A kind of acid / H2O based on porphyrin grafted carboxymethyl chitosan 2 O 2 Preparation and application of responsive nanoparticles

Abstract

The invention discloses the preparation and application of acid / H2O2-responsive nanospheres based on porphyrin-grafted carboxymethyl chitosan, belonging to the fields of advanced functional materials and biomedicine technology. The acid / H2O2-responsive nanospheres are self-assembled by nano-deposition method with modified porphyrin-grafted carboxymethyl chitosan and bis(2,4,5-trichlorosalicylato) pentyl oxalate (CPPO) as raw materials. The obtained nanospheres have the advantages of low drug leakage, microenvironment-responsive dissociation, intelligent bacterial targeting, photodynamic / photothermal therapy effects guided by microenvironment-specific spontaneous imaging, as well as good stability and biocompatibility, and have great application potential.

Description

Technical Field

[0001] The invention relates to the preparation and application of acid / H2O2 responsive nano-microspheres based on porphyrin-grafted carboxymethyl chitosan, and belongs to the technical fields of advanced functional materials and biomedicine. Background Art

[0002] Bacterial infection has always been a public health safety issue of global concern. Although the use of antibiotics has reduced the morbidity and mortality of bacterial infectious diseases to a certain extent, with the widespread use and even abuse of antibiotics, the problem of bacterial resistance to antibiotics has been exposed. Unlike traditional antibacterial therapies, phototherapy has the advantages of spatiotemporal controllability, low toxicity and no drug resistance, and has excellent application prospects in inactivating microorganisms and treating infectious diseases. The selection of photosensitizers during phototherapy is very important. Traditional photosensitizers have problems such as easy self-aggregation quenching, short cycle metabolism time, and lack of targeting, which makes their antibacterial treatment effect unsatisfactory. At the same time, since phototherapy is a local treatment method, accurate imaging guidance is also crucial to it. Therefore, it is particularly important to study and develop a new antibacterial strategy to improve the defects of traditional photosensitizers.

[0003] The development of nanotechnology has provided an effective solution for new antibacterial treatments that improve the defects of traditional photosensitizers. Encapsulating photosensitizers with natural hydrophilic carriers can improve their self-aggregation quenching phenomenon and prolong blood circulation metabolism time. In addition, the problem of local phototherapy can be solved through imaging guidance. However, most nano-diagnosis and treatment systems are based on simple physical encapsulation, and drug leakage is inevitable. In addition, most of them lack bacterial specificity and cause nonspecific damage to adjacent normal cells. At the same time, most of the existing imaging guidance is continuous irradiation, which has background fluorescence interference, and can also activate photosensitizers, causing nonspecific damage and endangering normal tissues. Therefore, it is urgent to develop a new type of nanomaterial that combines effective photosensitizer loading, responsiveness to the infection site, spontaneous imaging guidance at the infection site, and high-efficiency sterilization for the treatment of bacterial infections. Summary of the invention

[0004] [Technical issues]

[0005] Traditional nanomaterials are mostly physically encapsulated and directly applied to wounds, which are prone to drug leakage and lack responsiveness to infected areas. At the same time, traditional imaging guidance is mostly continuous irradiation, which has background fluorescence interference and can cause nonspecific damage and harm normal tissues.

[0006] [Technical solution]

[0007] To solve the above problems, the present invention designs and constructs bacterial microenvironment-responsive nanospheres with high-efficiency photosensitizer loading that have multiple functions including photodynamics, photothermal and autofluorescence imaging, and uses them for bacterial targeted phototherapy guided by autofluorescence imaging. This overcomes and solves the problems of traditional nanomaterials being prone to drug leakage, lacking responsiveness to the infection site, and background interference and nonspecific damage guided by traditional imaging.

[0008] The first object of the present invention is a method for preparing acid / H2O2 responsive nanospheres based on porphyrin grafted carboxymethyl chitosan, comprising the following steps:

[0009] (1) Synthesis of porphyrin grafted carboxymethyl chitosan (CMC-PC):

[0010] 5,10,15,20-tetrakis-(4-aminophenyl)porphyrin (TAPP), an alkali reagent, and N,N-dimethylformamide are mixed, chloroacetyl chloride is added dropwise after mixing, and the reaction is carried out at room temperature; after the reaction is completed, water is added to precipitate, the precipitate is separated and collected, and washed to obtain a reaction intermediate; then the reaction intermediate, an alkali reagent, and N,N-dimethylformamide are mixed, an aqueous solution of carboxymethyl chitosan (CMC) is added dropwise after mixing, and the reaction is carried out at room temperature; after the reaction is completed, dialyzed, centrifuged, the solid is collected, and dried to obtain porphyrin-grafted carboxymethyl chitosan, which is recorded as CMC-PC;

[0011] (2) Amino small molecule modification:

[0012] CMC-PC, alkaline reagent and N,N-dimethylformamide are mixed, and N,N,N',N'-tetraethyldiethylenetriamine is slowly added dropwise after mixing, and the reaction is carried out at room temperature. After the reaction is completed, the dialyzate is dialyzed, and the dialyzate is centrifuged, the solid is collected, and dried to obtain amino small molecule modified porphyrin molecule bonded carboxymethyl chitosan, which is recorded as CMC-PC-N;

[0013] (3) Preparation of acid / H2O2 responsive nanospheres (CPPO@CMC-PC-N):

[0014] CMC-PC-N and bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate (CPPO) were dissolved in tetrahydrofuran to obtain a mixed solution; the mixed solution was then slowly added into a phosphate buffer solution, mixed and reacted, and after the reaction was completed, tetrahydrofuran was removed, and the filtrate was collected by suction to obtain CPPO@CMC-PC-N.

[0015] In one embodiment of the present invention, in step (1), the molar ratio of TAPP, the alkaline reagent and chloroacetyl chloride is 1:(10-15):(10-15).

[0016] In one embodiment of the present invention, in step (1), the molar ratio of the reaction intermediate to the alkaline reagent is 1:(1-2).

[0017] In one embodiment of the present invention, in step (1), the mass ratio of the reaction intermediate to CMC is (3-5):1.

[0018] In one embodiment of the present invention, in step (1), the concentration of the aqueous solution of CMC is 1-5 mg / mL; specifically, 3 mg / mL may be selected.

[0019] In one embodiment of the present invention, in the process of preparing the reaction intermediate in step (1), the amount of N,N-dimethylformamide relative to TAPP is 0.03-0.05 mL / mg, and specifically 0.04 mL / mg.

[0020] In one embodiment of the present invention, in the process of preparing CMC-PC in step (1), the amount of N,N-dimethylformamide used relative to the reaction intermediate is 0.03-0.05 mL / mg, and specifically 0.04 mL / mg.

[0021] In one embodiment of the present invention, the alkaline reagent in step (1) is potassium carbonate.

[0022] In one embodiment of the present invention, the molecular weight cut-off for dialysis in step (1) is 3000-5000.

[0023] In one embodiment of the present invention, in step (2), the molar ratio of CMC-PC, the alkali reagent and N,N,N',N'-tetraethyldiethylenetriamine is 1:(10-15):(10-25).

[0024] In one embodiment of the present invention, in step (2), the amount of N,N-dimethylformamide relative to CMC-PC is 0.1-0.5 mL / mg, and specifically 0.25 mL / mg.

[0025] In one embodiment of the present invention, the alkaline reagent in step (2) is potassium carbonate.

[0026] In one embodiment of the present invention, the molecular weight cut-off of dialysis in step (2) is 3000-5000.

[0027] In one embodiment of the present invention, in step (3), the pH value of the phosphate buffer solution is 7.0-7.4.

[0028] In one embodiment of the present invention, the mass ratio of CMC-PC-N to CPPO in step (3) is 1:(1.5-2). Specifically, it can be 1:1.6.

[0029] In one embodiment of the present invention, the amount of tetrahydrofuran relative to CMC-PC-N is 1-3 mL / mg; specifically, 2 mL / mg may be selected.

[0030] In one embodiment of the present invention, the volume ratio of tetrahydrofuran to phosphate buffer solution is 1:(2-5); specifically, 1:3 may be selected.

[0031] In one embodiment of the present invention, in step (3), the content of amino small molecule modified porphyrin molecule (PC-N) in 1 mg / mL CPPO@CMC-PC-N is 0.1-0.5 mg / mL, and the content of CPPO is 0.4-0.8 mg / mL.

[0032] In one embodiment of the present invention, the method specifically comprises:

[0033] (1) Synthesis of porphyrin grafted carboxymethyl chitosan (CMC-PC):

[0034] 5,10,15,20-tetrakis-(4-aminophenyl)porphyrin (TAPP) was dissolved in 5-10 mL N,N-dimethylformamide, potassium carbonate was added, and chloroacetyl chloride was slowly added dropwise under stirring. The reaction was stirred at room temperature for 9-12 hours. After the reaction was completed, the reaction solution was added dropwise to a large amount of water. The product was precipitated and washed by centrifugation several times to obtain the reaction intermediate 1.

[0035] The reaction intermediate 1 was dissolved in 4-8 mL N,N-dimethylformamide, potassium carbonate was added, and 4-16 mL of an aqueous solution of carboxymethyl chitosan (CMC) was slowly dripped in a stirring state, and the reaction was carried out at room temperature for 9-12 hours. After the reaction was completed, the unreacted potassium carbonate, hydrochloric acid and other impurities were removed by dialysis for 3-4 days. The dialyzate was centrifuged and the supernatant was discarded. After freeze-drying, the unreacted reaction intermediate 1 was removed by Soxhlet extraction. After vacuum drying, porphyrin-grafted carboxymethyl chitosan was obtained, which was recorded as CMC-PC;

[0036] (2) Amino small molecule modification:

[0037] Dissolve CMC-PC in 5-10 mL N,N-dimethylformamide, add potassium carbonate and mix, slowly drop N,N,N',N'-tetraethyldiethylenetriamine under stirring, react at room temperature for 9-12 hours, dialyze for 3-4 days after the reaction to remove unreacted N,N,N',N'-tetraethyldiethylenetriamine, potassium carbonate, hydrochloric acid and other impurities, centrifuge the dialyzate and discard the supernatant, freeze-dry to obtain amino small molecule modified porphyrin molecule bonded carboxymethyl chitosan, recorded as CMC-PC-N;

[0038] (3) Preparation of acid / H2O2 responsive nanospheres (CPPO@CMC-PC-N):

[0039] CMC-PC-N and bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate (CPPO) dissolved in 1-3 mL of tetrahydrofuran were slowly injected into 5-9 mL of phosphate buffer solution. The mixture was vigorously stirred in a fume hood for about 9-12 hours to remove tetrahydrofuran. The CPPO not encapsulated in the mixed solution was removed by suction filtration. The obtained filtrate was transferred to a 10 mL volumetric flask and fixed to volume to obtain CPPO@CMC-PC-N.

[0040] In one embodiment of the present invention, the peak value of porphyrin in the ultraviolet spectrum of the washing liquid after Soxhlet extraction in step (1) is always lower than 0.2-0.4.

[0041] The second object of the present invention is to prepare acid / H2O2 responsive nano-microspheres CPPO@CMC-PC-N based on porphyrin grafted carboxymethyl chitosan using the method described in the present invention.

[0042] In one embodiment of the present invention, the structural formula of the structural unit of CMC-PC-N in the CPPO@CMC-PC-N is as follows:

[0043]

[0044] Wherein, n=20-2000.

[0045] The third object of the present invention is to provide an application of the above-mentioned acid / H2O2 responsive nano-microspheres in the preparation of sterilization materials for photodynamic / photothermal sterilization.

[0046] The fourth object of the present invention is to provide a photodynamic / photothermal sterilization method for non-disease diagnosis and treatment, wherein the method uses the above-mentioned acid / H2O2 responsive nano-microspheres as sterilization materials.

[0047] In one embodiment of the present invention, the photodynamic / photothermal sterilization method for non-disease diagnosis and treatment is to add CPPO@CMC-PC-N for light irradiation;

[0048] Specifically, the method includes the following steps: dissolving CPPO@CMC-PC-N in a solvent, then mixing it with a bacterial solution, irradiating it under an appropriate light source for 10 minutes, and then gradiently diluting and coating it on a solid culture medium. After culturing for 12-24 hours, the colonies are counted to obtain a sterilization rate. The solvent is a liquid culture medium, the bacterial solution includes Staphylococcus aureus and Escherichia coli, and the light source is a white LED lamp.

[0049] A fifth object of the present invention is to provide an application of the above-mentioned acid / H2O2 responsive nanospheres in the preparation of probes for autofluorescence imaging.

[0050] The sixth object of the present invention is to provide a method for guiding autofluorescence imaging for non-disease diagnosis and treatment, wherein the method uses the acid / H2O2 responsive nano-microspheres based on porphyrin-grafted carboxymethyl chitosan described in the present invention as imaging materials.

[0051] In one embodiment of the present invention, the autofluorescence imaging guidance method for non-disease diagnosis and treatment is to add CPPO@CMC-PC-N to the wound infection site;

[0052] The specific steps include: applying CPPO@CMC-PC-N to the wound of mice infected with Staphylococcus aureus, photographing the mice using an IVIS imaging system, and obtaining autofluorescence imaging of the wound.

[0053] [Beneficial Effects]

[0054] (1) The photosensitizer porphyrin in the CPPO@CMC-PC-N prepared by the present invention is fixed with the natural polymer carboxymethyl chitosan by chemical bonding, thereby avoiding the leakage problem of the photosensitizer and improving the stability, biocompatibility and blood circulation time of the single photosensitizer porphyrin.

[0055] (2) The CPPO@CMC-PC-N prepared by the present invention is protonated and converted into quaternary ammonium salts in the weakly acidic environment of bacteria due to its abundant internal amino groups, which leads to increased hydrophilicity of the nanospheres, specific dissociation and electrostatic binding with the negatively charged bacterial cell membrane, thereby achieving pH-responsive dissociation at the infection site and improving bacterial targeting.

[0056] (3) The unique CPPO in the CPPO@CMC-PC-N prepared by the present invention can react with H2O2 in the bacterial microenvironment to activate the photosensitizer to emit fluorescence, thereby achieving spontaneous fluorescence imaging guidance of the infection site and improving the photodynamic and photothermal effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the NMR image of the reaction intermediate 1 prepared in Example 1.

[0058] Figure 2 This is the mass spectrum of the reaction intermediate 1 prepared in Example 1.

[0059] Figure 3 Infrared spectra of reaction intermediate 1, CMC-PC and CMC-PC-N prepared in Example 1.

[0060] Figure 4This is the fitted fluorescence spectrum of CPPO@CMC-PC-N prepared in Example 1.

[0061] Figure 5 TEM images of CPPO@CMC-PC-N prepared in Example 1 at different pH values, scale: 100 nm.

[0062] Figure 6 This is the singlet oxygen production performance diagram of CPPO@CMC-PC-N prepared in Example 1, where ΔA0 and ΔA are the absorbance of ABDA at 380 nm before irradiation and at a certain moment of irradiation minus the original absorbance of the tested solution without adding ABDA.

[0063] Figure 7 This is the photothermal effect diagram of CPPO@CMC-PC-N prepared in Example 1.

[0064] Figure 8 This is a plate sterilization diagram of CPPO@CMC-PC-N prepared in Example 1.

[0065] Fig. 9 This is the cytotoxicity graph of CPPO@CMC-PC-N prepared in Example 1.

[0066] Fig.10 This is the in vivo autofluorescence imaging of CPPO@CMC-PC-N prepared in Example 1.

[0067] Fig.11 This is a time-dependent relative area diagram of infected wounds in different groups of mice with CPPO@CMC-PC-N prepared in Example 1; S0 and S are the areas of infected wounds before treatment and at a certain treatment point, respectively.

[0068] Fig.12 This is a graph of the spontaneous fluorescence intensity of CPPO@CMC-PC-N at 670 nm obtained with different CPPO feed amounts prepared in Example 3.

[0069] Fig.13 It is a schematic diagram of the working principle of the bacterial microenvironment responsive nanospheres of the present invention that have photodynamic, photothermal and autofluorescence imaging capabilities. DETAILED DESCRIPTION

[0070] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0071] Example 1

[0072] A method for preparing acid / H2O2 responsive nanospheres based on porphyrin grafted carboxymethyl chitosan specifically comprises the following steps:

[0073] (1) Synthesis of CMC-PC:

[0074] 500 mg TAPP (0.741 mmol), 1.3 g potassium carbonate (9.420 mmol) and 20 mL N,N-dimethylformamide were added to a 50 mL round-bottom flask, and 1.5 mL chloroacetyl chloride (9.348 mmol) was slowly dropped into the mixture under stirring. The mixture was stirred at room temperature for 12 h, and washed several times by centrifugation with a large amount of water. The precipitate was freeze-dried to obtain the reaction intermediate 1 with a yield of 77-83%.

[0075] 100 mg of reaction intermediate 1 (0.102 mmol), 14.1 mg of potassium carbonate (0.102 mmol) and 4 mL of N,N-dimethylformamide were added to a 25 mL round-bottom flask, and 8 mL of 24 mg of carboxymethyl chitosan (degree of substitution: ≥80%, molecular weight: 5.0×10 3 ~5.0×10 5 ) in an aqueous solution and react at room temperature for 12 hours. The resulting reaction mixture was placed in a dialysis bag (MWCO: 3000, molecular weight) and dialyzed for 3 days. The supernatant was discarded by centrifugation and freeze-dried. The product was washed with dichloromethane for multiple times using the Soxhlet extraction method. The product was dried in a vacuum drying oven at room temperature to obtain CMC-PC with a yield of 79-83% and a grafting rate of 80%-85%.

[0076] The grafting rate refers to the content of PC in the obtained CMC-PC, which is obtained by measuring the absorbance value of CMC-PC and referring to the PC standard curve.

[0077]

[0078] Where A is the absorbance of CMC-PC prepared with dimethyl sulfoxide as solvent, and ρ is the mass concentration of the prepared CMC-PC (g mL -1 ), M is the molar mass of CMC-PC under ideal conditions (1179 g mol -1 Each group of experiments was performed at least 3 times in parallel.

[0079] The structures of the synthesized reaction intermediate 1 and CMC-PC were characterized by NMR, mass spectrometry and Fourier transform infrared spectroscopy. Figure 1 ) and mass spectrum ( Figure 2 ) is the same as the theoretical value. -1 (stretching vibration of -NHCO-) ​​and 1061cm -1The appearance of characteristic peaks at (derived from -COC- stretching vibration of CMC) confirmed the successful preparation of reaction intermediate 1 and CMC-PC ( Figure 3 ).

[0080] (2) Synthesis of CMC-PC-N:

[0081] 40 mg CMC-PC (0.030 mmol), 50 mg potassium carbonate (0.362 mmol) and 10 mL N,N-dimethylformamide were added to a 25 mL round-bottom flask, and 0.1 mL N,N,N',N'-tetraethyldiethylenetriamine (0.624 mmol) was slowly dropped into the mixture under stirring. The mixture was reacted at room temperature overnight. The obtained reaction mixture was placed in a dialysis bag (MWCO: 3000, molecular weight) and dialyzed for 3 days. The supernatant was discarded after centrifugation and freeze-dried to obtain CMC-PC-N with a yield of 77-82%.

[0082] The structure of the synthesized CMC-PC-N was characterized by Fourier transform infrared spectroscopy. -1 The appearance of the characteristic peak at (-CH3 stretching vibration) confirmed the successful preparation of CMC-PC-N ( Figure 3 ).

[0083] (3) Preparation of CPPO@CMC-PC-N:

[0084] 1.5 mg CMC-PC-N and 2.4 mg CPPO were dissolved in 3 mL tetrahydrofuran and slowly injected into 9 mL phosphate buffer solution. The mixture was vigorously stirred in a fume hood for 12 h to remove tetrahydrofuran. The CPPO not encapsulated in the mixture was removed by suction filtration, and the filtrate was transferred to a 10 mL volumetric flask to obtain CPPO@CMC-PC-N. The content of PC-N in 1 mg / mL CPPO@CMC-PC-N was 0.359 mg / mL, and the content of CPPO was 0.589 mg / mL.

[0085] The contents of PC-N and CPPO in CPPO@CMC-PC-N are obtained by the following method:

[0086] The absorbance values ​​of CMC-PC-N and CPPO remaining on the filter paper were measured by UV-visible spectrophotometer, and the PC content and CPPO content in the CMC-PC-N remaining on the filter paper were calculated with reference to the standard curves of PC and CPPO. The PC-N content in the nanospheres was calculated by the molar mass ratio, and the CMC-PC-N content in the nanospheres was calculated by the molar mass ratio and the grafting rate. Among them, the sum of the contents of CMC-PC-N and CPPO is the content of the nanospheres, and then the amount of PC-N and CPPO in the nanospheres per unit concentration is calculated. When calculating, CMC in CMC-PC-N is considered to be an ideal single state.

[0087] The synthesized CPPO@CMC-PC-N was characterized by Fourier transform infrared spectroscopy at 1800-1700 cm -1 The appearance of the characteristic absorption peak of -COO- at -100 nm confirmed the successful coating of CPPO.

[0088] 1.5 mg CMC-PC-N was dissolved in 1 mL tetrahydrofuran and slowly injected into 9 mL phosphate buffer solution. The mixture was vigorously stirred in a fume hood for 12 h to remove tetrahydrofuran. The filtrate was transferred to a 10 mL volumetric flask and fixed to volume to obtain nanospheres 2. The content of PC-N in 1 mg / mL nanospheres 2 was 0.850 mg / mL.

[0089] Example 2

[0090] The photodynamic / photothermal therapeutic effects of CPPO@CMC-PC-N obtained in Example 1 were investigated:

[0091] The main research contents include the compound's optical properties, degradation performance, singlet oxygen production performance, photothermal performance, bacterial killing effect, biocompatibility, in vivo imaging performance and in vivo therapeutic effect evaluation.

[0092] (1) Optical properties investigation:

[0093] Take CPPO@CMC-PC-N (expressed as PC-N concentration, 1×10 -5 mol L -1 ), and collected fluorescence images at wavelengths of 570nm, 620nm, 670nm, 710nm, 790nm and 845nm on the IVIS imaging system and read the fluorescence intensity. The results showed that no fluorescence images were collected at the above wavelengths and the fluorescence intensity was basically the same as the background fluorescence intensity (ca.10 2 p / sec / cm 2 / sr).

[0094] CPPO@CMC-PC-N was activated by 1 mM H2O2 (expressed as PC-N concentration, 1×10 -5 mol L -1 ), fluorescence images at wavelengths of 570 nm, 620 nm, 670 nm, 710 nm, 790 nm and 845 nm were collected on the IVIS imaging system and the fluorescence intensity was read.

[0095] The results are as follows Figure 4 As shown, CPPO@CMC-PC-N can emit fluorescence, and the fluorescence spectrum of CPPO@CMC-PC-N fitted with peaks at 665nm and 714nm is consistent with the fluorescence spectrum of PC-N, proving that CPPO@CMC-PC-N can be activated by the energy generated by CPPO and H2O2 to emit fluorescence from PC-N.

[0096] (2) Degradation performance investigation:

[0097] CPPO@CMC-PC-N at pH=7.4, pH=6.5, pH=6.0, and pH=5.5 were prepared, and their states were observed using a transmission electron microscope (TEM).

[0098] The results are as follows Figure 5 As shown in the figure, under the condition of pH 7.4, the prepared CPPO@CMC-PC-N presents a regular spherical shape with an average diameter of 69.6±5.0nm. As the pH value changes from 7.4 to 5.5 (simulating normal physiological pH to bacterial acidic microenvironment), the morphology of CPPO@CMC-PC-N becomes irregular and the particle size becomes larger. It is proved that under the conditions of bacterial microenvironment, the amino group of N,N,N',N'-tetraethyldiethylenetriamine in CPPO@CMC-PC-N can be protonated and converted into quaternary ammonium salt, the hydrophilicity is improved, and specific dissociation occurs.

[0099] (3) Evaluation of singlet oxygen production performance:

[0100] Using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) as an indicator, 990 μL of CPPO@CMC-PC-N with different concentrations (expressed as PC-N concentration, 1×10 -6 mol L -1 , 5×10 -6 mol L -1 , 1×10 -5 mol L -1 ) and 10 μL ABDA solution (5×10 -3 mol L -1 ) were mixed. The mixed solution was then placed in a cuvette and heated at 100 mW cm -2The power density of the white LED light was irradiated for 10 min. At the same time, the UV absorption spectra of each solution were measured at different time points (0s, 10s, 20s, 30s, 40s, 50s, 60s, 1.5min, 2min, 3min, 5min, 10min) using a UV-visible near-infrared spectrophotometer. -6 mol L -1 ) and ABDA (5×10 -5 mol L -1 ) were used as the control group and blank group.

[0101] The results are as follows Figure 6 As shown in the figure, CPPO@CMC-PC-N has good singlet oxygen production performance and overcomes the self-aggregation quenching effect of a single photosensitizer. At the same time, the singlet oxygen production performance of CPPO@CMC-PC-N is concentration- and time-dependent. -6 mol L -1 , 5×10 -6 mol L -1 , 1×10 -5 mol L -1 The degradation rates of ABDA in the CPPO@CMC-PC-N system were 42%, 99%, and 99%, respectively. When the PC-N concentration in CPPO@CMC-PC-N was 5×10 -6 mol L -1 When , the singlet oxygen production performance reaches the maximum value within 10 minutes.

[0102] (4) Photothermal performance evaluation:

[0103] 1 mL of CPPO@CMC-PC-N with different concentrations (expressed as PC-N concentration, 1×10 -6 mol L -1 , 5×10 -6 mol L -1 , 1×10 -5 mol L -1 ) were placed in a 48-well plate. -2 The white LED light with a power density of 100 nm was irradiated for 10 min. The temperature of each solution was recorded by infrared thermal imager every 1 min. -6 mol L -1 ) and phosphate buffer solution were used as the control group and blank group.

[0104] The results are as follows Figure 7As shown, compared with phosphate buffer solution and reaction intermediate 1, the temperature change of CPPO@CMC-PC-N is obvious and shows time- and concentration-dependent hyperthermia. -6 mol L -1 , 5×10 -6 mol L -1 , 1×10 -5 mol L -1 The temperature changes of CPPO@CMC-PC-N were 3.0℃, 4.6℃, and 7.0℃, respectively. The temperature changes were not large, which is beneficial to avoid nonspecific damage to surrounding normal tissues due to excessively high temperature.

[0105] (5) Bacterial killing effect:

[0106] In order to explore the photodynamic antibacterial effect, the plate counting method was used to evaluate. CPPO@CMC-PC-N (expressed as PC-N concentration, 5×10 -6 mol L -1 ) was mixed with Escherichia coli or Staphylococcus aureus suspension and then incubated on a shaker at 37°C and 200 rpm for 4 h. -2 The cells were irradiated with a white LED light with a power density of 100 μL for 10 minutes, 100 μL of Escherichia coli or Staphylococcus aureus suspension was diluted gradiently and evenly spread on the LB agar plate, and the colonies were counted after culturing in an aerobic environment at 37°C for 24 hours.

[0107] The results are as follows Figure 8 As shown in the figure, CPPO@CMC-PC-N itself has certain bactericidal properties under dark conditions, and the singlet oxygen and heat it produces after illumination can further enhance its bactericidal effect. Under dark conditions, the bactericidal rate of CPPO@CMC-PC-N against Escherichia coli is 38%, and the bactericidal rate against Staphylococcus aureus is 34%, while after illumination, the bactericidal rates against Escherichia coli and Staphylococcus aureus are increased to 100% and 96%, respectively.

[0108] (6) Biocompatibility:

[0109] The cytotoxicity of CPPO@CMC-PC-N was determined by the standard tetrazolyl colorimetric method. Mouse embryonic fibroblasts (3T3) (100 μL, 10 5 cells, DMEM) were inoculated in 96-well plates and cultured for 24 h. Then, 100 μL of CPPO@CMC-PC-N with different concentrations (expressed as PC-N concentration, 1×10 -6 mol L -1 , 2.5×10 -6 mol L -1 , 5×10- 6 mol L -1 , 7.5×10 -6 mol L -1 , 1×10 -5 mol L -1 ) and continued incubation for 24 h. The cells incubated with fresh culture medium alone were used as blank controls. Then, 100 μL of tetramethylrhodamine solution (0.5 mg mL -1 ) to replace the original culture medium. After incubation for 4 h, the supernatant was removed and 100 μL of dimethyl sulfoxide was added to each well. Finally, the absorbance was measured at 570 nm using a microplate reader.

[0110] The results are as follows Fig. 9 As shown in Figure 2, CPPO@CMC-PC-N exhibits good biocompatibility. The high concentration of CPPO@CMC-PC-N (4.156×10 -2 mg mL -1 , PC content is 1×10 -5 mol L -1 ), 3T3 cells still maintained more than 80% cell viability after 24 h of co-culture.

[0111] (7) In vivo imaging performance:

[0112] To explore the imaging indication of infected wounds, a skin infection model was established using Staphylococcus aureus. First, mice were anesthetized with chloral hydrate, and then the hair on the back was shaved. Subsequently, a circular skin wound (d = ≈ 7 mm) was made on the back of each mouse. The mice were randomly divided into 3 groups (n = 3): (1) No treatment, CPPO@CMC-PC-N was added to the wound (expressed as PC-N concentration, 1×10 -5 mol L -1 ), (2) After the wound was infected with Staphylococcus aureus, nanospheres 2 (expressed as PC-N concentration, 1×10 -5 mol L -1 ), (3) After the wound was infected with Staphylococcus aureus, CPPO@CMC-PC-N was added to the wound (expressed as PC-N concentration, 1×10 -5 mol L -1 ). Fluorescence images of mice in each group before and after administration were collected on the IVIS imaging system.

[0113] The results are as follows Fig.10As shown, fluorescence can only be emitted at the wound infected with Staphylococcus aureus and using CPPO@CMC-PC-N, indicating that this process is caused by the activation of PC-N by CPPO and H2O2. The prepared CPPO@CMC-PC-N can be used for self-luminous imaging without background interference at the bacterial infection site.

[0114] (8) Evaluation of in vivo treatment effects:

[0115] Mice infected with Staphylococcus aureus were randomly divided into 4 groups (n=5): (1) no treatment, (2) phosphate buffer solution + white light (100 mW cm -2 , 10 min), (3) CPPO@CMC-PC-N, (4) CPPO@CMC-PC-N+white light (100 mW cm -2 , 10min). The wound size of each group of mice was recorded daily until the wounds of the experimental group mice were completely healed.

[0116] The results are as follows Fig.11 As shown, the wound healing speed of mice in group (4) was much faster than that of other control groups, and the degree of healing reached 90% after 9 days, indicating that CPPO@CMC-PC-N has a good in vivo photodynamic / photothermal therapeutic effect.

[0117] Example 3

[0118] The feeding amount of CPPO in CPPO@CMC-PC-N in step (3) of Example 1 was adjusted from 2.4 mg to 0.3 mg, 0.6 mg, and 1.2 mg, and other conditions were kept consistent with Example 1 to obtain the corresponding CPPO@CMC-PC-N.

[0119] The obtained CPPO@CMC-PC-N was tested for its autofluorescence performance. The specific steps included: activating each CPPO@CMC-PC-N (expressed in PC-N concentration, 1×10 -5 mol L-1), and the fluorescence imaging at a wavelength of 670 nm was collected on the IVIS imaging system and the fluorescence intensity was read.

[0120] The results are as follows Fig.12 As shown in the figure, the autofluorescence intensity of CPPO@CMC-PC-N is positively correlated with the concentration of CPPO, and the autofluorescence intensity of CPPO@CMC-PC-N obtained by adjusting the CPPO dosage to 0.3 mg, 0.6 mg or 1.2 mg at 670 nm is lower than 1.841×10 4 p / sec / cm 2 / sr, indicating that its spontaneous fluorescence effect is lower than that of CPPO@CMC-PC-N prepared when the CPPO dosage is 2.4 mg.

[0121] Comparative Example 1

[0122] 24 mg of the carboxymethyl chitosan in step (1) of Example 1 was directly weighed and added to a round-mouth flask without using water as a solvent to dissolve it in advance. Other conditions were the same as those in Example 1.

[0123] The results showed that there was almost no reaction product after the reaction was completed.

[0124] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing acid / H2O2 responsive nanospheres based on porphyrin grafted carboxymethyl chitosan, characterized in that: The steps include: (1) Synthesis of porphyrin grafted carboxymethyl chitosan: 5,10,15,20-tetrakis-(4-aminophenyl)porphyrin, an alkali reagent and N,N-dimethylformamide are mixed, chloroacetyl chloride is added dropwise after mixing, and the reaction is carried out at room temperature; after the reaction is completed, water is added to precipitate, the precipitate is separated and collected, and washed to obtain a reaction intermediate; then the reaction intermediate, an alkali reagent and N,N-dimethylformamide are mixed, an aqueous solution of carboxymethyl chitosan is added dropwise after mixing, and the reaction is carried out at room temperature; after the reaction is completed, dialyzed, centrifuged, the solid is collected, and dried to obtain porphyrin-grafted carboxymethyl chitosan, which is recorded as CMC-PC; (2) Amino small molecule modification: CMC-PC, alkaline reagent and N,N-dimethylformamide are mixed, and N,N,N',N'-tetraethyldiethylenetriamine is slowly added dropwise after mixing, and the reaction is carried out at room temperature. After the reaction is completed, the dialyzate is dialyzed, and the dialyzate is centrifuged, the solid is collected, and dried to obtain amino small molecule modified porphyrin molecule bonded carboxymethyl chitosan, which is recorded as CMC-PC-N; (3) Preparation of acid / H2O2 responsive nanospheres: CMC-PC-N and bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate (CPPO) were dissolved in tetrahydrofuran to obtain a mixed solution; the mixed solution was then slowly added into a phosphate buffer solution, mixed and reacted, and after the reaction was completed, tetrahydrofuran was removed, and the filtrate was collected by suction to obtain acid / H2O2 responsive nanoparticles, which were recorded as CPPO@CMC-PC-N; In the step (1), the molar ratio of 5,10,15,20-tetrakis-(4-aminophenyl)porphyrin, the alkaline reagent and chloroacetyl chloride is 1:(10-15):(10-15); In the step (1), the molar ratio of the reaction intermediate, the alkaline reagent and the carboxymethyl chitosan is 1:(1-2):(0.5-2); In the step (2), the molar ratio of CMC-PC, the alkaline reagent and N,N,N',N'-tetraethyldiethylenetriamine is 1:(10-15):(10-15); The mass ratio of CMC-PC-N to CPPO in the step (3) is 1:(1.5-2).

2. The method according to claim 1, characterized in that In the process of preparing the reaction intermediate in step (1), the amount of N,N-dimethylformamide used relative to 5,10,15,20-tetrakis-(4-aminophenyl)porphyrin is 0.03-0.05 mL / mg.

3. The method according to claim 1, characterized in that In the process of preparing CMC-PC in step (1), the amount of N,N-dimethylformamide used relative to the reaction intermediate is 0.03-0.05 mL / mg.

4. The acid / H2O2 responsive nano-microspheres CPPO@CMC-PC-N prepared by the method described in claim 1.

5. The acid / H2O2 responsive nanospheres according to claim 4 are used in the preparation of sterilization materials for photodynamic / photothermal sterilization.

6. Use of the acid / H2O2 responsive nanospheres according to claim 4 in preparing probes for autofluorescence imaging.

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