CN116172978A

CN116172978A - Composite cell membrane bionic targeted antibacterial nano drug delivery system and preparation method thereof

Info

Publication number: CN116172978A
Application number: CN202310114910.1A
Authority: CN
Inventors: 王伟; 刘贺宁; 唐璐
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2023-02-15
Filing date: 2023-02-15
Publication date: 2023-05-30

Abstract

The invention discloses a composite cell membrane bionic targeted antibacterial nano drug delivery system and a preparation method thereof, wherein the drug delivery system is formed by a liposome drug-carrying inner core composed of phospholipid, cholesterol, antibiotics and phototherapy agents and a composite membrane which is modified on the surface of the liposome drug-carrying inner core and is prepared from a heme cell membrane and a macrophage membrane, the cell membrane fusion modified liposome with different sources is further expanded and the function of the drug-carrying nano system is enhanced, the ideal long circulation time is obtained, the in vivo degradation rate is reduced, the drugs are more effectively delivered, the use dosage of the antibiotics is obviously reduced while the treatment efficiency is improved, the possibility of bacterial drug resistance is reduced, the systemic toxicity is reduced, the life cycle of the drug delivery system is prolonged, and the slow release of the antibiotics is realized; on the other hand, the liposome endows bionic performance, and has good biocompatibility and non-immunogenicity.

Description

Composite cell membrane bionic targeted antibacterial nano drug delivery system and preparation method thereof

Technical Field

The invention relates to a targeted antibacterial nano drug delivery system, in particular to a composite cell membrane bionic targeted antibacterial nano drug delivery system and a preparation method of the drug delivery system.

Background

Bacterial infections often induce a variety of diseases such as peritonitis, pneumonia, meningitis, sepsis, skin ulcers, etc., which pose a great threat to world economy and public health. Traditional antibiotic therapy is the primary means of controlling and treating bacterial infections. The emergence and prevalence of bacterial resistance caused by improper and excessive use of antibiotics can result in the loss of therapeutic effect of conventional antibiotic treatment, and the development of a brand new antibiotic is about 10-18 years, but the generation of resistance of antibiotics only requires 1-2 years, and the bacterial resistance speed is far higher than the development speed of new antibiotics. Reducing the amount of antibiotics can delay the time of bacteria resistant to the antibiotics, but the efficacy is greatly compromised. In recent years, development and research of nano-antibacterial agents for treating bacterial infectious diseases and overcoming bacterial resistance have been rapidly progressed. Nanomaterial (such as silver, zinc oxide, titanium dioxide, etc.) produces unique antibacterial activity through interfacial effect, small-sized effect, etc., and has excellent therapeutic effect in resisting bacterial infection. However, these nanomaterials also suffer from several drawbacks including non-specific cytotoxicity, poor biocompatibility, lack of targeting, etc.

Abuse and misuse of antibiotics are major factors leading to the development of bacterial resistance. Therefore, a reasonable drug delivery system is constructed, the time and space controllable release of the antibiotics is realized, the improper use of the antibiotics is avoided, and the method is an effective means for reducing the generation of bacterial drug resistance. The antibiotic improvement by the nano drug-carrying mode is one of the methods, however, the problems of poor targeting of the nano carrier, poor treatment effect by a single means and the like exist in the prior art; therefore, it is important to develop an alternative therapeutic strategy to enhance biosafety, therapeutic efficacy, prevent and reduce the development of bacterial resistance.

Disclosure of Invention

The invention aims to: the invention aims to provide a composite cell membrane bionic targeted antibacterial nano drug delivery system with strong bacterial chemotaxis, good biological safety and insensitive bacterial drug resistance, and also provides a preparation method of the drug delivery system.

The technical scheme is as follows: the invention relates to a composite cell membrane bionic targeted antibacterial nano drug delivery system which is formed by a liposome drug-carrying inner core composed of phospholipid, cholesterol, antibiotics and a phototherapy agent and a composite membrane RM which is modified on the surface of the liposome drug-carrying inner core and is prepared from a red cell membrane RBCM and a macrophage membrane Mm, wherein the antibiotics are one of Amikacin (AM), ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MXF), imipenem (IPM), aztreonam (AZT) and Tobramycin (TOB).

Preferably, the mass ratio of the phospholipid, the cholesterol, the phototherapy agent and the antibiotics is 200:50:16:1-5, the mass ratio of the heme membrane to the macrophage membrane is 3-1:1, and the mass ratio of the phospholipid to the composite membrane in the liposome drug-carrying inner core is 3-1:1.

Preferably, the phototherapy agent is black phosphorus nano-sheet (BP), black Phosphorus Quantum Dot (BPQD), molybdenum disulfide (MoS) ₂ ) Gold nanoparticles (AuNPs), gold nanorods (AuNRs), gold nanocages (AuNCs), indocyanine green (ICG), new indocyanine green (IR 820), polydopamine (PDA), graphene Quantum Dots (GQDs), or the like.

Preferably, the phospholipid is one of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, sphingomyelin, palmitoyl phosphatidylcholine.

Preferably, the macrophage membrane is derived from one of mouse mononuclear macrophage leukemia cells (RAW 264.7), bone marrow-derived macrophages (BMDM), peritoneal-derived macrophages (PM), spleen-derived macrophages (RSMa), monocyte-derived macrophages (MDM), and peripheral blood-derived macrophages.

Preferably, the bacterial type is one of pseudomonas aeruginosa (p. Aeromonas), staphylococcus aureus (s. Aureus), klebsiella pneumoniae (k. Peneumoniae), streptococcus pneumoniae (s. Pneumoniae), escherichia coli (e. Coli).

The preparation method of the drug delivery system comprises the following steps:

(1) Dissolving phospholipid and cholesterol in an organic solvent, evaporating to remove the organic solvent to form a film, adding a mixed aqueous solution of a phototherapy agent and an antibiotic, carrying out hydration reaction and ultrasonic dispersion, and finally filtering through a water-based filter membrane to obtain a liposome medicine carrying inner core loaded with the antibiotic and the phototherapy agent;

(2) Separating out red blood cells by low-speed centrifugation, performing ice bath pyrolysis, centrifuging at high speed, and freeze-drying to obtain red blood cell membrane;

(3) Resuspension of macrophages with Tris-magnesium salt buffer solution, standing, ice bath ultrasound, centrifugation, precipitation and freeze-drying to obtain macrophage membranes;

(4) Dissolving a red blood cell membrane and a macrophage membrane in phosphate buffer salt solution, and performing ultrasonic dispersion to prepare a composite membrane; and then mixing the mixture with liposome drug-carrying inner cores uniformly, extruding the mixture together, and centrifuging the mixture to obtain the composite cell membrane bionic targeted antibacterial nano drug delivery system.

Preferably, in the step (1), the mass ratio of the phospholipid, the cholesterol, the phototherapy agent and the antibiotic is 200:50:16:1-200:50:16:5; the antibiotic is AM, and the phototherapy agent is BPQD; the ultrasonic power is 200-300W; the ultrasonic time is 15-30 min.

Preferably, in the step (2), the time for ice bath lysis of the red blood cells is 20-30 min.

Preferably, in the step (3), the power of the macrophage ultrasonic disruption is 50-100W, and the ultrasonic time is 5-15min.

Preferably, in the step (4), the mass ratio of the phospholipid to the cell membrane is 3:1-1:1; the coextrusion times are 10 to 30 times.

Preferably, the step (1) specifically comprises: the phospholipid and cholesterol were precisely weighed out to a certain mass and dissolved in an organic solvent and added to a round bottom flask. And (3) performing rotary evaporation at 40 ℃ for 15min to form a layer of uniform film on the inner wall, adding a mixed aqueous solution of BPQD and AM, performing rotary hydration at 40 ℃ for 30min, performing ultrasonic dispersion on the aqueous dispersion through a probe, and finally filtering through a 0.45 mu m and 0.22 mu m aqueous filter membrane to obtain the liposome inner core AB@Lip loaded with AM and BPQD.

Preferably, the step (2) specifically comprises: fresh blood of the mice is collected in a centrifuge tube containing heparin sodium, lower red blood cells are collected by low-speed centrifugation, washed 3 times by 1 XPBS, and then added into a 10-time volume of precooled hypotonic lysate (2.5 mM PBS), and the mixture is subjected to ice bath lysis for 20-30 min. Centrifuging at 4deg.C and 2000 Xg for 15min in a high-speed low-temperature centrifuge to remove impurities. Centrifuging supernatant at 4deg.C and 10000 Xg for 1 hr, collecting precipitate, lyophilizing for 24 hr to obtain RBCM, and storing at-80deg.C.

Preferably, the step (3) specifically comprises: macrophage cell at 10 ⁶ ～10 ⁷ Inoculating into cell culture flask at density of one/mL, adding into DMEM medium containing 10% fetal calf serum and 1% green-streptomycin double antibody, and adding into CO ₂ The culture was carried out in an environment with a concentration of 5%, a temperature of 37℃and a humidity of 95%. Macrophages were isolated from the flask, washed 3 times with PBS, added to 4℃pre-chilled Tris-magnesium salt buffer, and allowed to stand at 4℃for 12h. Then the cell suspension is crushed for 5 to 15 minutes under the power of 50 to 100W, and 1M sucrose solution is added into the cell homogenate to prepare the cell suspension with the final concentration of sucrose of 0.25M. After centrifugation at 2000 Xg for 10min at 4℃the supernatant was taken and the precipitate was removed. Centrifuging the supernatant at 4deg.C for 30min at 3000 Xg, removing supernatant, adding Tris-magnesium salt buffer containing 0.25M sucrose, suspending, centrifuging for 30min at 3000 Xg again, collecting precipitate, lyophilizing for 24 hr to obtain Mm, and storing at-80deg.C.

Preferably, the step (4) specifically comprises: RBCm and Mm were dissolved in PBS solution (ph 7.4) and sonicated with a probe under ice bath conditions for 5min to prepare a fusion membrane RM. And then uniformly mixing the mixture with an AB@lip solution according to a certain proportion (the mass ratio of phospholipid to cell membrane is 3:1-1:1), manually extruding the mixture through a 400nm polycarbonate membrane by using a liposome extruder for 10-30 times, and centrifuging the extruded mixture for 10min by 6000 Xg to remove residual cell membranes to obtain the final composite bionic nano drug delivery system AB@LRM.

The principle of the invention: the invention adopts a composite membrane RM formed by RBCM and Mm to modify liposome carrying water-soluble phototherapy agent and antibiotics to form a composite bionic nano drug delivery system. The antibiotic AM is an aminoglycoside antibiotic, and the antibacterial mechanism of the aminoglycoside antibiotic is 30S subunit acting on ribosome in bacteria, inhibits the synthesis of bacterial protein, and damages the integrity of bacterial cell walls, so that bacterial cell membranes are damaged to die, but AM can cause cochlear nerve injury, nephrotoxicity and neuromuscular blocking. And with improper or excessive clinical use, strains that are resistant to AM are also increasing; the BPQD has the characteristics of high light absorption and photo-thermal conversion efficiency, biodegradability, good biocompatibility and the like, is a near infrared phototherapy agent with PTT/PDT effect, but is unstable in physiological environment, very sensitive to oxygen and water, and is oxidized into phosphorus oxide after being contacted with oxygen and water, and then degraded into phosphate ions and phosphite ions. While the smaller the size of the black phosphorus, the faster its degradation rate in aqueous solution.

The CD47 protein existing on the surface of RBCM can be used for preparing and directly playing a role with signal regulatory protein alpha expressed by macrophage to send out a signal, and can effectively avoid phagocytosis of immune system. The Mm surface expresses Toll-like receptor complex, and specifically recognizes pathogen related molecular patterns such as bacteria, so that the virus can be efficiently retained at an infected part, and the maximum accurate targeted delivery of the medicine can be realized.

The bionic nanometer drug delivery system formed by modifying the inner core of the drug-carrying liposome through the composite membrane solves the problems of no targeting property of AM, strong toxic and side effects, increased drug resistance risk of antibiotics, poor stability of BPQD, high degradation rate, short ROS half-life period and diffusion distance, thermal damage to healthy tissues and the like; realizes the targeted delivery of BPQD and AM, promotes the efficient retention of the BPQD and AM in bacterial infection microenvironment, and is further beneficial to fully playing the role of synergistic treatment by combining PTT/PDT and chemotherapy.

Phototherapy has been designed as an effective antimicrobial treatment modality, including photothermal therapy (PTT) and photodynamic therapy (PDT), with the advantage of space-time controllability, non-invasiveness, avoidance of bacterial resistance. However, there are several disadvantages to the monotherapy approach of light response: high temperatures, such as those caused by PTT, may cause localized collateral damage to normal cells and adjacent healthy tissue due to unavoidable thermal diffusion. The Reactive Oxygen Species (ROS) generated by PDT has a short half-life and diffusion distance, resulting in difficulty in exerting an ideal therapeutic effect. Multimode co-therapy has significant advantages in terms of safety and therapeutic efficacy over monotherapy. Phototherapy can be combined with antibiotics to realize synergistic treatment of bacterial infection, and can reduce toxic and side effects on normal tissues while treating. The local overheat caused by the photo-thermal effect of the material can make bacteria more sensitive to the antibacterial drugs in a mode of high bacterial membrane permeability and the like, and reduce the dosage of antibiotics while enhancing the drug action.

The composite membrane adopted by the invention takes the inherent long circulation characteristic of RBCM and the bacterial trend of Mm as the focus, and prepares the AB@Lip drug-loaded nanoparticle inner core modified by the composite membrane from the purposes of maintaining the cell membrane function and guaranteeing the treatment effect. The nano-delivery system is injected into the in vivo targeted bacterial infection microenvironment by intravenous injection and specifically binds to bacteria. The local overheating caused by the BPQD photo-thermal effect can make bacteria more sensitive to antibacterial drugs by improving the cell membrane permeability of bacteria and the like, and reduce the dosage of antibiotics while enhancing the drug action; and ROS are generated to destroy DNA and protein structures, promote lipid peroxidation, and accelerate bacterial lysis and death. The nanometer drug delivery system is used as a transport means for releasing drugs according to requirements, realizes multi-mode cooperative treatment of phototherapy and chemotherapy, and better carries the antibacterial drugs to the infection site in a directional way so as to generate obvious antibacterial effect, lower toxicity and greatly reduce the generation of bacterial drug resistance.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) Through the multi-mode synergistic antibacterial, the treatment efficiency is improved, meanwhile, the use dosage of antibiotics is obviously reduced, and compared with free medicines, the use amount of the antibiotics is reduced by 4 times, so that the possibility of bacterial drug resistance is reduced, and the systemic toxicity is reduced; (2) The composite cell membrane is fused and then modified on the surface of the liposome, so that long circulation in vivo is realized, the degradation rate in vivo is reduced, the drug is more effectively delivered, and the drug is delivered to the target part to the maximum extent; meanwhile, the liposome is protected from being damaged, so that the slow release of antibiotics is realized.

Drawings

FIG. 1 is a graph showing the results of particle size characterization of nanoparticles in examples 1 and 3, wherein A is a graph of the particle size of AB@lip nanoparticles and B is a graph of the particle size of AB@LRM complexes;

fig. 2 is a graph of the transmission electron microscope characterization result of ab@lrm in example 3, wherein a graph a is a transmission electron microscope graph of BPQD and B graph B is a transmission electron microscope graph of ab@lrm;

FIG. 3 is a graph of the flow characterization of the double fluorescent labels of the AB@LRM nanocomposite of example 3;

FIG. 4 is a diagram showing SDS-PAGE characterization of AB@LRM nanocomposites in example 3;

FIG. 5 is a graph showing the results of in vitro photothermal conversion of the AB@LRM nanocomposite of example 4, wherein graph A is a thermal imaging graph at different time points, and graph B is a temperature change line graph at different time points;

FIG. 6 is a graph depicting the photo-thermal conversion stability of the AB@LRM nanocomposite of example 4;

FIG. 7 is a graph of the results of RAW264.7 cells uptake of AB@LRM nanocomposites in example 5, wherein graph A is an inverted fluorescence microscopy image and graph B is an analysis image of the average fluorescence intensity of each group C6;

FIG. 8 is a graph of cytotoxicity results of AB@LRM nanocomposite of example 6;

FIG. 9 is a graph of the results of the minimum inhibitory concentration study of the AM and AB@LRM (+) groups of example 7;

FIG. 10 is a graph showing the statistical results of CFU after treatment of different formulation groups in example 8, wherein A is a photograph of LB solid plate, B is a graph showing the results of counting and analyzing CFU for each group, and C is a graph showing the results of counting and analyzing the antibacterial rate for each group;

FIG. 11 is a graph showing the growth of bacteria after treatment with different formulation groups of example 9;

FIG. 12 is a graph showing the in vitro organ tissue distribution of DiR@LRM in P.aeromonas pneumonia mice in example 10, wherein graph A is an in vitro organ fluorescence imaging graph, graph B is a graph showing the results of fluorescence quantitative analysis of each organ, and graph C is a graph showing the results of treatment of the fluorescence intensity ratios of each group of liver and lung tissues;

FIG. 13 is a graph showing ELISA results for proinflammatory factors in serum after treatment with different formulation groups of example 11, wherein A is IL-1β, B is IL-6, and C is TNF- α;

FIG. 14 is a graph showing the H & E staining results of lung tissue sections after treatment with different formulation groups in example 12;

FIG. 15 is a graph showing the results of H & E staining of heart, liver, spleen, lung, and kidney tissue sections of healthy mice, as assessed for in vivo safety in example 13.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

Example 1

Preparation and characterization of AB@lip nanoparticles

25mg of soybean phospholipid, 6.25mg of cholesterol were precisely weighed into 5mL of methanol solution and transferred to a round bottom flask. Rotary evaporation was carried out for 15min in a water bath at 40 c until a uniform lipid film formed at the bottom of the flask. Then preparing an aqueous solution containing a certain amount of BPQD and AM, adding the aqueous solution into a round bottom flask, hydrating for 30min under the water bath condition of 40 ℃ to obtain a suspension, guaranteeing that the film is completely wall-removed, transferring the suspension into a 10mL centrifuge tube, carrying out ultrasonic treatment for 15min by using a 300W power ice bath of an ultrasonic cell grinder, and sequentially filtering by using a 0.45 mu m water film and a 0.22 mu m water film to obtain the AB@Lip nanoparticle aqueous solution with opalescence. The particle size of the AB@lip nanoparticle aqueous solution is measured by a laser particle sizer, and the result is shown in a graph A in the attached figure 1, wherein the average particle size of the nanoparticles is 97.0+/-2.8 nm, and the PDI is 0.241+/-0.033.

Example 2

AB@LRM nanocomposite preparation

Fresh blood was collected from mice, lower red blood cells were collected by low-speed centrifugation, and washed 3 times with 1×pbs. Then 10 volumes of pre-chilled hypotonic lysate (2.5 mM PBS) was added and lysed in an ice bath. Centrifuging at 4deg.C and 2000 Xg for 15min in a high-speed low-temperature centrifuge to remove impurities. Centrifuging supernatant at 4deg.C and 10000 Xg for 1 hr, collecting precipitate, and freeze drying for 24 hr to obtain RBCM. Macrophages were cultured and collected, washed 3 times with PBS, added with 4℃pre-chilled Tris-magnesium salt buffer, and lysed overnight at 4 ℃. Then the cell suspension is crushed for 5 to 15 minutes under the power of 50 to 100W, and 1M sucrose solution is added into the cell homogenate to prepare the cell suspension with the final concentration of sucrose of 0.25M. After centrifugation at 2000 Xg for 10min at 4℃the supernatant was taken and the precipitate was removed. Centrifuging the supernatant at 4deg.C for 30min at 3000 Xg, removing supernatant, adding Tris-magnesium salt buffer solution containing 0.25M sucrose, re-centrifuging at 3000 Xg for 30min, collecting precipitate, and lyophilizing for 24 hr to obtain Mm. The RBCM and Mm were mixed and dissolved in PBS (pH 7.4) at a mass ratio of 3:1, and sonicated with a 200W probe for 5min to prepare a fusion membrane RM. And then uniformly mixing the solution with an AB@Lip solution according to the mass ratio of phospholipid to cell membrane of 3:1, manually extruding the solution through a 400nm polycarbonate membrane by using a liposome extruder for 30 times, and centrifuging the residual cell membrane for 10min by 6000 Xg to remove the residual cell membrane to obtain the final composite bionic nano drug delivery system AB@LRM.

Example 3

Characterization of ab@lrm nanocomposites

(1) Particle size and morphology investigation

The particle size of the AB@LRM nanocomposite aqueous solution is measured by a laser particle sizer, and the result is shown in a diagram B in the attached figure 1, wherein the average particle size of the nanocomposite is 122.3+/-2.4 nm, and the PDI is 0.206+/-0.017; the structure of the AB@LRM nanocomposite aqueous solution is observed by a transmission electron microscope, and the result is shown in a figure 2, wherein the size of the actual nanocomposite is similar to the detected particle size, the morphology is round and the distribution is uniform, a layer of film on the outer layer of the liposome can be clearly seen to form a shell-core structure, and the composite film RM is successfully modified on the surface of the drug-loaded liposome, so that the AB@LRM nanocomposite is successfully prepared. (A figure Scale bar:50nm, B figure Scale bar:100 nm)

(2) Double fluorescent labeling flow type investigation of AB@LRM nanocomposite

According to the characteristic of lipid solubility of coumarin 6 (C6) dye, AB@Lip labeled with C6 was prepared, free C6 was removed by centrifugation at 4000rpm/min for 10min through an ultrafiltration centrifuge tube, RM was labeled with DiI dye, and free DiI was also removed by ultrafiltration centrifugation.

And manually extruding through a 400nm polycarbonate film by using a liposome extruder, and extruding for proper times to obtain the double-fluorescence-labeled composite bionic nano drug delivery system (DiI-RM/C6-AB@Lip). The fluorescence intensity of the nanoparticle is measured by using a flow cytometer by taking the cell membrane biomimetic liposome drug-loaded nanoparticle (AB@LRM) which is not subjected to fluorescence labeling as a control group. As shown in figure 3, compared with the control group, the fluorescence intensity of C6 and DiI in the DiI-RM/C6-AB@Lip nano compound is obviously increased, so that 96.7% of AB@Lip is successfully modified by RM, and the preparation of the composite bionic nano drug delivery system AB@LRM is successful. (Scale bar:2 μm)

(3) Characterization of AB@LRM nanocomposites by polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was used to detect the protein composition of Red Blood Cells (RBC), macrophages (Ma), RBCm, mm, RM and composite membrane modified nanoparticles (RM/NPs). The results are shown in FIG. 4, where the RBC and RBCM histone strips are identical, indicating similar protein compositions, and similarly, ma and Mm also have similar protein compositions. The method shows that the membrane proteins on RBC and Ma can be completely reserved through the operations of hypotonic swelling and cracking, probe ultrasound and the like, and RBC and Mm can be successfully prepared; meanwhile, RM and RM/NPs histone strips are consistent, which shows that the composite membrane RM is successfully modified on the surface of the liposome and maintains the original biological characteristics of membrane protein.

Example 4

Property study of AB@OL nanocomposites

(1) Examine the photo-thermal conversion performance of AB@LRM nanocomposite

1.0mL of PBS, BPQD, AB@lip and AB@LRM are respectively placed in an EP tube, and irradiated with 808nm laser with the laser power set to 1.5W/cm ² . The temperature change of each sample was recorded over 10min. As shown in FIG. 5, the temperature of the PBS solution is almost unchanged under the irradiation of near infrared laser, while the temperature of each group of BPQD, AB@Lip and AB@LRM is continuously increased along with the extension of the irradiation time under the same irradiation condition, and no large difference exists, so that the photo-thermal conversion performance of the BPQD is not affected by whether the liposome or the cell membrane is coated or not.

(2) Investigation of photo-thermal stability of AB@LRM nanocomposites

Preparing AB@LRM solution with BPQD concentration of 50 μg/mL, irradiating with 808nm laser for 10min with power of 1.5W/cm ² Then the laser is turned off, and after the laser is naturally cooled to room temperature, the laser is turned on again to irradiate, thusAnd 5 times of circulation are carried out, and the temperature change condition of the sample solution is monitored and recorded by an infrared thermal imager in the whole process. As shown in figure 6, the AB@LRM solution can be stably heated to about 54 ℃ after each illumination, and the prepared AB@LRM has excellent and stable photo-thermal conversion performance, thereby laying a good foundation for the on-demand release of the subsequent photo-thermal excited medicament and the precise photo-thermal/antibiotic cooperative treatment.

Example 5

Investigation of ab@lrm nanocomposite uptake by RAW264.7 cells;

nanoparticles c6@lip and c6@lrm carrying coumarin 6 (C6) were prepared according to the prescription procedure of examples 1 and 2, respectively. RAW264.7 cells at 5X 10 ⁴ The individual/well concentrations were inoculated into 24 well plates, after 24h incubation the medium was discarded and c6@lip and c6@lrm (both C6 concentrations 25 ug/mL) were diluted with serum free medium and incubated for 6h. Washing 3 times with PBS after incubation; then 500. Mu.L of 4% (w/w) paraformaldehyde was added to each well, and the cells were fixed for 20min and washed 3 times with PBS; cells were incubated with 250 μl of DAPI staining solution for 10min and washed 3 times with PBS; finally, observing the uptake condition of RAW264.7 cells by using an inverted fluorescence microscope. As shown in figure 7, the fluorescence intensity of C6 in macrophages of the C6@LRM group is obviously lower than that of the C6@Lip group, which shows that the dosage taken by the macrophages can be obviously reduced after the inner core of the drug-loaded liposome is modified by cell membranes, thereby verifying that AB@LRM can realize long circulation in vivo and improving bioavailability.

Example 6

In vitro toxicity study of AB@LRM nanocomplex on L929, A549, HUVEC cells

Preparing cell suspension of human umbilical vein cells (HUVEC) in logarithmic phase, human non-small cell lung cancer cells (A549) and fibroblast cells (L929), adding into 96 wells, setting three compound wells at 100 μl each, standing at 37deg.C, maintaining temperature at 5% CO ₂ After culturing for 12 hours in a cell culture box, discarding the original culture solution, respectively adding 100 mu L of AB@LRM nano-composite (with AM concentration as standard) with different final concentrations diluted by serum-free culture medium into each hole, continuously incubating for 24 hours, then adding 20 mu L of MTT solution with concentration of 5mg/mL into each hole, and continuously culturing for 4 hours; then discard all of the upper partClear, add DMSO (100. Mu.L/well), shake with a micro-shaker for 5min to dissolve the crystals completely, measure absorbance at 490nm wavelength with a microplate reader and calculate survival.

As shown in figure 8, the AB@LRM nanocomposite has no obvious cytotoxicity in the administration concentration range, and the cell survival rate is more than 80%. The AB@LRM nanocomposite is proved to have good safety and biocompatibility in vitro.

Cell viability= (a _Sample -A _PBS )/(A _control -A _PBS )×100％

A _Sample Absorbance of groups of cells treated with different concentrations of nanocomposites

A _PBS Absorbance of PBS solution

A _control Absorbance of the cell group of the blank group

Example 7

Minimum inhibitory concentration investigation of AM and AB@LRM (+)

The minimum inhibitory concentration of AM and ab@lrm (+) was determined by a micro broth dilution method. Taking P.aeroginosa in logarithmic phase, and diluting the bacterial suspension to 10 ⁶ CFU/mL. The samples were diluted in LB medium gradient to concentrations (calculated as AM) of 16, 8, 4, 2, 1, 0.5 and 0.25. Mu.g/mL, respectively. And 100. Mu.L of the bacterial liquid was inoculated into a 96-well plate containing 100. Mu.L of the drug-containing broth, incubated at 37℃for 16 to 18 hours, and then absorbance values of each well were measured at 600nm using an enzyme-labeled instrument. As shown in FIG. 9, the minimum inhibitory concentration of AM was 4. Mu.g/mL, the minimum inhibitory concentration of AB@LRM (+) was 1. Mu.g/mL, and the antibiotic dosage was reduced by 4 times compared to the free drug. Therefore, the combination of a plurality of treatment modes proves that the dosage of antibiotics can be obviously reduced, the possibility of bacterial drug resistance is reduced, and the antibacterial curative effect is improved.

Example 8

Flat plate Colony Forming Unit (CFU) investigation of AB@LRM nanocomposites

Diluting P.aeromonas in logarithmic growth phase to 1X 10 ⁶ CFU/mL, respectively, is treated with physiological Saline (Saline), AM, BPQD (+) and free AMAnd BPQD mix (AB (+)) group, ab@lip (+) group and ab@lrm (+). The illumination group adopts 808nm infrared excitation light (1.5W/cm) ² ) Irradiating for 10min. Then the bacterial suspension is placed in a constant temperature incubator at 37 ℃ for 2 hours of culture. Diluting the cultured bacterial solutions of each group by 1X 10 ⁵ After doubling, 100. Mu.L of the diluted solution was dropped onto LB agar medium, and the solution was spread uniformly with an inoculating loop. Culturing in a constant temperature incubator at 37 ℃ for 18-24 hours, observing colony growth condition on LB agar medium, photographing and counting. As shown in FIG. 10, from the photographs of bacterial standard petri dish colonies and the counting results, the Saline treatment group had a large number of colonies formed, and the BPQD group had colonies formed similar to the Saline group, indicating that the BPQD itself had no effect on bacterial activity, but the colony count was significantly reduced after irradiation with NIR laser. At the same time, the AM treated group did not show a significant antibacterial effect, which may be related to the use of AM concentrations below the minimum inhibitory concentration (4. Mu.g/mL). When the bacterial liquid was treated with the AM and BPQD solutions, and simultaneously irradiated with the NIR laser, the colony formation numbers were less than those of BPQD (+) alone, indicating that AM and BPQD light had a synergistic effect. While ab@lrm light groups showed almost no bacterial colony formation, indicating complete inhibition of bacterial growth under the combined actions of antibiotics, phototherapy and Mm targeting.

Example 9

Investigation of bacterial growth conditions by AB@LRM nanocomposites

Diluting P.aeromonas in logarithmic growth phase to 1X 10 ⁶ CFU/mL, treated with different materials, i.e. including Saline group, AM group, BPQD (+) group, AB (+) group, ab@lip (+) group and ab@lrm (+) group, respectively. After the treatment, the culture was performed in a constant temperature incubator at 37 ℃. Each group of bacterial solutions was taken at a concentration of 100. Mu.L every 2 hours, added to a 96-well plate, and absorbance at 600nm (i.e., OD600 nm) of the bacterial suspension was measured by an enzyme-labeled instrument. The measurement was continued for 24 hours and a growth curve was drawn. As a result, as shown in FIG. 11, the Saline-treated group of bacteria had been grown for 6 hours and then entered the logarithmic phase, and the number of bacteria had been exponentially increased, and at the time of 12 hours of cultivation, the bacteria had grown in a typical S-type. The AM group can not inhibit bacterial growth, but the BPQD light treatment group has bacterial growth lag relative to PBS, and the growth of the bacteria is delayed after 10h of cultivation, which indicates that the BPQD light treatment group has a certain bacterial growthThe inhibition effect is superior to that of the BPQD illumination group, and meanwhile, the antibacterial effect of the AM and BPQD combined illumination group is superior to that of the BPQD illumination group, so that the AM and the BPQD illumination have a synergistic effect. In the AB@LRM illumination group, the bacterial growth curve is not increased in the whole 24h culture process, and the AB@LRM nanocomposite has a high-efficiency antibacterial effect.

Example 10

DiR@LRM in vitro organ tissue distribution of P.aeromonas pneumonia mice

And constructing a P.aerocosia pneumonia model of the mice, injecting DiR@Lip, diR@LR and DiR@LRM into tail veins respectively, taking off cervical vertebrae to kill the mice when the mice are administrated for 48 hours, and taking the heart, liver, spleen, lung and kidney of the mice to carry out shooting and data analysis by using a living imaging system. As shown in FIG. 12, the lung fluorescence intensity of the DiR@LR group is higher than that of the DiR@Lip group; the fluorescence intensity of the lung of the DiR@LRM group is obviously better than that of the DiR@lip group and the DiR@LR administration group. It shows that under the Mm targeting effect and the RBCM long-circulation synergistic effect, more medicines can reach the bacterial infection area of the lung, thereby achieving better treatment effect.

Example 11

Investigation of the influence of the AB@LRM nanocomposite on the proinflammatory cytokine level

Proinflammatory cytokines such as IL-1 beta, IL-6, and TNF-alpha play a key role in the development and progression of inflammatory diseases. After the end of the dosing period, blood samples of each group of mice were collected with an EP tube, left standing at room temperature for 2h after blood collection, and centrifuged at 4℃at 2500rpm/min for 10min. TNF-alpha, IL-1 beta and IL-6 inflammatory factor levels were detected using ELISA detection kits. As a result, as shown in FIG. 13, IL-1β, IL-6 and TNF- α levels were significantly higher in Saline group mice than in the treatment group. Compared with other groups, the AB@LRM (+) group inflammatory cytokine level is obviously reduced, so that the nano drug delivery system constructed by the invention is verified to be capable of effectively inhibiting inflammatory response in vivo.

Example 12

H & E staining investigation of bacterial infection lung tissue by AB@LRM nanocomposite

Healthy ICR female mice are selected, and 6 groups of mice are respectively dosed after a bacterial infection pneumonia model is established, wherein the mice are Saline, AM, AM@lip, AB@lip (+), AB@LR (+), and AB@LRM (+). The administration was by tail vein injection once every 2 days for a total of 7 administrations. 24h after the last administration, the lung tissue of the mice was taken after anaesthetizing the mice and fixed with 4% paraformaldehyde. Paraffin embedding-slicing-dewaxing to water-hematoxylin staining-eosin staining-dehydration sealing-microscopy. As a result, as shown in FIG. 14, the mouse treated with saline had serious alveolar tissue injury and had massive inflammatory cell infiltration. Of the near infrared light groups, the ab@lrm (+) group had the least alveolar tissue damage. The integrity of the alveoli of the mice after near infrared irradiation is obviously higher than that of other experimental groups, and the congestion and inflammatory cell infiltration degree of the alveoli are reduced.

Example 13

In vivo safety investigation of AB@LRM nanocomposites

Healthy ICR female mice of 8 weeks of age were randomly divided into 6 groups, respectively a Saline group, an AM group, an am@lip group, an ab@lip (+) group, an ab@lr (+) group, and an ab@lrm (+) group. After 1 week of intravenous administration, mice were sacrificed by cervical dislocation, and the main organs (heart, liver, spleen, lung, kidney) were HE stained, observed under a microscope and photographed. The results are shown in fig. 15, in which no histological abnormalities and inflammatory lesions were present in each organ in the ab@lrm group compared to the control group. The results show that at a given dose, intravenous injection of ab@lrm has good in vivo safety. (Scale bar:100 μm)

In-vivo and in-vitro drug effect evaluation, the dosages of antibiotics are kept consistent in each group, and the result shows that the effect of using a single antibiotic is far less good than that of the targeted antibacterial nano drug delivery system. This demonstrates that by synergy of the multiple modes of treatment, a good therapeutic effect can be achieved with reduced antibiotic usage. Therefore, a reasonable drug delivery system is constructed, the time and space controllable release of the antibiotics is realized, the concentration of the effective antibiotics is increased, the use dosage of the antibiotics is reduced, the improper use of the antibiotics is avoided, and the method is an effective means for reducing the generation of bacterial drug resistance.

Comparative example 1

In comparison to example 2, ab@lrm nanocomposite preparation, coating was performed with only red blood cell membranes:

fresh blood was collected from mice, lower red blood cells were collected by low-speed centrifugation, and washed 3 times with 1×pbs. Then 10 volumes of pre-chilled hypotonic lysate (2.5 mM PBS) was added and lysed in an ice bath. Centrifuging at 4deg.C and 2000 Xg for 15min in a high-speed low-temperature centrifuge to remove impurities. Centrifuging supernatant at 4deg.C and 10000 Xg for 1 hr, collecting precipitate, and freeze drying for 24 hr to obtain RBCM. And then uniformly mixing the mixture with an AB@Lip solution according to the mass ratio of phospholipid to cell membrane of 3:1, manually extruding the mixture through a 400nm polycarbonate membrane by using a liposome extruder for 30 times, and centrifuging the residual cell membrane for 10min by 6000 Xg to remove the residual cell membrane to obtain the final composite bionic nano drug delivery system AB@LR. The nanoparticle carrying coumarin 6 (C6) is prepared, and the fluorescence intensity is observed to be obviously lower than that of a C6@Lip group, so that the dosage taken by macrophages can be obviously reduced after the inner core of the drug-carrying liposome is modified by cell membranes, and the long circulation in vivo can be verified, and the bioavailability is improved; the in vivo experiment shows that the characteristics of long circulation of erythrocyte membrane are mainly reflected by tissue in vitro imaging, and the in vivo experiment result also proves that the treatment effect of singly wrapping the liposome by the erythrocyte membrane is not good as that of wrapping the liposome by the composite membrane.

At the same time, the synergy of phototherapy and chemotherapy; in the in vitro group setting, a single chemotherapeutic agent group, a single phototherapy agent BPQD group and a chemotherapeutic agent+phototherapy agent group are respectively arranged, and in vitro experimental results also show that the antibacterial effect of the chemotherapeutic agent+phototherapy agent group is obviously superior to that of the single chemotherapy or the single phototherapy group, so that the synergistic antibacterial treatment effect is shown.

Claims

1. The composite cell membrane bionic targeted antibacterial nanometer drug delivery system is characterized by comprising a liposome drug-carrying inner core composed of phospholipid, cholesterol, antibiotics and phototherapy agents and a composite membrane coated outside the liposome drug-carrying inner core and formed by a heme cell membrane and a macrophage membrane, wherein the antibiotics are one of amikacin, ciprofloxacin, levofloxacin, moxifloxacin, imipenem, aztreonam or tobramycin.

2. The drug delivery system of claim 1, wherein the mass ratio of phospholipid, cholesterol, phototherapeutic agent and antibiotic is 200:50:16:1-5, the mass ratio of heme membrane to macrophage membrane is 3-1:1, and the mass ratio of phospholipid to composite membrane in liposome drug-carrying inner core is 3-1:1.

3. The drug delivery system of claim 1, wherein the phototherapeutic agent is one of black phosphorus nanoplatelets, black phosphorus quantum dots, molybdenum disulfide, gold nanoparticles, gold nanorods, gold nanocages, indocyanine green, neoindocyanine green, polydopamine, or graphene quantum dots.

4. The drug delivery system of claim 1, wherein the macrophage membrane is one of a mouse mononuclear macrophage leukemia cell membrane, a bone marrow-derived macrophage membrane, an abdominal cavity-derived macrophage membrane, a spleen-derived macrophage membrane, a monocyte-derived macrophage membrane, or a peripheral blood-derived macrophage membrane.

5. A method of manufacturing a drug delivery system according to claim 1, comprising the steps of:

6. The method according to claim 5, wherein in step (1), the antibiotic is amikacin and the phototherapeutic agent is black phosphorus quantum dot; the ultrasonic power is 200-300W; the ultrasonic time is 15-30 min.

7. The method according to claim 5, wherein in the step (2), the red blood cells are lysed in an ice bath for 20 to 30 minutes.

8. The method according to claim 5, wherein in the step (3), the power of the ultrasonic disruption of the macrophage is 50-100W and the ultrasonic disruption time is 5-15min.

9. The method according to claim 5, wherein in the step (4), the number of coextrusion is 10 to 30.