CN115260204A

CN115260204A - Quaternized porphyrin derivative used as photodynamic antibacterial agent and preparation method and application thereof

Info

Publication number: CN115260204A
Application number: CN202210538566.4A
Authority: CN
Inventors: 张家婧; 袁晓茜; 班超; 孙文昊; 王晓涵; 侯桂革
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-11-01

Abstract

The invention provides a quaternized porphyrin derivative used as a photodynamic antibacterial agent, and a preparation method and application thereof. The quaternized porphyrin derivative provided by the invention has double antibacterial properties, makes up the defects of weak antibacterial and anti-inflammatory effects and strong stabbing pain sense of a single silica gel, and can achieve the functions of penetration hemostasis, deep water retention, antibacterial and anti-inflammatory, illumination self-purification, intelligent repair and the like.

Description

Quaternized porphyrin derivative used as photodynamic antibacterial agent and preparation method and application thereof

Technical Field

The invention relates to the technical field of materials, in particular to a medical antibacterial material, and particularly relates to a photodynamic antibacterial agent as well as a preparation method and application thereof.

Background

Porphyrins are composed of a substituted aromatic macrocycle consisting of four pyrrole residues, linked by four methyl groups, and the porphyrin nucleus possesses a total of 22 pi electrons, of which 18 pi electrons are delocalized over a large period. Because of their aromatic nature, porphyrins often participate in electrophilic substitution reactions at mesogenic positions. Porphyrins are a light-sensitive drug that has received approval from the U.S. Food and Drug Administration (FDA). At present, porphyrin has wide application in the fields of catalysts, semiconductors, electronic materials, superconducting materials, anti-cancer drugs, nuclear magnetic resonance image enhancers, nonlinear optical materials, DNA-binding or fragmentation reagents and the like. Porphyrins are ideal Photosensitizers (PS) due to their high chemical and photostability, ease of synthesis and modification, and high efficiency of ROS generation in the therapeutic window, and some have been clinically used in photodynamic therapy for cancer.

The basic principle of photodynamic antibacterial therapy is that light and Photosensitizers (PS) generate Reactive Oxygen Species (ROS) through electron transfer (type i reaction) or energy transfer (type ii reaction), and these reactive oxygen species interact with many internal components of bacteria to cause oxidative damage and death of the bacteria. The current field of application of this unique disease treatment combining light, photosensitizers and oxygen has extended from primary tumors to age-related macular degeneration and microbial infections. Particularly in the treatment of microbial infection diseases, the photodynamic therapy has the advantages of wide antibacterial spectrum and strong target selectivity, and can carry out structural modification on the photosensitizer to increase the affinity of the photosensitizer to microorganisms.

Photosensitizers of many different chemical structures have been used for photodynamic inactivation of microorganisms, including porphyrins, phthalocyanines, phenothiazines, and the like. In particular to porphyrin compounds, has good spectral characteristics, high singlet oxygen yield and better biocompatibility, and is an ideal candidate drug as an antibacterial photosensitizer^[13]. The cell outer wall membrane breaking agent ethylenediamine tetraacetic acid, the cationic polypeptide polymyxin B, the polymer, the nano-particles, the biological molecules and the like are combined with the photosensitizer, so that the affinity of the photosensitizer for microorganisms can be remarkably improved, and particularly, the photosensitizer with cationic charges can remarkably inactivate the microorganisms recently.

The prior photosensitive antibacterial compound has the problems of poor chemical stability, water insolubility and the like, the quaternary ammonium salt per se has the defect of high cytotoxicity, and in addition, most PS materials utilize complex compositions to covalently connect PS and polymers, adopt a non-telescopic packaging method and/or combine PS or a bracket, and have limited practical use. In existing contact and release sterilization strategies, more techniques utilizing surface decoration and the like often fail to produce materials with sufficient stability and practicality.

Disclosure of Invention

The invention creatively obtains the quaternized porphyrin derivative by performing quaternization modification on tetraphenylporphyrin, so that the prepared quaternized porphyrin derivative of the photodynamic antibacterial agent can achieve double antibacterial performance. One approach to this problem is to employ an anti-bacterial adhesion-sterilization (anti-kill) combination strategy, i.e., creating a surface with dual antimicrobial functions that both prevent bacterial attachment and kill bacteria.

The invention firstly provides a quaternized porphyrin derivative used as a photodynamic antibacterial agent, and the structural formula is as follows:

the invention further provides a preparation method of the quaternized porphyrin derivative, which comprises the following steps:

performing nucleophilic substitution reaction on TMP-OH and halogen substituted hexane to obtain TMPO (CH)₂)₆X, and then the TMPO (CH)₂)₆Carrying out quaternization reaction on the X and N-methyldiethanolamine to obtain the compound;

wherein the halogen is selected from any one of F, cl, br or I; the halogen-substituted hexane is preferably 1, 6-dichlorohexane, 1, 6-dibromohexane, 1, 6-difluorohexane or 1, 6-diiodohexane, more preferably 1, 6-dibromohexane.

In one embodiment according to the present invention, the TMP-OH is prepared by a method comprising the steps of:

adding pyrrole, trimesobenzaldehyde, p-hydroxybenzaldehyde and boron trifluoride diethyl etherate into trichloromethane as a solvent while stirring, wherein the molar ratio of trimesobenzaldehyde to p-hydroxybenzaldehyde to pyrrole is 3; stirring for 1 hour at normal temperature, then adding Tetrachlorobenzoquinone (TCQ), and stirring and refluxing for 1 hour at 65 ℃; cooling to room temperature after the reaction is finished, and separating and purifying to obtain TMP-OH;

preferably, the separation and purification is achieved by a method comprising the following steps: evaporating to remove solvent, dissolving with dichloromethane, vacuum filtering, and separating and purifying by silica gel column chromatography.

In one embodiment according to the invention, the TMPO (CH)₂)₆X is prepared by a method comprising the following steps:

dissolving TMP-OH by acetone, adding NaOH to carry out magnetic stirring, reacting for 4-8 hours, adding halogen-substituted hexane, reacting at 65 ℃ by using an oil bath, monitoring the reaction by TLC, stopping the reaction after 20 hours, and separating and purifying to obtain the compound;

preferably, the separation and purification is achieved by a method comprising the following steps:

evaporating to remove solvent, dissolving in methanol, vacuum filtering, and filtering to obtain residueDissolving out chloromethane; then, the dichloromethane layer was collected and spin-dried on the column, and after loading and washing the column with petroleum ether to remove unreacted 1, 6-dibromohexane, the column was replaced with dichloromethane/petroleum ether =1:3 as developing agent, collecting to obtain intermediate TMP (CH)₂)₆X。

In one embodiment according to the present invention, the quaternization reaction is effected by a process comprising the steps of:

the TMP (CH)₂)₆Dissolving X in DMF, slowly dripping the solution into N-methyldiethanolamine, setting the temperature of an oil bath at 80 ℃, condensing and refluxing, collecting a reaction mixture after 20 hours, and separating and purifying to obtain the quaternary ammonium porphyrin derivative;

evaporating DMF solvent, adding distilled water for redissolving, extracting with ethyl acetate until water layers are colorless, collecting ethyl acetate layers, spin-drying to obtain products, washing the products with ethyl acetate, collecting solids, dissolving the solids with methanol, spin-drying, adding methanol for suction filtration, collecting filtrate, and spin-drying to obtain the quaternary aminated porphyrin derivative.

In another aspect of the present invention, a method for preparing an electrospun nanofiber membrane is provided, comprising:

1) Weighing polycaprolactone particles and collagen, dissolving in hexafluoroisopropanol, uniformly stirring by magnetic force at room temperature, after completely dissolving, sealing, uniformly stirring by magnetic force, and standing for a period of time to obtain a homogeneous solution;

2) Adding the quaternized porphyrin derivative of claim 1 into a 6% solution with the PCL/COL mass ratio of 60; the quaternized porphyrin derivative accounts for 0.02-0.5% of the solution by mass percent;

3) Adding the spinning solution into an electrostatic spinning device, setting spinning parameters to be 16KV, setting the flow rate to be 0.0016mm/s, and setting the receiving distance to be 18cm; the ambient temperature is 28 +/-3 ℃, and the relative humidity is 40 +/-10%. Vacuum drying the PCL/COL/quaternary ammonium salt composite nanofiber membrane at room temperature for three days to obtain the PCL/COL/quaternary ammonium salt composite nanofiber membrane;

preferably, an injector of the electrostatic spinning device is fixed on a main pump of the micro-injection pump, the positive pole of a high-voltage power supply is connected to a metal needle head of the injector to serve as a spinning nozzle, the negative pole of the high-voltage power supply is connected to cylindrical receiving aluminum foil paper and is connected with a grounding wire, and fibers are collected on the aluminum foil to form a fiber membrane; more preferably, the spinneret has a needle of 22G.

In a further aspect of the invention, there is provided a nanofiber membrane comprising the quaternized porphyrin derivative described above.

The invention further provides the application of the quaternized porphyrin derivative in the preparation of antibacterial agents or antibacterial materials; or the application of the nanofiber membrane in preparing antibacterial materials.

In one embodiment according to the invention, the antimicrobial material is a dressing for wound healing, scar repair or tissue engineering.

The technical scheme of the invention has the following beneficial effects:

the quaternized porphyrin provided by the invention can efficiently generate ROS under an optical treatment window, and can kill various microbial cells under the conditions of relatively low concentration and low optical power. The functional polymer nanofiber prepared by adding the functional polymer nanofiber into spinning raw materials by utilizing an electrostatic spinning technology has the advantages of good biocompatibility, low cytotoxicity and the like, can be used as a high-quality antibacterial dressing, realizes light-operated sterilization and can promote wound healing.

Drawings

FIG. 1: TMPOH, TMPO (CH)₂)₆Br、

(ii) infrared spectroscopy;

FIG. 2 is a schematic diagram: TMPOH, TMPO (CH)₂)₆Br、

Is/are as follows¹HNMR spectrogram;

FIG. 3 is a NMR hydrogen spectrum of 2-2TMP-O- (CH 2) 6 Br;

FIG. 4 is a mass spectrum of 2-2TMP-O- (CH 2) 6 Br;

FIG. 5 is a NMR spectrum of 2-5 quaternized porphyrins;

FIG. 6 is a mass spectrum of 2-5 quaternized porphyrins;

FIG. 7 is a mass spectra comparison of tetraphenylporphyrin, 2-2TMP-O- (CH 2) 6Br and 2-5 quaternized porphyrin;

FIG. 8 is a comparison infrared spectrum of tetraphenylporphyrin, 2-2TMP-O- (CH 2) 6Br and 2-5 quaternized porphyrin;

FIG. 9A is a scanning electron microscope and diameter distribution diagram of PCL + COL film;

FIG. 9B shows an infrared spectrum and common characteristic peaks of a drug-containing film;

FIG. 10 is a scanning electron microscope and diameter distribution diagram of a nanofiber membrane with a drug concentration of 0.02%;

FIG. 11 is a scanning electron microscope and diameter distribution of the nanofiber membrane with 0.05% drug concentration;

FIG. 12 is a scanning electron microscope and diameter distribution diagram of the nanofiber membrane with 0.1% drug concentration;

fig. 13 is a graph showing the results of testing cytotoxicity of nanofiber membranes using CCK 8;

FIG. 14 is a graph showing the results of the bacteriostatic rate of nanofiber membranes against Staphylococcus aureus;

FIG. 15 is a graph showing the results of the bacteriostatic rate of nanofiber membranes on Escherichia coli;

FIG. 16 is a graph showing the results of a contact angle test;

FIG. 17 is a graph showing the results of a hemolysis rate test for nanofiber membranes of different concentrations;

FIG. 18 is a TGA and DSC measurements of nanofiber membranes with different concentrations of drugs, wherein a is a graph of the TGA measurements of nanofiber membranes with different concentrations of drugs; b is a spectrum of DSC measurement result of the nanofiber membrane containing the drugs with different concentrations. .

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

Practice ofExample 1:

preparation of (2)

(1) Laboratory preparation of TMP-OH:

adding a proper amount of trichloromethane into an experimental device, and sequentially adding pyrrole, trimesic benzaldehyde, p-hydroxybenzaldehyde and boron trifluoride diethyl etherate while stirring, wherein the adding amount of trimesic benzaldehyde, p-hydroxybenzaldehyde and pyrrole is 3. After stirring at room temperature for 1 hour, tetrachlorobenzoquinone (TCQ) was added and the mixture was refluxed at 65 ℃ for 1 hour. Cooling to room temperature after the reaction is finished, evaporating to remove the solvent, dissolving with dichloromethane, performing suction filtration, and performing separation and purification by silica gel column chromatography twice to obtain TMP-OH;

(2)TMP(CH₂)₆laboratory preparation of Br:

dissolving the TMP-OH obtained by the above steps by using acetone, adding NaOH to carry out magnetic stirring, reacting for 4-8 hours, adding 1, 6-dibromohexane, reacting at 65 ℃ in an oil bath, monitoring the reaction by TLC, and stopping the reaction after 20 hours. After the solvent is removed by evaporation, the mixture is dissolved in methanol and filtered, and filter residue is dissolved out by dichloromethane. The dichloromethane layer was collected and spin-dried on the column, and after loading the column was washed with petroleum ether to remove unreacted 1, 6-dibromohexane, the column was replaced with dichloromethane/petroleum ether =1:3 as developing agent, and collecting purple product TMP (CH)₂)₆Br；

(3)

The laboratory preparation of (a):

the intermediate TMP (CH) obtained above₂)₆Br is dissolved in a small amount of DMF and slowly added into N-methyldiethanolamine dropwise, the temperature of an oil bath is set to be 80 ℃, reflux condensation is carried out, and the reaction progress is monitored by TLC. The reaction was collected at 20 hours. Evaporating solvent DMF, adding small amount of distilled water, extracting with ethyl acetate for several times until water layer is colorless, collecting ethyl acetate layer, spin-drying, adding small amount of ethyl acetate to wash product, collecting solid (solid is dissolved with methanol and then spin-dried) (repeating for 2 times), spin-drying, adding methanol, vacuum-filtering, collecting filtrate, and spin-drying to obtain final productTarget product

Example 2:

preparation of

(1) Pilot plant preparation of TMP-OH:

1000ml of chloroform was put into a 2L round-bottomed flask, 1388. Mu.l of pyrrole, 2213.6. Mu.l of mesitylaldehyde, 0.8188g of p-hydroxybenzaldehyde and 864. Mu.l of boron trifluoride diethyl ether were added in this order by means of a disposable syringe (pipette gun), and stirred at normal temperature for 1 hour, followed by addition of 3688mg of chloranil (TCQ), and then stirred at 65 ℃ under reflux for 1 hour. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solvent was distilled off. Dissolving with dichloromethane, vacuum filtering, and separating with silica gel column chromatography twice to obtain TMP-OH;

TMP(CH₂)₆pilot plant preparation of Br:

dissolving the porphyrin TMP-OH (300mg, 0.40mmol) obtained above in 10ml of acetone, adding NaOH (0.2934g, 0.007mol) for magnetic stirring, reacting for 4-8 hours, adding 1ml of 1, 6-dibromohexane, reacting in an oil bath at 65 ℃, monitoring by TLC, and stopping reaction after 20 hours. After the solvent was evaporated, the residue was dissolved in methanol and filtered, and the residue was dissolved in dichloromethane. Collecting dichloromethane layer, spin-drying, passing through a column, loading, washing the column with petroleum ether to remove unreacted 1, 6-dibromohexane, and then changing the column with dichloromethane/petroleum ether =1: 3. collecting purple product TMP (CH) as developing agent₂)₆Br；

Pilot plant preparation of (1):

mixing TMPO (CH)₂)₆Br (0.1g, 0.11mmol) in DMF (1 ml) was added dropwise to 1ml of N-methyldiethanolamine at 80 ℃ under reflux with heating, and the reaction was collected after 20 hours. Distilling to remove DMF, adding small amount of distilled water, extracting with ethyl acetate for several times until the water layer is colorless, collecting ethyl acetate layer, spin drying, addingAnd (3) adding a small amount of ethyl acetate to wash the product, collecting a solid (the solid is dissolved by methanol and then is dried in a spinning mode) (repeating for 2 times), adding methanol after the solid is dried in the spinning mode, carrying out suction filtration, collecting filtrate, and carrying out the spinning drying to obtain the target product.

Example 3 validation of detection of quaternized porphyrin derivatives

(1) Infrared spectroscopic analysis

Firstly, TMPO (CH) is synthesized by introducing H in long-chain alkyl substituted hydroxyl by nucleophilic substitution reaction between TMP-OH as base and 1, 6-dibromohexane₂)₆Br, which in turn reacts with tertiary amine to produce porphyrin quaternary ammonium salt. As shown in FIG. 1, 3390cm-1 of TMP-OH can be assigned as NH stretching vibration peak, 3310cm-1 can be assigned as-OH stretching vibration peak, 2919cm-1 is-CH₃The stretching vibration peak is 2855cm-1 is-CH₂A stretching vibration peak. TMPO (CH)₂)₆1242cm-1 in Br map can be attributed to aromatic ether bond v_C-OThe stretching vibration peak of the porphyrin quaternary ammonium salt also has aromatic ether bond v_C-OThe peak of stretching vibration of 1043cm-1 in FIG. 1 can be ascribed to the peak of stretching vibration of the primary alcoholic hydroxyl group.

(2)

¹H NMR spectroscopic analysis

Tetraphenylporphyrin derivatives (TMPOH) and bromoalkanes (TMPO (CH)₂)₆Br) of¹The HNMR analysis test selects d-chloroform as solvent and quaternary ammonium salt¹H NMR analysis was performed using deuterated DMSO on a Bruker 600 instrument. The analysis results are shown in FIG. 2, which are represented by TMPOH, and the results are represented by NMR hydrogen spectra₂)₆Br, 8.80ppm, 8.70ppm, 8.68ppm, 7.25ppm, 7.01ppm of the benzene ring and the C-H bond in the pyrrole ring and 2.62ppm, 1.84ppm of-CH of the benzene ring₃. Substituted TMPO (CH) with bromoalkane₂)₆4.24ppm and 3.49ppm of-OCH appear in Br₂-、-CH₂A C-H bond of Br. The 3.20ppm assignment appearing in the spectrum of the quaternary ammonium salt is-N-CH₃And newly appearing 3.94ppm, 3.58ppm are assigned-N-(CH₂)₂The C- -H bond in- -was assigned at 4.28ppm as- -OH bond.

Example 4 synthetic procedure

1) Synthesis of 2-1TMP (OH)

The reaction process is shown as the above reaction formula:

1000ml of chloroform was added to a 2L round bottom flask, 1388. Mu.l of pyrrole, 2213.6 (553.4). Mu.l of trimesobenzaldehyde, 0.8188g (0.2049 g) of p-hydroxybenzaldehyde and 864. Mu.l of boron trifluoride etherate were added by means of a one-shot syringe (pipette gun), and stirred at room temperature for 1h (the solution turned yellow and subsequently orange-red), then 3688mg of chloranil (TCQ) were added and stirred at 72 ℃ under reflux for 1h.

After the reaction is finished, cooling to room temperature, and performing spin drying on a rotary evaporator.

2) And (3) post-treatment of crude TMP (OH):

selecting the coarsest column, loading dichloromethane into the column, eluting with dichloromethane, collecting the eluate with a column length of 15cm, loading amount of about 8-9g, collecting the first purple band as TMP, the yellow-green band, and the second purple TMP (OH), washing the first purple band, washing the yellow band with a small amount of the second purple band mixed with the first band, collecting the first purple band and the mixed band, drying the first purple band, washing with dichloromethane, and vacuum filtering to obtain a large amount of yellow solid.

With EA: PE =1: the 5-point plate can be separated

The PE was packed in a column with a length of 12cm, and two purple bands were clearly visible on the column, the lighter of which was a small amount of TMP, and the second purple band was the point of TMP (OH).

The optimized post-processing mode is as follows:

and (3) filling the column with dichloromethane about 6cm in length, dissolving a sample to be used as a developing agent, collecting a second purple color band, performing spin-drying, and performing spin-drying by using dichloromethane, methanol and EA (ethylene oxide) PE =1:5 washing, collecting the filtrate, spin-drying, washing with EA: PE =1:5 as developing agent, dissolving the sample, carrying out column chromatography separation with the column length of 10cm, collecting the purple color band, and carrying out column chromatography separation with the following steps of EA: PE =1:8, spot plate observation is carried out to obtain a pure product.

3)2-2TMP-O-(CH₂)₆Synthesis of Br

The reaction process is as follows:

porphyrin TMP-OH (300mg, 0.40mmol) was dissolved in 10ml acetone and NaOH (0.2934 g,0.007 mol) was added and magnetic stirring was carried out, after 4h-8h of reaction 1, 6-dibromohexane 1ml was added, at which time an oil bath was used instead for 65 ℃ reaction, TLC was used to monitor the reaction, dichloromethane: petroleum ether =1: the reaction was stopped after 20h by 2-point plate observation.

And (3) post-treatment:

after the solvent is removed by evaporation, methanol is used for dissolving and filtering, filter residue is dissolved out by dichloromethane, filtrate and filter residue are all collected by dichloromethane: PE =1: 3-point plate observation, small amount of product in methanol layer (discard)

The dichloromethane layer was spin-dried on the column, dichloromethane: petroleum ether =1:3 column packing, column length 12cm, dichloromethane: petroleum ether =1:3 as developing agent, dissolving the sample, flushing the column with petroleum ether after loading the sample, and removing the unreacted 1, 6-dibromohexane.

Color band distribution:

a light purple impurity band (abandoned) is arranged in front of the product band, a purple color band (impurities) is arranged behind the product band, then a small amount of reactant color bands are arranged, finally a black impurity color band is arranged, the impurity band is close to the product band in front of and behind the product color band, and dichloromethane is used instead after the light purple band in front of the product band is washed down: PE =1:2 as developing agent, collecting the product spot plate.

The NMR spectrum is shown in FIG. 3, and the mass spectrum results are shown in FIG. 4. Hydrogen nuclear magnetic resonance spectroscopy was as follows:

¹H NMR(600MHz,CDCl₃)δ8.79(d,J＝4.6Hz,2H),8.65(d,J＝4.6Hz,2H), 8.61(s,4H),8.08(d,J＝8.5Hz,2H),7.24(dd,J＝8.6,3.0Hz,8H),4.24(t,J＝6.3 Hz,2H),3.49(t,J＝6.8Hz,2H),2.61(d,J＝4.2Hz,9H),1.84(d,J＝4.4Hz,18H), 1.53(s,6H),1.25(s,2H),-2.56(s,2H).

δ 8.79ppm, 8.65ppm, 8.61ppm are assigned to the C-H bond in the β -position of the pyrrole ring; β 08.08ppm and δ 7.24ppm are assigned to the C-H bond on the benzene ring, and the δ 4.24ppm peak is assigned to the chemical shift of hydrogen on the oxygen α -position C; the peak at delta 3.49ppm is assigned as the chemical shift of hydrogen on the carbon at the alpha-position of Br, and the peaks at delta 2.61ppm and delta 1.84ppm are assigned as the chemical shifts of methyl hydrogen on a benzene ring; delta 1.53ppm as-OCH₂CH₂CH₂CH₂CH₂CH₂CH in Br₂Chemical shift of (a); delta 1.25ppm as-OCH₂CH₂CH₂CH2CH₂CH₂CH in Br₂Chemical shift of (a); -2.56ppm chemical shift attributed to-NH.

4) 2-5 quaternized porphyrins

The reaction process is as follows:

TMPO (CH 2) 6Br (0.13mmol, 120mg) was dissolved in 1.5ml DMF and was slowly added dropwise to N-methyldiethanolamine (2 ml), the oil bath temperature was set at 80 ℃, reflux was condensed, and the progress of the reaction was monitored by TLC.

When the spot plate is observed by using dichloromethane and ethyl acetate as developing agents for 16 hours, a purple point is formed at the origin, and a small amount of impurities and incomplete reactants are arranged in front of the purple point. The reaction was collected at 20 h.

And (3) post-treatment:

evaporating DMF solvent, adding small amount of distilled water, extracting with ethyl acetate for several times until water layer is colorless, collecting ethyl acetate layer, spin-drying, adding small amount of ethyl acetate to wash product, collecting solid (solid is dissolved with methanol and then spin-dried) (repeating for 2 times), adding methanol after spin-drying, suction-filtering, collecting filtrate, spin-drying, and spotting with ethyl acetate and methanol as developing agent respectively.

Process for preparing 2-5 quaternized porphyrins¹The HNMR result spectrum and the mass spectrum are respectively shown in figure 5 and figure 6.

¹H NMR(600MHz,DMSO)δ8.83(s,2H),8.61(d,J＝29.6Hz,6H),8.10(s, 2H),7.31(d,J＝16.1Hz,8H),4.24(s,3H),3.91(s,30H),3.50(t,J＝23.4Hz,20H), 3.20(d,J＝27.1Hz,8H),2.53(s,9H),2.26(s,3H),1.92(d,J＝13.9Hz,3H),1.85(s, 3H),1.76(s,18H),1.65(s,2H),1.47(s,2H),-2.64(s,2H).

Δ 8.83ppm, 8.61ppm assigned to the C-H bond at the β -position of the pyrrole ring; beta 08.10ppm and delta 7.31ppm belong to C-H bonds on a benzene ring, and a peak of delta 4.24ppm belongs to chemical shifts of hydrogen on an oxygen alpha-position C; the peak at delta 3.20ppm is attributed to the chemical shift of hydrogen on the carbon at Br alpha-position, and the peaks at delta 2.53ppm and delta 1.76ppm are attributed to the chemical shift of methyl hydrogen on a benzene ring; delta 1.53ppm as-OCH₂CH₂CH₂CH₂CH₂CH₂CH in Br₂Chemical shift of (a); delta 1.25ppm was assigned to-OCH₂CH₂CH₂CH2CH₂CH₂CH in Br₂Chemical shift of (a); -2.56ppm chemical shift attributed to-NH.

FIG. 7 is a mass spectra comparison of tetraphenylporphyrin, 2-2TMP-O- (CH 2) 6Br and 2-5 quaternized porphyrin.

FIG. 8 is a comparison infrared spectrum of tetraphenylporphyrin, 2-2TMP-O- (CH 2) 6Br and 2-5 quaternized porphyrin; the analysis was as follows:

firstly, TMPO (CH) is synthesized by H which is introduced into long-chain alkyl substituted hydroxyl by nucleophilic substitution reaction on TMP-OH as the basis and 1, 6-dibromohexane₂)₆Br, and further reacts with tertiary amine to generate porphyrin quaternary ammonium salt. 3390cm in TMP-OH^-1Can be attributed to NH stretching vibration peak, 3310cm^-1Can be classified as-OH stretching vibration peak, 2919cm^-1is-CH₃Peak of stretching vibration, 2855cm^-1is-CH₂A stretching vibration peak. TMPO (CH)₂)₆1242cm in Br map^-1Can be ascribed to aromatic ether bond v_C-OThe stretching vibration peak of the porphyrin quaternary ammonium salt also has aromatic ether bond v_C-OThe peak of stretching vibration of (3), 1043cm in FIG. 8^-1Can be assigned as the stretching vibration peak of primary alcohol hydroxyl.

Example 5 electrospinning nanocomposite film preparation

Preparing a PCL/COL solution with a PCL/COL mass ratio of 60 to 6 percent.

Weighing a certain amount of polycaprolactone particles and collagen, dissolving in hexafluoroisopropanol, uniformly stirring by magnetic force at room temperature, after complete dissolution, uniformly stirring by closed magnetic force, and standing for a period of time to obtain a homogeneous solution. Adding the quaternized porphyrin according to a certain mass fraction (0.02%, 0.05%, 0.1%, 0.2%, 0.5%) accounting for the percentage content of the solid content in the solution into a 6% PCL/COL solution according to a mass ratio of 60.

The spinning solution is added into an injector which is fixed on a main pump of a micro-injection pump, the positive pole of a high-voltage power supply is connected to a metal needle head (the needle head is 22G) of the injector to be used as a spinning nozzle, the negative pole of the high-voltage power supply is connected to cylindrical receiving aluminum foil paper and is connected with a grounding wire, and fibers are collected on the aluminum foil to form a fiber membrane. The spinning parameters are set to be 16KV, the flow rate is 0.0016mm/s, and the receiving distance is 18cm; the ambient temperature is 28 +/-3 ℃, and the relative humidity is 40 +/-10%. The PCL/COL/quaternary ammonium salt composite nanofiber membrane was vacuum dried at room temperature for three days to remove residual solvent.

FIG. 9A shows the scanning electron microscope and diameter distribution of PCL + COL and drug-containing films of different concentrations.

FIG. 9B shows the infrared spectrum and common characteristic peaks of the medicated membrane, wherein 3300cm^-1The peak is O-H, N-H characteristic peak, 2940 and 2860cm^-1Peaks at (B) can be respectively attributed to asymmetric and symmetric stretching vibration of methylene, 1720cm^-1The peak is the peak of the C = O for the absorption of the stretching vibration, 1650cm^-1Bending vibration peak assignable as hydroxyl group, 1240cm^-1And 1180cm^-1The peak is respectively the expansion vibration absorption peak of C-C and C-O.

FIG. 11 is a scanning electron microscope and diameter distribution diagram of a nanofiber membrane with a drug concentration of 0.05%;

FIG. 12 is the scanning electron microscope and diameter distribution diagram of the nanofiber membrane with 0.1% drug concentration.

According to the experimental results, the PCL/COL/quaternized porphyrin nanofiber membrane is prepared by adding quaternized porphyrins with different contents into spinning solutions with the concentration of 6% and the concentration of PCL: COL = 60. FIGS. 9 to 12 are SEM photographs of fibrous membranes containing 0.02%, 0.05%, 0.1%, 0.2% and 0.5% of PCL + COL and the drugs, respectively. Observing the fiber structure on the surface of the spinning membrane, the fiber appearance is better when the addition amount of the quaternized porphyrin is within 0.5 percent. With the increase of the content of the quaternized porphyrin, a plurality of tiny fibers begin to appear, because the addition of the quaternary ammonium salt increases the conductivity of the spinning solution, the electric field force of the spinning solution drops in the electric field is increased, the spinning solution drops are continuously stretched, and the spinning process is unstable.

Under the same conditions, the diameter of PCL + COL membrane fibers is 81.6nm, the diameter of membrane fibers containing 0.02% of the drug is 139.2nm, the diameter of membrane fibers containing 0.05% of the drug is 140.2nm, the diameter of membrane fibers containing 0.1% of the drug is 278.7nm, the diameter of membrane fibers containing 0.2% of the drug is 376.8nm, and the diameter of membrane fibers containing 0.5% of the drug is 458.0nm, so that the diameters of the fibers are gradually increased.

Example 6 TGA and DSC measurements of nanofiber membranes containing varying concentrations of drug

Measured by thermogravimetric analysis (TGA) and a synchronous thermal analyzer instrument, a sample (7.5 mg) is placed in a platinum disc sample holder to be heated at the heating speed of 20-500 ℃ and 10 ℃/min, and the cooling speed is 20 ℃/min. An aluminum sealed pot was used as a sample holder.

The thermal decomposition performance is tested by adopting a thermal analysis method, various transformations and reactions of substances can occur under the control of a temperature program, and the mass or energy of the substances changes along with the temperature or time.

As shown in FIG. 18, the DSC curve shows an endothermic peak at 59.6 ℃ where the nanofiber membrane shows an endothermic peak associated with the melting temperature of the crystalline phase, and also shows an endothermic phenomenon at 87.2 ℃ which corresponds to the highest temperature at which the collagen sample is completely denatured, and a broader peak at 407.04 ℃ shows a strong endothermic phenomenon due to the decomposition and destruction of the PCL structure. The TGA curve shows that the weight loss of collagen in the first stage is 3.35%, the weight loss occurs at 30-140 ℃, the weight loss is caused by water evaporation, the weight loss in the second stage occurs at 230-500 ℃, the weight loss is 77.02%, the weight loss is caused by thermal decomposition of PCL and collagen macromolecular chains, and when the weight loss begins to occur, the weight of the PCL + COL membrane is reduced firstly, so that compared with the PCL + COL membrane, the thermal stability of the drug-containing membrane is better.

Example 7ROS detection

The amount of ROS generated by nanofiber membranes containing different concentrations of quaternary ammonium porphyrin under light irradiation was measured by using 1, 3-Diphenylisobenzofuran (DPBF) as a chemical sensing probe. The detection mechanism is based on the generated¹O₂The absorbance of DPBF can be irreversibly quenched at 410nm under light irradiation. Containing DPBF (14. Mu. GmL)^-1) And nanofiber membranes (1.0X 1.0 cm) containing different concentrations of quaternary ammonium salts²) The methanol solution of (1) (660 nm, 3.2 mWcm)^-2) The lamp(s) is continuously illuminated for different times (0-80 min). Subsequently, the absorbance value at 410nm of the solution was recorded by an ultraviolet-visible spectrometer. Meanwhile, the DPBF solution containing the nanofiber membrane under the dark condition is used as a control.

After the pure DPBF and DPBF/PCL + COL blank films are continuously irradiated for 80min, the absorbance at 410nm is not changed, which shows that¹O₂The amount of the product is negligible. However, in the nanofiber membrane doped with quaternized porphyrin, the adsorption amount of DPBF at 410nm gradually decreased with the increase of the light irradiation time. In addition, the absorbance of the film DPBF was most reduced during the course of light irradiation at a quaternized porphyrin concentration of 0.5%.

The light quaternization porphyrin drug solution has the advantages that although the absorbance is increased, the peak shape is not changed, so that the drug is not changed after long-time light irradiation.

Example 8 cytotoxicity assay

L929 cytotoxicity (non-toxicity)

Cytotoxicity was assessed using the CCK-8 assay.

Quaternary alkylated porphyrin preparation solution

The quaternized porphyrin was dissolved in methanol to prepare a solution of 10mg/ml, and the solution was diluted 100 times to measure the absorbance.

L929 mouse fibroblast (DMEM) supplemented with 10vol% Fetal Bovine Serum (FBS) and 1vol% cyanA solution of streptomycin and streptomycin. Cells were digested from the petri dish by adding 0.25% trypsin-edta solution and resuspended in fresh medium for subsequent experiments. The nanofiber membrane (1.0X 1.0 cm)²) Gently placed at the bottom of a 24-well plate, and then 5X 10 wells per well⁴After cells in a cell density medium (10. Mu.L) were inoculated on a membrane and incubated for 2 hours, 500. Mu.L of the medium was added, incubated in a humidified atmosphere of 5% carbon dioxide for 24 hours, and the 24-well plate was taken out of the incubator and left to stand for 3 hours. Parallel experiments without sample were used as controls. After standing for 3 hours, the medium was taken out, and 500. Mu.L (medium: CCK-8= 1) of the solution was added and incubated for 2 hours. After 2h, 200. Mu.L of the supernatant was pipetted into a 96-well plate and the OD at 450nm was measured using a microplate reader. The results are in terms of OD values obtained relative to the control.

The illumination condition is as follows:

l929 mouse fibroblast cells (DMEM) supplemented with 10vol% Fetal Bovine Serum (FBS) and 1vol% penicillin-streptomycin solution. Cells were digested from the petri dish by adding 0.25% trypsin-edta solution and resuspended in fresh medium for subsequent experiments. The nanofiber membrane (1.0X 1.0 cm)²) Gently placed at the bottom of a 24-well plate, and then 5X 10 wells per well⁴Cells in a cell density medium (10. Mu.L) were inoculated on a membrane and incubated for 2 hours, 500. Mu.L of the medium was added, and after incubation for 24 hours in a humidified atmosphere of 5% carbon dioxide, the 24-well plate was removed from the incubator and illuminated for 3 hours (9 mw/cm)²). Parallel experiments without sample were used as controls. After the light irradiation, the medium was aspirated, and 500. Mu.L (medium: CCK-8= 1) of the solution was added and incubated for 2 hours. After 2h, 200. Mu.L of the supernatant was pipetted into a 96-well plate and the OD at 450nm was measured using a microplate reader. The results are relative to the OD values obtained for the control group.

Dark conditions:

the nanofiber membrane (1.0X 1.0 cm)²) Gently placed at the bottom of a 24-well plate, and then 5X 10 wells per well⁴Cells in cell density media (10. Mu.L) were seeded onto the membrane and incubated for 2h, then 500. Mu.L of media was added and incubated for 24h in a humidified atmosphere of 5% carbon dioxide. Parallel experiments without sample were used as controls. After 24h, the medium was aspirated,500. Mu.L (medium: CCK-8= 1) of the solution was added and incubated for 2 hours. After 2h, 200. Mu.L of the supernatant was pipetted into a 96-well plate, and OD at 450nm was measured using a microplate reader. The results are relative to the OD values obtained for the control group.

The results are shown in FIG. 13, in which the OD values of the PCL + COL blank membrane and the sample membrane group are increased compared with the control group under the dark condition, which indicates that the addition of collagen is beneficial to the growth and proliferation of cells. However, as the concentration of quaternized porphyrin was increased, cytotoxicity increased. Referring to the cytotoxicity evaluation grade in GB/T16886.5, the membrane cytotoxicity evaluation grade is 0 grade under dark conditions, and the membrane is qualified. The illumination condition is limited by the experimental condition, and the cells are cultured under the natural condition when the illumination is carried out for 3 hours. When the culture is carried out under natural conditions, the content of the quaternized porphyrin is increased from 0.1% to 0.5% by comparing with the culture without light, the cytotoxicity is over 75%, the cytotoxicity grade is 1, and the quaternary ammonium porphyrin is evaluated to be qualified.

Example 9 antibacterial property test

Gram-positive staphylococcus aureus and gram-negative escherichia coli are taken as representative strains, and the photodynamic inactivation of surface bacteria is detected.

Staphylococcus aureus or E.coli cells were plated onto separate agar plates and incubated overnight at 37 ℃. Using a single colony of each bacterium, 30mL of the medium was inoculated at 37 ℃ for 12h, and then centrifuged to remove the supernatant. The bacterial concentration was then determined by measuring the absorbance of the cell dispersion at 600nm using a microplate reader. An optical density at 600nm of 1.0, corresponding to-10⁹Cell mL^-1. These active cultures were diluted in sterile Phosphate Buffered Saline (PBS) to a final concentration of 10⁶Individual cell mL^-1. Test membranes (1 cm. Times.1 cm) were sterilized and plated flat on the bottom of a 24-well plate, and 100. Mu.L (106 cells/mL) was taken^-1) The test membrane was incubated at 37 ℃ for 5 hours in the dark and then with light for 3 hours, followed by addition of 900. Mu.L of sterile PBS and removal of the bacteria on the membrane surface by sonication (3 min). Then, 100. Mu.L of each bacterial suspension (Staphylococcus aureus and Escherichia coli) was plated on sterile LB agarThe plates were incubated overnight at 37 ℃ to give visible colonies. The number of colonies on each plate was counted and the total number of viable bacteria adhered to each sample was evaluated.

The result of the bacteriostatic rate of the nanofiber membrane on staphylococcus aureus is shown in fig. 14, and under a dark condition, the nanofiber membrane containing quaternized porphyrin has a certain inhibitory effect on staphylococcus aureus but the bacteriostatic effect is not obvious. After illumination, the bacteriostatic rate is increased along with the increase of the content of the quaternized porphyrin, wherein the bacteriostatic rate of the film with the content of the quaternized porphyrin of 0.5 percent is 97.15 percent, and the bactericidal effect is obvious.

The results of the bacteriostatic rate of the nanofiber membrane on escherichia coli are shown in fig. 15, and under a dark condition, the nanofiber membrane containing quaternized porphyrin has a certain inhibitory effect on staphylococcus aureus but the bacteriostatic effect is not obvious. After illumination, the bacteriostatic rate is increased along with the increase of the content of the quaternized porphyrin, wherein the bacteriostatic rate of a membrane with the content of the quaternized porphyrin being 0.5 percent is 93.63 percent.

The comparison of results of escherichia coli and staphylococcus aureus shows that the antibacterial nanofiber membrane containing quaternized porphyrin is more sensitive to staphylococcus aureus and has a more remarkable sterilization effect.

Example 10 contact Angle test

And measuring the contact angle of the fiber membrane by using a contact angle measuring instrument, and inspecting the influence of the water-oil ratio and the emulsifier on the hydrophilicity and hydrophobicity of the fiber surface. The measurement parameters are: the nanofiber membrane was laid flat on a glass slide, flattened with a cover glass, and placed on a stage, with the water drop volume set to 1 μ l per time, and the instantaneous contact angle image of the water drop on the fiber membrane was taken, as shown in fig. 16. Test results show that the water contact angle of the PCL + COL blank membrane is 77.00 degrees, while the water contact angle of the nano-fiber membrane containing 0.5 percent of quaternized porphyrin is 85.38 degrees. Compared with a PCL + COL blank membrane, the quaternized porphyrin/collagen nanofiber membrane has certain hydrophobicity, and the angle of water contact angle is increased along with the increase of the concentration of the quaternized porphyrin.

Example 11 hemolytic rate test:

cutting the nano-fiber membranes containing quaternized porphyrins with different concentrations prepared by electrospinning into squares of 1cm multiplied by 1cm, and placing the squares at the bottom of a 24-hole culture plate after ultraviolet sterilization. Balb/C mice were bled from the eyeball, 1mL of blood was centrifuged to remove supernatant to isolate red blood cells, washed twice, resuspended and diluted with 30mL of LPBS (10mM, pH = 7.2-7.4), 1mL of diluted red blood cells was added to a nanocoating on a 24-well plate, incubated at 37 ℃ for 1h, each well extract was collected, centrifuged at 1000rpm for 10min, the supernatant was separated, the absorbance of the supernatant at 540 nm was measured with a microplate reader, 0.1 Triton X-100 was added to red blood cells as a positive control (n = 4), and 0.1 PBS was added to red blood cells as a blank control.

The results of the hemolysis rate test for nanofiber membranes of different concentrations are shown in fig. 17. In the test, the liquid of the positive control group is dark red, because the red blood cells are broken due to the addition of the distilled water, so that the hemolysis phenomenon occurs, the supernate of the test sample group and the supernate of the negative control group are clear, colorless and transparent, and the unbroken red blood cells are deposited at the bottom of the centrifuge tube, so that the hemolysis phenomenon is not seen.

According to the data, the hemolysis rate of PCL + COL blank membrane is 0.343, and the hemolysis rate increases with the increase of quaternized porphyrin. The hemolysis rates of the PCL + COL membrane and the nanofiber membrane containing 0.02%, 0.05%, 0.1%, 0.2% and 0.5% do not exceed 5%, and the results show that the membranes can not produce hemolysis when applied to the body, have good biocompatibility and meet the requirement of hemolysis rate.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A quaternized porphyrin derivative for use as a photodynamic antimicrobial agent, characterized by the following structural formula:

2. the method of preparing a quaternized porphyrin derivative of claim 1, comprising:

wherein the halogen is selected from any one of F, cl, br or I; the halogen-substituted hexane is preferably 1, 6-dichlorohexane, 1, 6-dibromohexane, 1, 6-difluorohexane or 1, 6-diiodohexane, and more preferably 1, 6-dibromohexane.

3. The method of claim 2, wherein the TMP-OH is prepared by a method comprising the steps of:

adding pyrrole, trimesic benzaldehyde, p-hydroxybenzaldehyde and boron trifluoride diethyl etherate into chloroform as a solvent while stirring, wherein the molar ratio of trimesic benzaldehyde to p-hydroxybenzaldehyde to pyrrole is 3.2; stirring for 1 hour at normal temperature, then adding Tetrachlorobenzoquinone (TCQ), and stirring and refluxing for 1 hour at 65 ℃; cooling to room temperature after the reaction is finished, and separating and purifying to obtain TMP-OH;

4. The method of claim 2, wherein the TMPO (CH)₂)₆X is prepared by a method comprising the following steps:

dissolving TMP-OH by acetone, adding NaOH to carry out magnetic stirring, reacting for 4-8 hours, adding halogen-substituted hexane, reacting at 65 ℃ in an oil bath, monitoring the reaction by TLC (thin layer chromatography), stopping the reaction after 20 hours, and separating and purifying to obtain the compound;

after the solvent is removed by evaporation, methanol is used for dissolving and filtering, and filter residue is dissolved out by dichloromethane; then, the dichloromethane layer was collected and spin-dried on the column, and after loading the column was washed with petroleum ether to remove unreacted 1, 6-dibromohexane, the column was replaced with dichloromethane/petroleum ether =1:3 as developing agent, collecting to obtain intermediate TMP (CH)₂)₆X。

5. The method of claim 2, wherein the quaternization reaction is carried out by a process comprising the steps of:

the TMP (CH)₂)₆Dissolving X in DMF, slowly dripping the solution into N-methyldiethanolamine, setting the temperature of an oil bath at 80 ℃, condensing and refluxing, collecting a reaction mixture after 20 hours, and separating and purifying to obtain the quaternized porphyrin derivative;

evaporating to remove a solvent DMF, adding distilled water for redissolving, then extracting by ethyl acetate until a water layer is colorless, collecting the ethyl acetate layer, spin-drying to obtain a product, washing the product by ethyl acetate, collecting a solid, dissolving the solid by methanol, spin-drying, then adding methanol for suction filtration, collecting a filtrate, and spin-drying to obtain the quaternized porphyrin derivative.

6. The preparation method of the electrostatic spinning nanofiber membrane is characterized by comprising the following steps:

1) Weighing polycaprolactone particles and collagen, dissolving in hexafluoroisopropanol, magnetically stirring uniformly at room temperature, sealing, magnetically stirring uniformly and standing for a period of time after complete dissolution to obtain a homogeneous solution;

2) Adding the quaternized porphyrin derivative of claim 1 into a solution with a PCL/COL mass ratio of 60; the quaternized porphyrin derivative accounts for 0.02-0.5% of the solution by mass percent;

3) Adding the spinning solution into an electrostatic spinning device, setting spinning parameters to be 16KV, setting the flow rate to be 0.0016mm/s, and setting the receiving distance to be 18cm; the environmental temperature is 28 +/-3 ℃, the relative humidity is 40 +/-10%, and the PCL/COL/quaternary ammonium salt composite nanofiber membrane is dried in vacuum at room temperature for three days to obtain the PCL/COL/quaternary ammonium salt composite nanofiber membrane;

preferably, an injector of the electrostatic spinning device is fixed on a main pump of the micro-injection pump, the positive pole of a high-voltage power supply is connected to a metal needle head of the injector to serve as a spinning nozzle, the negative pole of the high-voltage power supply is connected to cylindrical aluminum foil receiving paper and is connected with a grounding wire, and fibers are collected on the aluminum foil to form a fiber membrane; more preferably, the needle of the spinning nozzle is 22G.

7. A nanofiber membrane, comprising the quaternized porphyrin derivative of claim 1.

8. The nanofiber membrane according to claim 7, which is prepared by the preparation method according to claim 6.

9. Use of a quaternized porphyrin derivative according to claim 1 for the preparation of an antibacterial agent or an antibacterial material; or the use of a nanofibrous membrane according to claim 7 or 8 for the preparation of an antibacterial material.

10. The use of claim 8, wherein the antibacterial material is a dressing for wound healing, scar repair or tissue engineering.