Near-infrared light response nanometer preparation for photo-thermal / photodynamic synergistic ablation of artery plaques

Abstract

The invention relates to the technical field of biological medicines, and discloses a near-infrared light response nano preparation for photothermal / photodynamic synergistic ablation of artery plaques, which is prepared from the following raw materials: a poly (L-glutamic acid)-g-methoxy polyethylene glycol grafted copolymer, methylene blue and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone. Through a low-temperature limited impinging stream micro-mixing technology, methylene blue and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone are assembled in a polymer hydrophobic microcell to form the charge transfer compound. The near-infrared light response nano preparation for photo-thermal / photodynamic synergistic ablation of artery plaques has near-infrared absorption characteristics and pH responsiveness, can specifically activate photo-thermal and photodynamic effects in an arterial plaque slightly acidic environment, solves the problems that 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone is easy to hydrolyze and a compound is unstable, and has good application prospects. Cooperative ablation of deep plaques is realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nano-formulation for the synergistic ablation of arterial plaques by near-infrared light-responsive photothermal / photodynamic therapy. Background Technology

[0002] Atherosclerosis is the main pathological basis for cardiovascular and cerebrovascular diseases such as myocardial infarction and stroke. Compared with systemic drug therapy that requires long-term use and invasive vascular stent implantation, phototherapy strategies based on photothermal therapy and photodynamic therapy have shown application potential in the field of arterial plaque ablation due to their advantages such as high spatiotemporal controllability, low systemic toxicity and side effects, and minimal invasiveness.

[0003] Existing photosensitizers used in current technologies primarily utilize excitation wavelengths concentrated in the ultraviolet or visible light regions, limiting their penetration depth into biological tissues and making it difficult to effectively reach deep vascular plaques. Furthermore, conventional photodynamic therapy mainly relies on type II photochemical mechanisms, i.e., converting ground-state oxygen into singlet oxygen through energy transfer to kill diseased cells. Arterial plaques are typically in a hypoxic state, limiting the efficiency of singlet oxygen generation and therapeutic efficacy. While constructing organic charge-transfer complexes using the interaction between electron donor and electron acceptor molecules can effectively narrow the molecular orbital band gap, redshift the absorption spectrum to the near-infrared region with stronger tissue penetration, and endow the material with oxygen-independent type I photodynamic activity, charge-transfer complexes face the technical bottleneck of poor physiological environmental stability in practical applications.

[0004] In particular, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, as a strong electron acceptor, is chemically reactive and readily undergoes irreversible hydrolysis or nucleophilic substitution degradation in aqueous media, leading to structural dissociation of the charge transfer complex and quenching of its photothermal / photodynamic functions. Simple physical mixing or conventional nanoprecipitation methods are insufficient to effectively protect the structural integrity of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in aqueous solutions, and it is also difficult to achieve a specific response to the microenvironment of arterial plaques. How to construct chemically stable near-infrared charge transfer complex nanoparticles that are resistant to physiological environmental interference and possess lesion-targeting activation functions in aqueous systems is a pressing technical problem that needs to be solved to achieve efficient ablation of deep arterial plaques.

[0005] Therefore, the purpose of this invention is to provide a near-infrared light-responsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques, in order to overcome the shortcomings of the prior art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a near-infrared photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques. This solves the problems in existing technologies, such as the easy hydrolysis and failure of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in aqueous media, the difficulty in maintaining the stability of organic charge transfer complexes under physiological conditions, and the low ablation efficiency caused by the difficulty of traditional phototherapy strategies in reaching deep blood vessels and the limitation of the hypoxic microenvironment of plaques.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoparticle formulation for arterial plaques, prepared from raw materials comprising the following parts by weight: 150-250 parts of poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer; 25-40 parts of methylene blue; 20-40 parts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0008] By employing the above-mentioned technical solution, a charge-transfer complex core with near-infrared absorption properties is constructed by molecularly assembling the electron donor methylene blue and the electron acceptor 2,3-dichloro-5,6-dicyano-1,4-benzoquinone within a hydrophobic microdomain formed by a poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer. The mechanism of action lies in the orbital hybridization between the highest occupied molecular orbital of the methylene blue molecule and the lowest unoccupied molecular orbital of the 2,3-dichloro-5,6-dicyano-1,4-benzoquinone molecule, which reduces the band gap and establishes an intermolecular charge-transfer transition channel in the 780 nm to 850 nm region. This endows the nano-formulation with photothermal conversion capability and type I photodynamic activity under 808 nm excitation. Furthermore, the side chain carboxyl groups of the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer are highly ionized under physiological conditions of pH 7.4 to form a negative charge layer, which shields the activity of the charge transfer complex. Under slightly acidic conditions of pH 6.5, the side chain carboxyl groups undergo protonation, which causes a conformational change and charge reversal in the polymer, thereby activating the responsive photothermal and photodynamic effects on the lesion microenvironment.

[0009] Preferably, in the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer, the grafting rate of polyethylene glycol is 5%-25%.

[0010] By employing the above technical solution, the grafting rate is controlled between 5% and 25% to balance the water solubility and pH response sensitivity of the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer. When the grafting rate is below 5%, the colloidal stability of the nano-formulation is insufficient; when the grafting rate is above 25%, the steric hindrance effect of the polyethylene glycol segments will hinder the conformational transformation of the poly(L-glutamic acid) segments, reducing the response rate to acidic environments.

[0011] Preferably, the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer is prepared by the following steps: ring-opening polymerization of γ-benzyl-L-glutamic acid ester-N-carboxylic acid anhydride is initiated by n-hexylamine to obtain poly(γ-benzyl-L-glutamic acid ester); the side chain protecting groups of poly(γ-benzyl-L-glutamic acid ester) are removed, and the poly(L-glutamic acid) is acidified and precipitated to obtain poly(L-glutamic acid); the side chain carboxyl groups of poly(L-glutamic acid) are activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as activators, and grafted with methoxy polyethylene glycol amine.

[0012] Preferably, in the ring-opening polymerization, the molar ratio of γ-benzyl-L-glutamate-N-carboxylic anhydride to the initiator is controlled at 50:1 to 70:1; the removal of the side chain protecting group is carried out by reacting with HBr / acetic acid solution, and then purified by neutralization with sodium bicarbonate aqueous solution and adjustment of pH to 3.0 with hydrochloric acid.

[0013] Preferably, in the grafting reaction, the molar ratio of the amino group of methoxy polyethylene glycolamine to the carboxyl group of poly(L-glutamic acid) is 0.05-0.25.

[0014] By adopting the above technical solution and controlling the ratio of monomer to initiator and the grafting feed ratio, a poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer with a specific molecular weight and grafting density is synthesized, so that the copolymer forms a dense core-shell structure during self-assembly, thereby achieving effective loading of methylene blue and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0015] Preferably, the raw materials also include sodium hydroxide for adjusting the pH value and solvents for dissolving the raw materials, the solvents including anhydrous N,N-dimethylformamide and deionized water.

[0016] Preferably, the sodium hydroxide is an aqueous solution with a concentration of 0.1 M, and the amount of sodium hydroxide solution added is the amount required to adjust the pH of the aqueous phase containing the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer to 7.60-7.80.

[0017] By employing the above technical solution, anhydrous N,N-dimethylformamide is used to dissolve hydrophobic raw materials, preventing the hydrolysis of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The pH of the aqueous phase is adjusted to 7.60-7.80 to maintain the poly(L-glutamic acid) segments in a deprotonated, extended state, facilitating rapid capture of the hydrophobic core while avoiding nucleophilic substitution degradation of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone caused by localized strong alkaline environments.

[0018] Preferably, the molar ratio of methylene blue to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is 1:1.0-1.5.

[0019] By adopting the above technical solution, an excess of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is used to shift the charge transfer equilibrium toward the formation of the complex, thereby improving the absorption coefficient and photothermal conversion efficiency in the near-infrared region.

[0020] Preferably, the near-infrared light-responsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques is prepared from raw materials comprising the following parts by weight: 200 parts of poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer; 32 parts of methylene blue; and 22.7-34.0 parts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0021] Preferably, the nano-formulation is prepared by the following steps: Methylene blue and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone were dissolved in anhydrous N,N-dimethylformamide and cooled to 1-3 °C to obtain an organic phase. The poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer was dissolved in water, and sodium hydroxide solution was added dropwise at 1-3℃ to adjust the pH to 7.60-7.80 to obtain the aqueous phase. The organic phase and the aqueous phase are pumped into a micro mixer at a volume flow ratio of 1:10-15 for impact mixing, with the Reynolds number controlled above 4000, and the effluent is collected to obtain a nano suspension. The nano-suspension was subjected to tangential flow ultrafiltration to remove the organic solvent and replace the dispersion medium with a buffer solution. The retentate was collected and filtered to obtain an aqueous dispersion of the nano-formulation.

[0022] By employing the above technical solution, combined with low-temperature control and confined impingement flow micromixing technology, the problems of easy hydrolysis of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and easy dissociation of charge-transfer complexes were solved. The specific preparation principle includes: (1) Control the temperature of the organic phase and the aqueous phase at 1-3℃ to reduce the rate constant of the hydrolysis reaction of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and maintain its structural integrity; (2) A turbulent field with a Reynolds number of over 4000 is constructed by a micro-mixer to achieve millisecond-level mixing of the two phases, resulting in high supersaturation, which makes the nucleation rate of methylene blue and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone much greater than the crystal growth rate. (3) At the moment of mixing, the extended poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer rapidly encapsulates the nascent charge transfer complex micronucleus, using hydrophobic and electrostatic interactions to stabilize the metastable charge transfer complex in the dense polymer core, blocking water molecule contact and preventing complex dissociation. (4) Low-temperature tangential flow ultrafiltration technology was used to remove anhydrous N,N-dimethylformamide, and purification was achieved under the premise of avoiding drug leakage and structural rearrangement, so as to obtain nano-formulation with uniform particle size and stable chemical properties.

[0023] This invention provides a near-infrared photoresponsive nano-formulation for synergistic photothermal / photodynamic ablation of arterial plaques. It possesses the following beneficial effects: 1. This invention utilizes the intermolecular charge transfer interaction between the electron donor methylene blue and the electron acceptor 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in the hydrophobic microregion of a polymer to construct a charge transfer complex with a near-infrared absorption peak of 780-850 nm. Under 808 nm laser excitation, the complex generates thermal energy through non-radiative transitions and simultaneously induces electron transfer to generate superoxide anion free radicals, achieving a synergistic output of photothermal and type I photodynamic effects. The near-infrared light-responsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques avoids the oxygen dependence of traditional photosensitizers and improves the ablation efficiency of deep arterial plaques by utilizing the deep penetration capability of near-infrared light in tissues.

[0024] 2. The nano-formulation prepared in this invention utilizes the pKa characteristics of the carboxyl groups on the side chains of poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer to achieve intelligent response to the lesion microenvironment. Under normal physiological pH 7.4, the carboxyl groups on the side chains are highly ionized to form a negative charge layer, maintaining the stability of the nanostructure and inhibiting drug activity. Under the slightly acidic pH 6.5 environment of arterial plaques, the protonation of the carboxyl groups on the side chains leads to polymer conformational contraction and charge flipping, specifically activating photothermal and photodynamic properties at the lesion site, reducing non-specific damage to normal vascular tissue.

[0025] 3. This invention employs low-temperature confined impinging flow micromixing technology combined with tangential flow ultrafiltration to solve the preparation challenges of easily hydrolyzed 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and easily dissociated charge-transfer complexes. By controlling a low-temperature environment of 1-3℃ and a Reynolds number greater than 4000, millisecond-level mixing of the organic and aqueous phases is achieved, rapidly capturing and anchoring the metastable charge-transfer complex within the polymer core. The near-infrared photothermal / photodynamic synergistic ablation nanoparticle formulation for arterial plaques exhibits uniform particle size distribution and maintains long-term chemical stability in aqueous media, meeting the quality requirements for intravenous injection formulations. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Main raw materials and reagents: The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0028] Methoxylated polyethylene glycolamine (mPEG-NH2, CAS No.: 80506-64-5, linear structure, average molecular weight Mn of 2000 Da and 5000 Da, dispersity ≤1.05); dialysis bags (molecular weight cutoff MWCO of 3500 Da, 8000-14000 Da and 50 kDa, respectively).

[0029] Preparation Examples 1-4: Preparation Example 1: This preparation example provides a method for preparing poly(L-glutamic acid)-g-methoxy polyethylene glycol (PLG-g-mPEG) with a grafting rate of 15%, comprising the following steps: (1) Synthesis of poly(γ-benzyl-L-glutamate) (PBLG): In a dry, nitrogen-filled glove box, 5.0 g of γ-benzyl-L-glutamate-N-carboxylic anhydride (BLG-NCA, approximately 19.0 mmol) was dissolved in 100 mL of anhydrous 1,4-dioxane. 32 mg of n-hexylamine (approximately 0.316 mmol) was added as an initiator to maintain a monomer-to-initiator molar ratio of 60:1. The reaction was stirred at 30 °C for 72 hours. After the reaction was completed, the reaction solution was slowly added dropwise to 1000 mL of anhydrous ethanol to settle. The white flocculent precipitate was collected by filtration and dried under vacuum for 24 hours to obtain the PBLG homopolymer.

[0030] (2) Preparation of poly(L-glutamic acid) (PLG): 3.0 g of the prepared PBLG was dissolved in 30 mL of trifluoroacetic acid (TFA), and 10 mL of 33 wt% HBr / acetic acid solution was added under ice bath cooling. The mixture was stirred at room temperature for 1 hour to remove the side chain protecting groups. The reaction solution was poured into excess anhydrous diethyl ether to precipitate, and the solid was collected by centrifugation. The solid was redissolved in saturated sodium bicarbonate aqueous solution and stirred for 1 hour to fully neutralize and hydrolyze the residual ester groups. Then, the pH was adjusted to 3.0 with 1 M HCl solution, at which point a large amount of white precipitate (acidic form of PLG) was produced. The precipitate was collected by centrifugation, washed three times with deionized water to remove inorganic salts, and freeze-dried to obtain PLG (acidic form) solid.

[0031] (3) Synthesis of PLG-g-mPEG graft copolymer: 1.0 g of the above-mentioned PLG acidic solid (glutamic acid repeating unit MW≈129, containing about 7.75 mmol of carboxyl groups) was dissolved in 50 mL of anhydrous DMSO, and 0.22 g of EDC·HCl (about 1.15 mmol) and 0.13 g of NHS (about 1.13 mmol) were added. The mixture was activated at room temperature for 1 hour. Subsequently, 2.33 g of methoxy polyethylene glycolamine (mPEG-NH2, Mn=2000 Da, about 1.16 mmol) was added to make the molar ratio of [-NH2] / [-COOH] about 0.15. After reacting at room temperature for 48 hours, the reaction solution was transferred to a dialysis bag with a MWCO 8000-14000 Da. Dialysis was performed against deionized water for 5 days, with the dialysate changed every 12 hours. Finally, the solution was freeze-dried to obtain the target product PLG-g-mPEG. The PEG grafting rate was calculated to be 14.8% by 1H NMR spectroscopy. Preparation Example 2: This preparation example provides a method for preparing poly(L-glutamic acid)-g-methoxy polyethylene glycol (PLG-g-mPEG) with a grafting rate of 25%, comprising the following steps: (1) The synthesis steps of PBLG and PLG are the same as those in steps (1) to (2) of Preparation Example 1, to obtain PLG (acidic) solid.

[0032] (2) Synthesis of PLG-g-mPEG graft copolymer: 1.0 g of PLG acidic solid (containing about 7.75 mmol of carboxyl groups) was dissolved in 50 mL of anhydrous DMSO, and 0.37 g of EDC·HCl (about 1.93 mmol) and 0.22 g of NHS (about 1.91 mmol) were added. The mixture was activated at room temperature for 1 hour. Then, 3.88 g of methoxy polyethylene glycolamine (mPEG-NH2, Mn=2000 Da, about 1.94 mmol) was added to make the molar ratio of [-NH2] / [-COOH] about 0.25. The reaction conditions and post-treatment were the same as in step (3) of Preparation Example 1. The target product was obtained, and the PEG grafting rate was calculated to be 24.5% by 1H NMR spectroscopy.

[0033] Preparation Example 3: This preparation example provides a method for preparing poly(L-glutamic acid)-g-methoxy polyethylene glycol (PLG-g-mPEG) with a grafting rate of 5%, comprising the following steps: (1) The synthesis steps of PBLG and PLG are the same as those in steps (1) to (2) of Preparation Example 1, to obtain PLG (acidic) solid.

[0034] (2) Synthesis of PLG-g-mPEG graft copolymer: 1.0 g of PLG acidic solid (containing about 7.75 mmol of carboxyl groups) was dissolved in 50 mL of anhydrous DMSO, and 0.075 g of EDC·HCl (about 0.39 mmol) and 0.045 g of NHS (about 0.39 mmol) were added. The mixture was activated at room temperature for 1 hour. Then, 0.78 g of methoxy polyethylene glycolamine (mPEG-NH2, Mn=2000 Da, about 0.39 mmol) was added to make the molar ratio of [-NH2] / [-COOH] about 0.05. The reaction conditions and post-treatment were the same as in step (3) of Preparation Example 1. The target product was obtained, and the PEG grafting rate was calculated to be 5.2% by 1H NMR spectroscopy.

[0035] Preparation Example 4: This preparation example provides a method for preparing a non-pH-responsive methoxy polyethylene glycol-b-poly(γ-benzyl-L-glutamate) (mPEG-b-PBLG) amphiphilic block copolymer (used as a comparative example), comprising the following steps: In a dry, nitrogen-filled glove box, 1.0 g of methoxy polyethylene glycol amine (mPEG-NH2, Mn=5000Da, 0.2 mmol) as a macromolecular initiator is dissolved in 20 mL of anhydrous 1,4-dioxane. 2.63 g of BLG-NCA (approximately 10.0 mmol) is added to maintain a monomer to initiator molar ratio of 50:1. The reaction is stirred at 35°C for 72 hours. After the reaction is complete, the reaction solution is slowly added dropwise to excess anhydrous diethyl ether to settle, the white precipitate is collected by filtration, and vacuum dried to obtain the mPEG-b-PBLG block copolymer. This polymer has benzyl ester side chains, no free carboxyl groups, and does not possess pH-responsive charge-flipping ability.

[0036] Examples 1-6: Example 1: This embodiment provides a near-infrared photoresponsive nano-formulation for synergistic photothermal / photodynamic ablation of arterial plaques. The specific preparation steps are as follows: In the following embodiments, all "parts" refer to parts by weight. The proportion of each component is calculated based on parts by weight. When liquid solvents are involved, the ratio of liquid solvent volume (mL) to solid weight parts (g) is 1:1 (i.e., when 1 part is 1g, 1 volume part is 1mL). In actual operation, the ratio can be scaled up according to the production scale.

[0037] (1) Preparation of the low-temperature organic phase (W1): Under strictly light-protected conditions, accurately weigh 32.0 parts of methylene blue (MB, about 0.1 mmol) and 27.2 parts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, about 0.12 mmol), making the molar ratio of MB to DDQ 1:1.2. Add the above solids to 10 mL of anhydrous N,N-dimethylformamide (DMF) and sonicate for 5 minutes in an ultrasonic cleaner until the solids are completely dissolved, forming a clear, deep blue-purple solution. After filtering the solution through a 0.22 μm organic PTFE syringe filter to remove potential insoluble particles, immediately place it in an ice-water bath to cool it, stabilizing the solution temperature at 2℃±1℃.

[0038] (2) Preparation and adjustment of the low-temperature aqueous phase (W2): Accurately weigh 200 parts of the PLG-g-mPEG (PEG grafting rate 15%) graft copolymer obtained from Preparation Example 1, add it to 100 mL of deionized water, and stir magnetically until completely dissolved to prepare a 0.2% (w / v) polymer aqueous solution. Cool the solution to 2℃±1℃ in an ice-water bath. While maintaining the low temperature and magnetic stirring, immerse the precision pH meter probe in the solution and slowly add 0.1M NaOH solution to precisely adjust the pH of the aqueous phase to 7.80. After adjustment, continue to keep it in the ice-water bath for later use.

[0039] (3) Instantaneous mixing and self-assembly: A microfluidic reaction device was constructed using a stainless steel T-type micromixer (channel inner diameter 0.5 mm). The inlet pipes of two high-pressure constant flow pumps were inserted into the low-temperature organic phase (W1) and low-temperature aqueous phase (W2) storage bottles, respectively, and the outlet pipes were connected to the two inlets of the T-type mixer. The flow rate of the organic phase pump was set to 15 mL / min, and the flow rate of the aqueous phase pump was set to 150 mL / min (volume flow ratio of 1:10, total flow rate of 165 mL / min, calculated Reynolds number Re>4000). The pumps were started, and the two-phase fluids underwent violent turbulent collision mixing in the micromixer chamber. The effluent was directly introduced into a receiving beaker containing 20 mL of 2℃ deionized water under magnetic stirring at 500 rpm through a short polytetrafluoroethylene tube, resulting in a dark-colored nano-suspension.

[0040] (4) Purification and Post-processing: After the reaction, the nano-suspension in the receiving beaker was immediately transferred to the storage tank of the tangential flow ultrafiltration (TFF) system. The system was equipped with a modified polyethersulfone (mPES) membrane with a molecular weight cutoff (MWCO) of 50 kDa. The ultrafiltration pump was started, and the temperature of the feed solution in the storage tank and pipeline was maintained at 4-8℃ by circulating the solution through an external chiller. A pH 7.4 phosphate buffer (PBS, 0.01 M) pre-cooled to 4℃ was used as the dialysis wash solution, and filtration was performed in constant volume mode, with a replacement volume of 10 times the original sample volume, until no DMF residue was detected in the permeate by gas chromatography. The retentate was collected and filtered through a 0.22 μm PES aqueous filter membrane for sterilization to obtain an aqueous dispersion of the target nano-formulation, which was stored at 4℃ in the dark.

[0041] Example 2: This embodiment provides a near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques. The specific preparation steps are as follows: (1) Preparation of the low-temperature organic phase (W1): Under light-protected conditions, accurately weigh 32.0 parts of methylene blue (MB, about 0.1 mmol) and 22.7 parts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, about 0.1 mmol), so that the molar ratio of MB to DDQ is 1:1.0. Dissolve the solid in 10 mL of anhydrous DMF, sonicate to aid dissolution, filter through a 0.22 μm organic filter, and cool to 2 °C in an ice-water bath for later use.

[0042] (2) Preparation and adjustment of low temperature aqueous phase (W2): Same as step (2) in Example 1, use the PLG-g-mPEG obtained in Preparation Example 1 to prepare a 0.2% (w / v) aqueous solution, adjust the pH to 7.80, and keep it at 2℃.

[0043] (3) Instantaneous mixing and self-assembly: The operation is the same as step (3) in Example 1. Set the flow rate of the organic phase to 15 mL / min and the flow rate of the aqueous phase to 150 mL / min, and mix them by impact in a T-type mixer.

[0044] (4) Purification and post-processing: The operation is the same as step (4) in Example 1. The organic solvent is removed by tangential flow ultrafiltration and replaced with pH 7.4 PBS buffer.

[0045] Example 3: This embodiment provides a near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques. The specific preparation steps are as follows: (1) Preparation of the low-temperature organic phase (W1): Under light-protected conditions, accurately weigh 32.0 parts of methylene blue (MB, about 0.1 mmol) and 34.0 parts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, about 0.15 mmol), so that the molar ratio of MB to DDQ is 1:1.5. Dissolve the solid in 10 mL of anhydrous DMF, sonicate to aid dissolution, filter through a 0.22 μm organic filter, and cool to 2 °C in an ice-water bath for later use.

[0046] (2) Preparation and adjustment of low temperature aqueous phase (W2): Same as step (2) in Example 1, use the PLG-g-mPEG obtained in Preparation Example 1 to prepare a 0.2% (w / v) aqueous solution, adjust the pH to 7.80, and keep it at 2℃.

[0047] (3) Instantaneous mixing and self-assembly: The operation is the same as step (3) in Example 1. Set the flow rate of the organic phase to 15 mL / min and the flow rate of the aqueous phase to 150 mL / min, and mix them by impact in a T-type mixer.

[0048] (4) Purification and post-processing: The operation is the same as step (4) in Example 1. The organic solvent is removed by tangential flow ultrafiltration and replaced with pH 7.4 PBS buffer.

[0049] Example 4: This embodiment provides a near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques. The specific preparation steps are as follows: (1) Preparation of low-temperature organic phase (W1): Same as step (1) in Example 1, weigh 32.0 parts of MB and 27.2 parts of DDQ and dissolve them in 10 mL of anhydrous DMF and cool to 2°C.

[0050] (2) Preparation and adjustment of the low-temperature aqueous phase (W2): Accurately weigh 200 parts of the PLG-g-mPEG (PEG grafting rate 25%) graft copolymer obtained from Preparation Example 2, dissolve it in 100 mL of deionized water, and prepare a 0.2% (w / v) polymer aqueous solution. Cool the solution to 2°C in an ice-water bath, and precisely adjust the pH to 7.80 by adding 0.1 M NaOH solution dropwise while stirring. Keep it at a low temperature until use.

[0051] (3) Instantaneous mixing and self-assembly: The operation is the same as step (3) in Example 1. The above-mentioned high grafting rate polymer solution is used as the aqueous phase and mixed with the organic phase in a T-type mixer at a volume flow ratio of 10:1 (150 mL / min: 15 mL / min).

[0052] (4) Purification and post-processing: The operation is the same as step (4) in Example 1.

[0053] Example 5: This embodiment provides a near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques. The specific preparation steps are as follows: (1) Preparation of low-temperature organic phase (W1): Same as step (1) in Example 1, weigh 32.0 parts of MB and 27.2 parts of DDQ and dissolve them in 10 mL of anhydrous DMF and cool to 2°C.

[0054] (2) Preparation and adjustment of the low-temperature aqueous phase (W2): Accurately weigh 200 parts of the PLG-g-mPEG (PEG grafting rate 15%) polymer obtained from Preparation Example 1 and dissolve it in 100 mL of deionized water. Cool the solution to 2°C in an ice-water bath, and use a precision pH meter to monitor the pH value while stirring. Add 0.1 M NaOH solution dropwise to precisely adjust the pH value to 7.60. Keep it at a low temperature until use.

[0055] (3) Instantaneous mixing and self-assembly: The operation is the same as step (3) in Example 1. The aqueous phase adjusted to pH 7.6 above is used for impact mixing with the organic phase.

[0056] (4) Purification and post-processing: The operation is the same as step (4) in Example 1.

[0057] Example 6: This embodiment provides a near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoformulation for arterial plaques. The specific preparation steps are as follows: (1) Preparation of low-temperature organic phase (W1): Same as step (1) in Example 1, weigh 32.0 parts of MB and 27.2 parts of DDQ and dissolve them in 10 mL of anhydrous DMF and cool to 2°C.

[0058] (2) Preparation and adjustment of low temperature aqueous phase (W2): Same as step (2) in Example 1, use the PLG-g-mPEG obtained in Preparation Example 1 to prepare a 0.2% (w / v) aqueous solution, adjust the pH to 7.80, and keep it at 2℃.

[0059] (3) Instantaneous mixing and self-assembly: A stainless steel T-type micromixer was used. The flow rate of the organic phase W1 was set to 10 mL / min, and the flow rate of the aqueous phase W2 was set to 150 mL / min (the volumetric flow rate ratio was adjusted to 1:15 to increase the instantaneous supersaturation during precipitation and reduce the proportion of organic solvent). The pump was started, and the two-phase fluids were mixed by high-speed impact in the micromixer. The effluent was introduced into a pre-cooled receiving container.

[0060] (4) Purification and post-processing: The operation is the same as step (4) in Example 1. The organic solvent is removed by tangential flow ultrafiltration and replaced with pH 7.4 PBS buffer.

[0061] Comparative Examples 1-7: Comparative Example 1: Compared with Example 1, the difference is that component B (DDQ) is not added when preparing the organic phase (W1), and only 32.0 parts of methylene blue (MB) are dissolved. The rest of the steps and parameters are the same.

[0062] Comparative Example 2: Compared with Example 1, the difference is that component A (MB) is not added when preparing the organic phase (W1), only 27.2 parts of DDQ are dissolved, and the rest of the steps and parameters are the same.

[0063] Comparative Example 3: Compared with Example 1, the difference is that when preparing the organic phase (W1), an equimolar amount of the conventional photothermal agent indocyanine green (ICG) was used to replace component B (DDQ) in an attempt to construct a physical co-encapsulation system of MB and ICG. The remaining steps and parameters are the same.

[0064] Comparative Example 4: Compared with Example 1, the difference is that the non-pH responsive block copolymer (mPEG-b-PBLG) obtained in Preparation Example 4 was used instead of PLG-g-mPEG when preparing the aqueous phase (W2). The side chain of this polymer is a benzyl ester structure with no free carboxyl groups. The remaining steps and parameters are the same.

[0065] Comparative Example 5: Compared with Example 1, the difference lies in the change of temperature control throughout the process. Specifically, the organic phase preparation, aqueous phase preparation, mixing process, and ultrafiltration process are all carried out at room temperature of 25°C, without the use of ice-water baths and cold water circulation for temperature control, and the remaining steps and parameters are the same.

[0066] Comparative Example 6: Compared to Example 1, the difference lies in the change of mixing method. Specifically, instead of using a T-type micro mixer, a conventional dropwise addition method is adopted. That is, under magnetic stirring at 500 rpm, the organic phase (W1) is slowly added dropwise to the aqueous phase (W2) beaker at a rate of 1.5 mL / min using a syringe (the dropwise addition process lasts for about 10 minutes), and the remaining steps and parameters are the same.

[0067] Comparative Example 7: Compared to Example 1, the difference lies in the change of the pH adjustment parameter of the aqueous phase. Specifically, when preparing the aqueous phase (W2), the pH value is adjusted to 6.50 (simulating a slightly acidic environment, close to or slightly higher than PLGpKa, but lower than the strongly negatively charged range required for electrostatic adsorption), and the remaining steps and parameters are the same.

[0068] Test Examples 1-6: Test Example 1: Spectroscopic Verification of Charge-Transfer Complex Formation Experimental steps: (1) Sample preparation: The aqueous dispersion of the nano-formulation prepared in Example 1 was diluted with deionized water to a final concentration of methylene blue (MB) equivalent to 20 μg / mL. The preparation solutions of Comparative Example 1 (without DDQ) and Comparative Example 2 (without MB) were also diluted with deionized water to the equivalent molar concentration of the corresponding components. The raw materials methylene blue (MB) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) were accurately weighed and dissolved in N,N-dimethylformamide (DMF) to prepare single-component control solutions with the same molar concentration as in Example 1.

[0069] (2) Baseline calibration: Turn on the UV-Vis and near-infrared spectrophotometer (UV-3600Plus) and preheat for 30 minutes. Use cuvettes filled with deionized water (for aqueous samples) and DMF (for organic samples) to perform dual-beam baseline scanning and background subtraction.

[0070] (3) Spectral Scanning: Place each of the above-mentioned sample solutions in a quartz cuvette, set the scanning wavelength range to 200 nm to 1000 nm, the sampling interval to 1 nm, and the scanning speed to medium. Record the position of the characteristic absorption peak of each sample, i.e., the maximum absorption wavelength (denoted as λ). ), and simultaneously record the absorbance values ​​in the 600nm to 900nm region (especially at 808nm).

[0071] Experimental data: Table 1 Comparison of spectral characteristic parameters and absorbance at 808 nm for each component and formulation Experimental conclusion: Based on the data in Table 1 and the spectral scanning results, the main absorption peak of the raw material methylene blue (MB) in DMF solution and the single-supported system of Comparative Example 1 is concentrated in the visible light region around 662-665 nm. This is due to the typical π transition of the phenothiazine chromophore. Furthermore, the absorbance at 808 nm is extremely low (A<0.06), indicating that the MB molecule itself does not possess the ability to respond to 808 nm near-infrared laser light. Similarly, the electron acceptor DDQ shows almost no absorption in the visible and near-infrared regions.

[0072] The nanoformulation of Example 1 exhibited a significant change in spectrum. In addition to retaining the inherent visible light absorption peak of MB, a new, broad, and strong absorption band appeared in the long wavelength region from 780 nm to 850 nm, with an absorbance at 808 nm (0.687) that was approximately 11.8 times higher than that of Comparative Example 1 (0.058). This spectral redshift was not a simple physical superposition of the spectra of the components, but rather stemmed from the formation of a charge-transfer complex (CTC) between the electron donor MB and the electron acceptor DDQ within the nano-confined space.

[0073] Mechanistically, orbital hybridization occurs between the highest occupied molecular orbital (HOMO) of the MB molecule and the lowest unoccupied molecular orbital (LUMO) of the DDQ molecule, significantly reducing the band gap and creating a low-energy intermolecular charge transfer transition channel. Comparative example 5 data (A808 is only 0.143) further confirms that without cryogenic control and a confined impact flow process, DDQ is highly susceptible to hydrolysis or fails to form a tight packing structure with MB, resulting in the inability to effectively construct the CTC energy level.

[0074] In summary, Example 1 successfully constructed a CTC core with near-infrared absorption characteristics. This spectroscopic evidence directly supports the feasibility of using an 808nm laser to excite the CTC state for photothermal / photodynamic synergistic therapy.

[0075] Test Example 2: pH-responsive charge flipping and particle size change test Experimental steps: (1) Prepare phosphate-buffered saline (PBS, 10 mM) with different pH values ​​as simulation media. The pH value of the buffer was precisely adjusted using 1 M HCl or 1 M NaOH solution to obtain a pH 7.4 solution simulating the physiological blood environment, a pH 6.5 solution simulating the slightly acidic environment of atherosclerotic plaques, and a pH 5.0 solution simulating the intracellular lysosomal environment.

[0076] (2) Take an appropriate amount of the aqueous dispersion of the nano-formulation prepared in Example 1 and add it to the three groups of PBS buffers with different pH values ​​to dilute the final concentration of the nanoparticles to 0.5 mg / mL. Incubate the sample in a constant temperature shaker at 37°C for 4 hours to reach the equilibrium state of protonation / deprotonation.

[0077] (3) After incubation, samples from each group were added to disposable folded capillary sample cells (for potential testing) and polystyrene cuvettes (for particle size testing). Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements were performed using a Zetasizer Nano ZS90 instrument at 25°C. Each sample was measured three times, and the hydrodynamic diameter (Z-Average), polydispersity index (PDI), and Zeta potential data were recorded.

[0078] Experimental data: Table 2. Particle size distribution and Zeta potential test data of the nanoformulation in Example 1 under different pH conditions.

[0079] Experimental conclusion: Based on the test data in Table 2, the nanoformulation prepared in Example 1 exhibited significant pH-dependent structural evolution characteristics, confirming the effectiveness of the PLG layer shielding and acid-responsive activation mechanism.

[0080] Under simulated normal physiological conditions at pH 7.4, the nano-formulations exhibited a small hydrodynamic diameter (approximately 92-94 nm) and a narrow particle size distribution (PDI < 0.12), while maintaining a strongly negative Zeta potential of around -26 mV. This is because the carboxyl groups on the PLG side chain are in a highly ionized state (-COO) at this pH value. ⁻ The polymer chains extend and form a dense hydration layer, which imparts good colloidal stability to the particles through electrostatic repulsion. This highly negatively charged surface can effectively reduce the non-specific adsorption of plasma proteins, which is beneficial for prolonging the blood circulation time of the formulation in vivo.

[0081] When the ambient pH decreased to 6.5 (simulating a plaque microenvironment), the Zeta potential changed drastically, rising significantly from -25.8 mV to -5.2 mV, approaching electroneutrality. This data indicates an increase in the protonation degree of the carboxyl groups in the PLG side chains, resulting in the failure of the electrostatic shielding layer. Accompanying the decrease in potential, the hydrodynamic diameter increased to 148.6 nm and the PDI increased significantly (0.264), indicating that after losing electrostatic repulsion protection, the nanoparticles underwent a certain degree of hydrophobic aggregation or swelling and loosening of the polymer shell.

[0082] When the pH was further reduced to 5.0, the Zeta potential flipped to a positive value (+11.7 mV), and the particle size increased dramatically to over 450 nm. This is because the PLG layer was completely protonated and detached or precipitated from the surface, exposing the positively charged MB / DDQ core inside.

[0083] In summary, this formulation maintains its structural integrity and electronegative stealth state at pH 7.4, while undergoing charge reversal and structural dissociation in the acidic microenvironment of plaques. This transformation of physicochemical properties not only enhances the electrostatic interaction between nanoparticles and lesion sites (typically negatively charged cell membranes or matrix), promoting deeper drug penetration and cellular uptake, but also provides a structural basis for the subsequent specific restoration of photothermal / photodynamic activity at the lesion site.

[0084] Test Example 3: Effect of Preparation Process on Drug Encapsulation Stability and Chemical Stability Experimental steps: (1) Take 1 mL of each of the freshly prepared nano-formulation stock solutions from Example 1 (low temperature + confined impingement flow), Comparative Example 5 (room temperature preparation), and Comparative Example 6 (conventional dropwise addition method). Add 9 mL of acetonitrile to demulsify and vortex thoroughly for 2 minutes to disintegrate the polymer micelles and release the internal loadings. After filtration through a 0.22 μm microporous membrane, inject the filtrate into a high-performance liquid chromatograph (HPLC). Use a C18 reversed-phase column, with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, gradient elution, and a detection wavelength set to 280 nm. Calculate the retention rate of DDQ and the relative content of its main hydrolysis product (2,3-dichloro-5,6-dicyano-1,4-benzenediol, HQ) in each sample based on the standard curve to assess chemical stability.

[0085] (2) Take 5 mL of each of the three preparations mentioned above and place them into dialysis bags with a molecular weight cutoff (MWCO) of 3500 Da, and tie both ends tightly. Immerse the dialysis bags in 50 mL of release medium (PBS buffer, pH 7.4, containing 0.5% Tween-80 to maintain leak conditions). Place the beaker in a 37°C constant temperature shaker and set the speed to 100 rpm to shake in the dark.

[0086] (3) At 0.5, 2, 12, and 24 hours after the start of the release experiment, 2 mL of solution was drawn from the release medium outside the dialysis bag, and an equal volume of fresh preheated medium was immediately added. The absorbance of methylene blue (MB) in the release solution was measured at 664 nm using a UV spectrophotometer, and the cumulative release percentage of MB was calculated using a standard curve to evaluate the physical locking ability of the nanostructure for the drug and its anti-burst release performance. Each sample was measured in triplicate.

[0087] Experimental data: Table 3 Comparison of chemical stability and physical release characteristics of active ingredients under different preparation processes

[0088] Experimental conclusion: Based on the chromatographic analysis and in vitro release data in Table 3, the thermodynamic and kinetic parameters of the preparation process have a decisive influence on the quality attributes of the final product.

[0089] Regarding chemical stability, the hydrolysis byproduct (HQ) content of DDQ in Example 1 was only 1.82%, while that of Comparative Example 5, prepared at room temperature, was as high as 42.67%. The strong electron-withdrawing groups in the DDQ molecule make it extremely sensitive to water molecules, readily undergoing nucleophilic substitution reactions to generate phenolic derivatives without CTC activity. Example 1 employed a low-temperature process of 0-4°C, which significantly reduced the hydrolysis reaction rate constant according to the Arrhenius equation; combined with confined impingement flow technology, the microscopic mixing time between the organic and aqueous phases was shortened to the millisecond level, allowing the hydrophobic CTC cores to be instantly encapsulated by the polymer shell upon contact with water molecules, physically blocking water attack. In contrast, although Comparative Example 6 also used a low temperature, due to the low mixing efficiency of the dropwise method (macroscopic mixing), DDQ was exposed to the aqueous phase for a long time before nucleation, resulting in an HQ content of 16.94%.

[0090] Regarding physical encapsulation stability, Example 1 exhibited excellent sustained-release characteristics, with a cumulative release rate of less than 10% after 24 hours. This is attributed to the high supersaturation created by high Reynolds number (Re>4000) turbulence, which spurred explosive nucleation of nanoparticles, forming dense and uniformly sized nanospheres that effectively confined the small molecule drug. In contrast, Comparative Example 6, with its low supersaturation environment caused by the conventional dropwise addition method, resulted in a nucleation rate lower than the crystal growth rate. The formed particles had a loose structure, wide particle size distribution, and a significant burst release effect (22.51% release in 0.5 hours), indicating that the drug was not effectively encapsulated in the polymer core but rather adsorbed on the surface or in a loose aggregated state.

[0091] In summary, the low-temperature coupled process with confined impact flow is a necessary condition for ensuring the integrity of the DDQ chemical structure and constructing a dense core-shell nanostructure.

[0092] Test Example 4: pH-responsive photothermal conversion performance (PTT) comparison test Experimental steps: (1) Sample preparation: Take the stock solutions of the formulations from Example 1 (intelligent responsive type), Comparative Example 3 (ICG physically loaded type), and Comparative Example 4 (non-responsive polymer type), respectively. Dilute each group of samples with phosphate-buffered saline (PBS) at pH 7.4 and pH 6.5, and uniformly adjust the molar concentration of the effective photothermal component (MB or ICG) to 50 μM. Set up pure PBS buffer as a blank control group. Add the prepared samples to quartz cuvettes, and prepare 3 replicates for each group.

[0093] (2) Experimental setup: Place the cuvette containing the sample on a magnetic stirring table and maintain low-speed stirring to ensure uniform heating. Fix the 808nm near-infrared semiconductor laser probe, adjust the spot diameter to completely cover the liquid surface, and calibrate the output power density to 1.0W / cm² using an optical power meter. ²Fix the FLIR infrared thermal imager so that its lens is aligned with the center of the side of the cuvette for real-time temperature data recording.

[0094] (3) Photothermal testing process: The laser was turned on and continuously irradiated for 300 seconds (5 minutes). The infrared thermal imager was set to record the highest temperature point every 10 seconds. After irradiation, the laser was turned off and the temperature change during the natural cooling process of the solution was recorded. For the groups examining photostability (Example 1 and Comparative Example 3), the above heating-cooling cycle was repeated 3 times after cooling to room temperature.

[0095] Experimental data: Table 4. Photothermal temperature rise characteristics of each formulation under different pH conditions (end of 5 minutes)

[0096] Experimental conclusion: Based on the photothermal test data analysis in Table 4, the nano-formulation prepared in Example 1 exhibited significant pH-dependent photothermal conversion behavior, verifying the intelligent response mechanism of cyclic shielding and lesion activation.

[0097] In a pH 7.4 environment simulating physiological blood circulation, the temperature increment (ΔT) in Example 1 was only 4.2°C, and the final temperature did not exceed 30°C. This is because, under these pH conditions, the carboxyl groups of the PLG side chains are highly ionized, and the negatively charged polymer segments are tightly adsorbed onto the CTC core surface through strong electrostatic interactions. This dense polymer shell restricts the movement of water molecules and energy transfer, and the high-density charge environment may inhibit the heat generation efficiency of the excited state of CTCs by inducing changes in non-radiative decay pathways, thus keeping them in a photothermal quiescent or locked state. This characteristic is of great significance for reducing systemic side effects during treatment and avoiding thermal damage to normal vascular endothelium.

[0098] When the environment was adjusted to pH 6.5, the same as the simulated plaque, the ΔT in Example 1 surged to 27.8°C, eventually reaching 52.7°C, sufficient to induce irreversible thermal ablation of the biological tissue. The mechanism lies in the carboxyl grouping under acidic conditions, which reduces the charge density of the PLG shell, weakens electrostatic adsorption, and causes the polymer layer to swell or partially dissociate, thus removing the shielding effect on the CTC core and restoring its efficient photothermal conversion capability.

[0099] In contrast, Comparative Example 4 (non-responsive mPEG-b-PBLG) exhibited a strong heating effect at both pH 7.4 and pH 6.5 (ΔT 24.8℃ and 24.9℃, respectively). Because its side chain is a benzyl ester, it lacks pH-sensitive ionizing groups and cannot form a shielding layer through electrostatic interaction at physiological pH, resulting in the formulation remaining in a photothermal on-state. This lack of selectivity in photothermal agents poses a significant risk of off-target thermal damage in clinical applications.

[0100] Furthermore, although Comparative Example 3 (ICG group) also showed a good heating effect (ΔT approximately 18.8℃), its heating rate slowed down significantly after 3 minutes, and the final temperature was lower than that of Example 1 (activated state). This not only reflects that the photothermal conversion efficiency of ICG itself is lower than that of CTC, but also suggests that traditional organic dyes are prone to photobleaching under strong laser irradiation, leading to performance degradation. In contrast, the CTC structure of this invention has superior photothermal stability through an intermolecular charge transfer mechanism.

[0101] Test Example 5: Identification of Reactive Oxygen Species (ROS) Generation Types and pH Dependence Test Experimental steps: (1) Preparation of detection system: A 50 μM anhydrous ethanol solution of 1,3-diphenylisobenzofuran (DPBF) was prepared as a reactive oxygen species capture probe. The nano-formulation prepared in Example 1 was diluted with PBS buffer at pH 7.4 and pH 6.5 respectively to make the final concentration of methylene blue (MB) uniform at 10 μg / mL.

[0102] (2) Quenching agent grouping: The experiment was divided into four groups to identify the ROS mechanism: Group A (pH 7.4 control group): Formulation of Example 1 (pH 7.4) + DPBF.

[0103] Group B (pH 6.5 Activation Group): Formulation of Example 1 (pH 6.5) + DPBF.

[0104] Group C (singlet oxygen quenching group): Preparation of Example 1 (pH 6.5) + DPBF + sodium azide (NaN3, final concentration 50 μM, specific scavenging).

[0105] Group D (Superoxide anion quenching group): Preparation of Example 1 (pH 6.5) + DPBF + superoxide dismutase (SOD, final concentration 200 U / mL, specific clearance).

[0106] (3) Irradiation and Detection: The above mixture was placed in a quartz cuvette and allowed to stand in the dark for 2 minutes to reach adsorption equilibrium. Then, an 808nm laser (power density 1.0W / cm²) was used. 2The sample was vertically irradiated. Irradiation was paused every 60 seconds, and the sample was immediately placed in a UV-Vis spectrophotometer to measure the absorbance of DPBF at 410 nm. The total experimental time was 6 minutes. The relative absorbance (A / A0) at each time point was calculated based on the initial absorbance (A0) to reflect the degradation rate of DPBF and ROS production. Each experiment was repeated in triplicate.

[0107] Experimental data: Table 5. DPBF degradation kinetics data under different pH conditions and in the presence of quenchers (normalized absorbance A / A0)

[0108] Experimental conclusion: Based on the photodegradation kinetics data of DPBF in Table 5, the nano-formulation prepared in Example 1 exhibits clear pH dependence and type I photodynamic mechanism characteristics in terms of ROS generation.

[0109] First, comparing the data from group A (pH 7.4) and group B (pH 6.5), it is evident that under physiological pH conditions, after 6 minutes of continuous light exposure, the relative absorbance of DPBF remained at 0.942, with a degradation rate of less than 6%. This indicates that under pH 7.4 conditions, the PLG polymer shell is in a deprotonated, extended state, tightly shielding the CTC core through electrostatic interactions, effectively blocking the contact between dissolved oxygen and the photosensitive core or inhibiting the transfer of excited-state energy, thus confirming the photoinertia and safety of the formulation in blood circulation. Conversely, in the simulated lesion environment at pH 6.5, the absorbance of DPBF dropped sharply to 0.185 within 6 minutes, with a degradation rate exceeding 80%, demonstrating that the acidic environment induced charge reversal and dissociation of the polymer shell, exposing the CTC core and restoring its highly efficient photodynamic activity.

[0110] Secondly, the specific types of ROS were further clarified through quencher experiments. In group C, which added the singlet oxygen quencher NaN3, the degradation curve of DPBF (value at the end of 6 minutes was 0.204) was highly similar to that of group B without quencher (0.185), with no statistical difference. This indicates that the proportion of singlet oxygen produced in this system is extremely low, i.e., it does not follow the traditional type II photodynamic mechanism (energy transfer). In group D, which added the superoxide anion quencher SOD, the degradation of DPBF was significantly inhibited, and the absorbance at the end of 6 minutes remained at 0.873, returning to a level close to that of the control group A.

[0111] The above results confirm that the CTC nanoformulation of the present invention exerts its effects primarily through a type I photodynamic pathway (electron transfer) under 808 nm photoexcitation. The mechanism involves intramolecular or intermolecular electron transfer occurring in the charge-transfer complex formed by MB and DDQ in the excited state, directly transferring electrons to surrounding ground-state oxygen molecules to generate highly cytotoxic superoxide anion radicals, rather than relying on oxygen molecule energy level transitions to generate singlet oxygen. Given that arterial plaques are often in a hypoxic state due to abnormal lipid metabolism, this type I mechanism, with its low oxygen dependence (not requiring oxygen molecules to be at a specific energy level) and ability to utilize subsequent cascade reactions (such as the Haber-Weiss reaction to generate hydroxyl radicals), has superior plaque ablation potential compared to traditional type II photosensitizers.

[0112] Test Example 6: In vitro cell safety and specific killing test Experimental steps: (1) Cell Culture and Seeding: Mouse mononuclear macrophage leukemia cells (RAW264.7) were selected as a model of atherosclerotic plaque macrophages. Cells were passaged in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a constant temperature incubator at 37℃ and 5% CO2 saturated humidity. Cells in the logarithmic growth phase were digested with trypsin and counted, then seeded at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1,000 cells per well in 96-well plates, with PBS added to the edge wells to eliminate edge effects, and cultured for 24 hours to allow them to adhere.

[0113] (2) Drug treatment and pH environment simulation: Two sets of DMEM culture media were prepared, and the pH values ​​were adjusted to 7.4 (simulating the physiological environment) and 6.5 (simulating the slightly acidic environment of plaques) using 1M HCl and 1M NaOH, respectively, and sterilized by passing the medium through a 0.22μm filter membrane. The experiment was set as a blank control group (Control), Example 1 group (Ex-1), and Comparative Example 4 group (Comp-4). The old culture medium in the 96-well plate was discarded, and 100μL of fresh culture medium containing the above-mentioned different pH values ​​and corresponding nano-preparations was added. The concentration of each preparation was uniformly calculated based on the effective concentration of methylene blue (MB) and set at 20μg / mL. Each treatment was set with 6 replicates. The cells were incubated in an incubator for 4 hours to allow the cells to take up the nanoparticles through endocytosis.

[0114] (3) Illumination treatment: After incubation, the 96-well plate of the illumination group was placed under an 808nm near-infrared laser, the light spot was adjusted to cover the entire plate, and the power density was set to 1.0W / cm². 2Irradiate continuously for 5 minutes. During irradiation, the ambient temperature must be controlled to avoid nonspecific thermal damage. Cell plates in the dark toxicity group are placed in the dark under the same conditions without laser irradiation. After light treatment, the drug-containing culture medium is aspirated, and the cells are washed twice with PBS to remove unabsorbed drug. 100 μL of fresh, standard pH 7.4 complete culture medium is added, and the cells are cultured for another 24 hours.

[0115] (4) Cell viability assay: Add 10 μL CellCounting Kit-8 (CCK-8) assay reagent to each well, gently shake to mix, and incubate in an incubator for 2 hours. Measure the absorbance (OD value) at 450 nm using a microplate reader. Zero the instrument with cell-free wells and use the control group (control) viability as 100% to calculate the relative cell viability of each experimental group.

[0116] Experimental data: Table 6. Relative survival rate (%) of RAW264.7 cells under different pH and light conditions.

[0117] Experimental conclusion: Based on the CCK-8 cytotoxicity test data in Table 6, the nano-formulation prepared in Example 1 perfectly replicated the designed pH-controlled synergistic killing mechanism at the cellular level, demonstrating excellent biocompatibility and therapeutic specificity.

[0118] First, under light-free conditions (Dark column), regardless of whether the environment was pH 7.4 or pH 6.5, the cell viability of both Example 1 and Comparative Example 4 remained above 94%, with no statistically significant difference compared to the blank control group. This indicates that the PLG-g-mPEG carrier material constructed in this invention and the encapsulated MB / DDQ complex have good biocompatibility in the unactivated state, exhibit no endogenous cytotoxicity, and meet the basic safety requirements for biomedical materials.

[0119] The key difference lies in the light conditions. Under simulated normal physiological conditions (pH 7.4), the cell survival rate in Example 1 group was as high as 91.2%, showing only a very slight decrease in viability. This is attributed to the deprotonation of the carboxyl groups on the PLG side chains, which makes the nanoparticle surface negatively charged, forming a dense polymer hydration layer. This layer reduces non-specific phagocytosis of nanoparticles by cells through electrostatic repulsion (although macrophages have a phagocytic instinct, the highly negatively charged surface can effectively inhibit adsorption in a short time), and also shields the photothermal / photodynamic activity (locked state) of the CTC core. This result confirms that the formulation can effectively avoid accidental damage to normal vascular endothelial cells or non-targeted immune cells in the bloodstream.

[0120] Conversely, in the simulated plaque microenvironment (pH 6.5), the cell survival rate of the Example 1 group plummeted to 14.3% after light irradiation, demonstrating extremely strong cell-killing ability. The acidic environment induced charge flipping in the PLG layer, promoting electrostatic adsorption between nanoparticles and negatively charged cell membranes, enhancing endocytosis efficiency; simultaneously, further acidification within the lysosome (pH 5.0) led to complete dissociation of the polymer shell, fully releasing the CTC core. Under 808nm laser excitation, the released high-temperature heat (PTT) and a large amount of superoxide anions (Type-IPDT) produced a synergistic effect, inducing acute necrosis or apoptosis in macrophages.

[0121] In contrast, Comparative Example 4 (non-pH-responsive mPEG-b-PBLG) showed severe cell death after light exposure at both pH 7.4 (survival rate 21.5%) and pH 6.5 (survival rate 19.8%). Due to the lack of a pH-sensitive shielding layer, the photothermal / photodynamic activity of this formulation cannot be shut off with changes in ambient pH, and will produce severe non-specific phototoxicity if exposed to light during in vivo circulation.

[0122] In summary, the formulation of Example 1 achieves precise spatial regulation of therapeutic efficacy by specifically recognizing the acidic microenvironment of plaques, that is, maintaining low activity in normal tissues to ensure safety, and specifically activating at the lesion site to improve efficacy.

Claims

1. A near-infrared photoresponsive nano-formulation for photothermal / photodynamic synergistic ablation of arterial plaques, characterized in that, It is prepared from raw materials comprising the following parts by weight: 150-250 parts of poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer; 25-40 parts of methylene blue; 20-40 parts of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

2. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 1, characterized in that, In the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer, the grafting rate of polyethylene glycol is 5%-25%.

3. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 1, characterized in that, The poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer was prepared by the following steps: The ring-opening polymerization of γ-benzyl-L-glutamate-N-carboxylic anhydride was initiated by n-hexylamine to obtain poly(γ-benzyl-L-glutamate). Poly(γ-benzyl-L-glutamic acid ester) was deprotected by side chain protecting groups and then acidified and precipitated to obtain poly(L-glutamic acid). The side chain carboxyl groups of poly(L-glutamic acid) were activated using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as activators, and then grafted onto methoxy polyethylene glycol amine.

4. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 3, characterized in that, In the ring-opening polymerization, the molar ratio of γ-benzyl-L-glutamate-N-carboxylic anhydride to the initiator is controlled between 50:1 and 70:1; the removal of the side-chain protecting group is carried out by reacting with HBr / acetic acid solution, and then precipitated and purified by neutralization with sodium bicarbonate aqueous solution and adjustment of pH to 3.0 with hydrochloric acid.

5. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 3, characterized in that, In the grafting reaction, the molar ratio of the amino group of methoxy polyethylene glycolamine to the carboxyl group of poly(L-glutamic acid) is 0.05-0.

25.

6. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 1, characterized in that, The raw materials also include sodium hydroxide for adjusting the pH value and solvents for dissolving the raw materials, the solvents including anhydrous N,N-dimethylformamide and deionized water.

7. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 6, characterized in that, The sodium hydroxide is a 0.1 M aqueous solution, and the amount of sodium hydroxide solution added is the amount required to adjust the pH of the aqueous phase containing the poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer to 7.60-7.

80.

8. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 1, characterized in that, The molar ratio of methylene blue to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is 1:1.0-1.

5.

9. The nano-formulation for near-infrared photoresponsive photothermal / photodynamic synergistic ablation of arterial plaques according to claim 1, characterized in that, The nano-formulation is prepared from raw materials comprising the following parts by weight: 200 parts of poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer; 32 portions of methylene blue; 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone 22.7-34.0 parts.

10. The near-infrared photoresponsive photothermal / photodynamic synergistic ablation nanoformation for arterial plaques according to claim 1, characterized in that, The nano-formulation was prepared through the following steps: Methylene blue and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone were dissolved in anhydrous N,N-dimethylformamide and cooled to 1-3 °C to obtain an organic phase. The poly(L-glutamic acid)-g-methoxy polyethylene glycol graft copolymer was dissolved in water, and sodium hydroxide solution was added dropwise at 1-3℃ to adjust the pH to 7.60-7.80 to obtain the aqueous phase. The organic phase and the aqueous phase are pumped into a micro mixer at a volume flow ratio of 1:10-15 for impact mixing, with the Reynolds number controlled above 4000, and the effluent is collected to obtain a nano suspension. The nano-suspension was subjected to tangential flow ultrafiltration to remove the organic solvent and replace the dispersion medium with a buffer solution. The retentate was collected and filtered to obtain an aqueous dispersion of the nano-formulation.

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