maximum drug loading, and SAL@AKK was prepared under this condition. At the same time, part of the prepared SAL@AKK was taken for scanning electron microscopy and transmission electron microscopy, and DLS and Zeta potential were measured to determine the bacterial morphology and the dispersion coefficient in PBS.

[0096] Results: The standard curve of 50%:50% sterile PBS-methanol solution of SAL and the representative HPLC images of 0.5 mg / mL SAL are shown in Figures 1 and 2. Figure 8 , Fig. 9As shown. The HPLC peak area, drug loading, and encapsulation efficiency of AKK and SAL after vacuum co-incubation for 0.5, 1, 2, 4, 6, 8, and 10 hours and the original SAL solution are shown in Table 1. It can be seen that the maximum drug loading was reached at 4 hours, and the peak area increased after 8 hours. On the one hand, it may be due to the large-scale death of AKK at 6 hours, resulting in drug leakage, which is consistent with the results of the previous activity experiment; on the other hand, it can be attributed to the error of the measurement. Subsequently, the HPLC peak area, drug loading, and encapsulation efficiency of the original SAL solution after co-incubation of SAL and AKK at different concentrations at the time point of 4 hours are shown in Table 1. 1mL of AKK with a final concentration of OD value = 1.0 and 1mL of 5mg / mL SAL vacuum co-incubation has the maximum drug loading, that is, the optimal drug-to-mass ratio is 5.

[0097] SAL@AKK's SEM, TEM and flat coating test results are as follows Fig.10 As shown, no significant difference was observed between the SEM and TEM images and the vacuum AKK, and the plate coating activity did not change significantly compared with the AKK treated with vacuum alone. However, the DLS particle size increased from an average of 949.4 nm for the vacuum AKK to an average of 1295.0 nm, see Fig.11 This can be attributed to the increase in bacterial volume after SAL loading. Zeta potential and DLS particle size of SAL Fig.12 , Fig.13 As shown, its average Zeta potential is -24.8 mV, while the average Zeta potential of SAL@AKK is -17.4 mV. Fig.14 The Zeta potential of SAL@AKK is not much different from that of vacuum AKK, which indicates that the SAL carried is not adsorbed on the surface of AKK, but constitutes a delivery system in an internal manner.

[0098] Table 1. HPLC peak area, drug loading rate and encapsulation efficiency of SAL@AKK treated with different time and drug-to-mass ratio in vacuum co-incubation

[0099] Processing Peak area Drug loading rate Encapsulation efficiency 5mg / mL SAL 3182801 / / 5mg / mL SALvacuum 0.5h 2952047 31.58％ 9.23％ 5mg / mL SALvacuum 1h 2945936 32.14％ 9.47％ 5mg / mL SALvacuum 2h 2947714 31.98％ 9.40％ 5mg / mL SALvacuum 4h 2914981 34.88％ 10.71％ 5mg / mL SALvacuum 6h 3009690 25.72％ 6.92％ 5mg / mL SALvacuum 8h 3185260 -0.49％ -0.10％ 5mg / mL SALvacuum 10h 3191401 -1.75％ -0.34％ 1mg / mL SAL 3206548 / / 1mg / mL SALvacuum 4h 3206404 0.29％ 0.01％ 10mg / mL SAL 3217168 / / 10mg / mL SALvacuum 4h 3211231 0.24％ 10.61％

[0100] (4) Intestinal fluid release of SAL@AKK

[0101] Since the solvent of the test sample is artificial intestinal fluid, the concentration of 0.5 mg / mL SAL solution with 50%:50% methanol-artificial intestinal fluid was determined by HPLC under the same conditions to prove that the change of solvent would not affect the normal peak of SAL. Subsequently, 20 mL of the prepared SAL@AKK was centrifuged to remove the supernatant, mixed with 40 mL of artificial intestinal fluid, and incubated on a shaker at 50×rpm / min and 37°C for 6 hours. After taking 1 mL of the mixed solution at time points 1, 1.5, 2, 3, 4, and 5, 1 mL of fresh artificial intestinal fluid was added. After the mixed solution was centrifuged, 0.5 mL of the supernatant was taken and mixed with 0.5 mL of methanol (HPLC grade), and the SAL content was determined by high-performance liquid chromatography to obtain the intestinal fluid release of SAL@AKK.

[0102] Results: The amount of SAL released from the SAL@AKK sample co-incubated with artificial gastric juice after artificial intestinal juice is shown in Table 2. It can be concluded that SAL@AKK can slowly release SAL in intestinal juice and continue to release SAL at 5 h, with a release rate of 8.89%.

[0103] Table 2. Time gradient HPLC peak area, release amount and release rate of SAL@AKK co-incubated with artificial intestinal fluid

[0104]

[0105]

[0106] Example 2: Loading EGCG using EcN vacuum method

[0107] (1) Cultivation, management and detection of 1917 Escherichia coli

[0108] Prepare a liquid culture medium at a ratio of 25g / L in a conical flask and seal it with a sealing film. Sterilize at 121℃ with high pressure steam for 20min, cool naturally and place in a clean bench. Take the frozen or previously cultured full bacterial solution and inoculate it in the newly sterilized liquid culture medium with EcN, and culture it in a shaker at 37℃ for 12 hours until the logarithmic growth phase. Collect the bacteria by centrifugation at 8000rpm and wash three times with sterile saline. After resuspending in saline, measure the OD value of the bacterial solution at 600nm. And dilute the bacterial solution to a concentration of 0.5×109CFU / mL. At the same time, take some bacteria and fix them overnight with 4% glutaraldehyde, and wash them three times with sterile saline. Then use 30%, 50%, 70%, 90%, and 100% ethanol solutions for gradient dehydration. After the last centrifugation, a proper amount of 100% ethanol suspension was dropped onto the silicon wafer and copper grid, and the appearance was observed by scanning electron microscope and transmission electron microscope respectively. The particle size (DLS) and Zeta potential were measured by laser particle size analyzer.

[0109] Results: The SEM test results of EcN are as follows Fig.15 -A, the TEM test results of EcN are as follows Fig.15 -B, it can be seen from the SEM results that the EcN in the present invention is an oval fungus with some irregular structures on the surface, which is plump and complete, consistent with the structure observed by TEM. The DLS particle size and Zeta potential detection results of EcN are shown in Figure 1. Fig.16 , Fig.17 As shown, the average DLS particle size is 1542.1 nm and the average Zeta potential is -23.5 mV.

[0110] (2) EcN vacuum treatment

[0111] The concentration was 0.5×10 9 Take 2mL of EcN bacterial solution with CFU / mL and place it in a 10mL centrifuge tube, and put it in a vacuum drying oven, set to 37℃ and perform vacuum treatment. Take it out at 0, 2, 4, 6, 8, and 10h, respectively, dilute it in parallel to the appropriate concentration by the same multiple, measure its activity with CCK-8 and take 10μL for plate coating, and culture it in a 37℃ incubator. After 24h, observe the number of colonies to estimate the activity of EcN after vacuum treatment, so as to determine the vacuum treatment conditions that retain the activity of EcN.

[0112] Results: The EcN vacuum-treated flat plate was coated as Fig.18 As shown in the figure, it can be seen that the activity of AKK is almost unaffected before 10 hours after vacuum treatment. Therefore, vacuum treatment within 10 hours is selected as the vacuum condition for subsequent experiments. The TEM, SEM, and DLS particle sizes of EcN after vacuum treatment are shown in Figure 2. Fig.19 -A. Fig.19 -B. Fig. 20 As shown. In the results of SEM and TEM, the morphology of vacuum AKK reflected by TEM is consistent with SEM, proving that after vacuum treatment, many wrinkles appeared on the surface of AKK. These wrinkles increase the specific surface area and the pore size of the membrane, which may be the main reason why bacteria can carry more drugs after vacuum treatment. After vacuum treatment, the DLS particle size of EcN decreased from an average of 1542.1nm to 1029.3nm, which is consistent with the bacterial shrinkage observed by SEM and TEM.

[0113] (3) Determination of EGCG antibacterial concentration

[0114] First, EGCG was weighed and prepared into a high concentration stock solution of 20 mg / mL, and then gradiently diluted to 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL. Then, 1×10 9After incubating the bacterial solution with CFU / mL and the EGCG solution for 24 hours, 10 μL was taken for plate coating after dilution by the same multiple, and the plate was plated three times in parallel. After 24-48 hours, the colony was observed.

[0115] The results are as follows Fig.21 As shown in the figure, the colony counts at concentrations of 20 mg / mL and 10 mg / mL were significantly reduced, showing a significant antibacterial effect, while the colony counts at concentrations of 5 mg / mL and below were not significantly different from those in the normal group. Therefore, 5 mg / mL was used as the drug loading concentration.

[0116] (4) Preparation and characterization of EGCG@EcN

[0117] First, the standard curve of EGCG was obtained by high performance liquid chromatography under the following conditions: the mobile phase composition was methanol: water: acetic acid = 23:75:2, the flow rate was 0.5 mL / min, the wavelength was 276 nm, the column temperature was 24 °C, the sample collection volume was 20 μL, and the analysis time was 35 min.

[0118] 1) Determination of drug loading time

[0119] A certain amount of EcN with a final concentration of OD value of 0.5 was centrifuged (wet weight was about 10 mg) and mixed with EGCG solution to prepare a final concentration of 5 mg / mL. Then, co-incubated under vacuum conditions, centrifuged at 8000 rpm after the treatment, and the supernatant was taken. The supernatant was diluted by the same multiple (5 times) for easy comparison, and the concentration of the remaining EGCG solution in the supernatant was measured by high performance liquid chromatography, so as to obtain the vacuum treatment time with the best drug loading.

[0120] 2) Determination of drug-to-mass ratio

[0121] The drug loading time was determined as above and EcN was co-incubated with different concentrations of EcN (10 mg / mL and 1 mg / mL) at this time point. After the treatment, the supernatant was taken after centrifugation at 8000 rpm, and the supernatant was diluted by the same multiple (10 times) for easy comparison to obtain the drug-to-mass ratio and vacuum treatment conditions for the maximum drug loading amount. EGCG@EcN was prepared under these conditions, and a portion of the prepared EGCG@EcN was taken for scanning electron microscopy and transmission electron microscopy, and DLS and Zeta potential were measured to determine the bacterial morphology and the dispersion coefficient in physiological saline.

[0122] Results: The HPLC peak area, drug loading, and encapsulation efficiency of EcN and EGCG after vacuum co-incubation for 0.5, 1, 2, 3, 4, 5, and 6 hours are shown in Table 3. It can be seen that the maximum drug loading was reached at 2 hours, and the peak area increased after 2 hours. On the one hand, it may be due to the death of EcN at 2 hours, resulting in drug leakage; on the other hand, it can be attributed to the measurement error. Subsequently, the HPLC peak area, drug loading, and encapsulation efficiency of EGCG co-incubated with different amounts of EcN were measured at 2 hours. The results are shown in Table 4. 2 mg / mL EcN and 5 mg / mL EGCG co-incubated in vacuum for 2 hours have the maximum drug loading, so the optimal drug-to-mass ratio is 2.5.

[0123] Table 3. HPLC peak area, drug loading rate and encapsulation efficiency of EGCG@EcN in vacuum co-incubation at different time

[0124] Processing Peak area (average) Residual concentration Drug loading rate Encapsulation efficiency 5mg / mLEGCGvacuum0.5h 45719080.5 0.842 7.30％ 15.76％ 5mg / mLEGCGvacuum1h 34065906.5 0.637 15.33％ 36.21％ 5mg / mLEGCGvacuum2h 29876825.5 0.564 17.89％ 43.56％ 5mg / mLEGCGvacuum3h 30436909.5 0.574 17.55％ 42.58％ 5mg / mLEGCG vacuum 4h 36635407 0.683 13.68％ 31.70％ 5mg / mLEGCGvacuum5h 38474042 0.715 12.46％ 28.47％ 5mg / mLEGCG vacuum 6h 40051576 0.743 11.39％ 25.71％

[0125] Table 4. HPLC peak area, drug loading rate and encapsulation efficiency of EGCG@EcN in vacuum co-incubation with different drug-to-mass ratios

[0126] Processing Peak area (average) Residual concentration (mg / mL) Drug loading rate Encapsulation efficiency 8mgEcNvacuum2h 15992219 0.321 18.3％ 35.9％ 6mgEcNvacuum2h 20026973 0.351 19.8％ 29.7％ 4mgEcNvacuum2h 21482792.5 0.377 23.5％ 25.0％ 2mgEcNvacuum2h 22354773.5 0.392 35.0％ 21.5％

[0127] The SEM and TEM test results of EGCG@EcN are shown in Figure 2. Fig. 22 -A. Fig. 22 -B, the DLS particle size and Zeta potential of EcN after vacuum treatment are shown in Fig.23 , Fig.24 As shown. It can be seen that the DLS particle size increased from an average of 1029.3nm in EcN to an average of 1600.8nm, which can be attributed to the increase in bacterial volume after EGCG was loaded. The average Zeta potential of EcN is -23.5mV, while the average Zeta potential of EGCG@EcN is -23.9mV, which is not much different, indicating that the EGCG loaded is not adsorbed on the surface of EcN, but constitutes a delivery system in an internal loading manner.

Claims

1. A drug-loaded complex, characterized in that: The drug-carrying complex is composed of Gram-negative bacteria and loaded drugs, wherein the Gram-negative bacteria is a carrier of the drugs; the drugs include any one or more of salidroside and epigallocatechin gallate.

2. The drug-loaded complex according to claim 1, characterized in that: The Gram-negative bacteria include any one or more of 1917 Escherichia coli and Akkermansia mucinosa.

3. A drug-loaded bacterium, characterized in that: The bacteria are Akkermansia mucinosa containing salidroside and / or Escherichia coli type 1917 containing epigallocatechin gallate.

4. A method for loading drugs using a Gram-negative bacteria vacuum method, characterized in that: Gram-negative bacteria were incubated with the loaded drug under vacuum conditions.

5. The method according to claim 4, characterized in that The Gram-negative bacteria include any one or more of 1917 Escherichia coli and Akkermansia mucinosa; the loaded drugs include any one or more of salidroside and epigallocatechin gallate.

6. The method according to claim 4, characterized in that Vacuum treatment for 0.5-6 hours.

7. The method according to claim 4, characterized in that The drug mass ratio of the internal drug to the Gram-negative bacteria is 0.5-5.

8. The method according to claim 4, characterized in that The final concentration of the loaded drug is 3-7 mg / mL.

9. The method for preparing drug-loaded bacteria according to claim 3, characterized in that: The method is used for preparation.

10. The method according to claim 9, characterized in that The mucin Akkermansia and the salidroside are co-incubated under vacuum conditions for 0.5-4 hours to obtain the mucin Akkermansia loaded with salidroside, and the drug-to-drug ratio of the salidroside to the mucin Akkermansia is 0.5-5. And / or the 1917 type Escherichia coli and the epigallocatechin gallate are co-incubated under vacuum conditions for 1-4 hours to obtain the 1917 type Escherichia coli loaded with epigallocatechin gallate, and the drug-to-drug ratio of the epigallocatechin gallate to the 1917 type Escherichia coli is 0.5-2.5.

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