A nano-drug, a preparation method and application thereof

Abstract

The present application belongs to the technical field of nanometer material preparation, and relates to a kind of nanometer medicine, including medicine carrier and active ingredient, medicine carrier adopts amphiphilic polymer, and active ingredient includes photosensitizer Ce6 and analgesic drug LC. The preparation method of the nanometer medicine is also disclosed, comprising the following steps: weighing Ce6, analgesic drug LC and amphiphilic polymer, dissolving in solvent to obtain a mixed solution; the mixed solution is stirred uniformly, and dialyzed in ultrapure water; the solution obtained by dialysis is constant volume, and filtered to obtain nanometer medicine. The nanometer medicine particle size morphology is regular, the particle size is uniform, has good stability within 7 days, can have good enrichment effect at tumor site, and has good photodynamic therapy effect. Lidocaine and photosensitizer reach the lesion site at the same time through intravenous injection, not only reduce the operation steps, but also target the lesion site, reduce the anxiety of patients because they feel that anesthesia will affect intelligence.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology, specifically relating to a nanomedicine, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) has been approved by the U.S. Food and Drug Administration (FDA) for the clinical treatment of various diseases and has become a treatment method for many skin diseases (including port-wine stains) and malignant tumors. However, in clinical practice, patients often experience burning, stinging, or pain. Although patients' pain tolerance varies, these sensations significantly affect their ability to accept treatment. Some patients even forcibly terminate treatment or ultimately abandon it due to discomfort, greatly impacting the treatment progress and reducing the effectiveness of PDT to some extent. Therefore, alleviating the pain experienced by patients during PDT is crucial.

[0003] Currently, common methods for pain relief during clinical photodynamic therapy include: applying analgesic gel to the treatment site preoperatively, oral tramadol; intraoperative cold spraying of the treatment site; and general anesthesia. However, preoperative oral analgesics and preoperative gel application have not proven to be very effective in clinical practice. Intraoperative cold spraying of the treatment site only alleviates pain to a certain extent, but this reduction is largely due to the patient's psychological reduction of pain perception by medical staff. This method requires active cooperation between the patient and medical staff to achieve pain relief, placing high demands on both. General anesthesia is a high-risk and costly clinical procedure and is rarely used in dermatological photodynamic therapy, primarily due to considerations of patient safety and comfort. Therefore, based on current clinical photodynamic therapy procedures, it is essential to develop a novel method that effectively relieves pain during treatment with minimal side effects.

[0004] Lidocaine is a commonly used analgesic in clinical practice. Its analgesic mechanism mainly involves binding to the VGSCs of nerve endings, preventing sodium ion influx, and inhibiting nerve impulse generation. Additionally, lidocaine has an angiogenic inhibitory effect, which may help suppress tumor growth. Clinically, it is often used as a local anesthetic via infiltration anesthesia, adding an extra step to the photodynamic therapy process and creating an additional burden on the patient due to its lack of targeting function. Intravenous injection is the commonly used method for delivering photosensitizers in clinical practice. However, because different drugs have different pharmacokinetics, the time it takes for the two drugs to reach the lesion site after direct intravenous injection of photosensitizers and lidocaine differs, failing to achieve the desired pain relief effect during treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a nanomedicine, its preparation method, and its application, which solves the problem of relieving pain during photodynamic therapy.

[0006] This invention is achieved through the following technical solution: This invention discloses a nanomedicine comprising a drug carrier and an active ingredient. The drug carrier is an amphiphilic polymer, and the active ingredient comprises a photosensitizer Ce6 and an analgesic drug LC. The photosensitizer Ce6 is used to generate reactive oxygen species, providing cytotoxicity. The analgesic drug LC is used to block the influx of sodium ions, relieve pain, and inhibit angiogenesis.

[0007] Furthermore, by weight, it includes 1-2 parts of photosensitizer Ce6, 1-2 parts of analgesic drug LC, and 5-10 parts of amphiphilic polymer; wherein the photosensitizer Ce6 and the analgesic drug LC are encapsulated in the amphiphilic polymer.

[0008] Furthermore, the amphiphilic polymer is polyethylene glycol-polypropylene sulfur.

[0009] Furthermore, the nanomedicines exhibited uniform spherical shapes and regular morphology under a transmission electron microscope.

[0010] This invention also discloses a method for preparing the nanomedicine, comprising the following steps: (1) Weigh Ce6, analgesic drug LC and amphiphilic polymer, dissolve them in solvent to obtain a mixed solution; (2) Stir the mixed solution until homogeneous, and then dialyze it in ultrapure water; (3) The solution obtained by dialysis was diluted to a certain volume and filtered to obtain nanomedicine.

[0011] Furthermore, in step (1), the solvent used is dimethyl sulfoxide.

[0012] Furthermore, in step (2), the dialysis process is as follows: the mixed solution is packaged into a dialysis bag and dialyzed in ultrapure water for several days, during which the ultrapure water is replaced every 8 hours; In step (3), the nanomedicine is refrigerated after it is obtained.

[0013] The present invention also discloses the application of the nanomedicine in the preparation of tumor-targeting drugs.

[0014] The present invention also discloses the application of the nanomedicine in photodynamic therapy to relieve pain during photodynamic therapy.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses a nanomedicine using an amphiphilic polymer as a drug carrier and a photosensitizer Ce6 and an analgesic LC as active ingredients. The photosensitizer Ce6 generates reactive oxygen species, providing cytotoxicity; the analgesic LC blocks sodium ion influx, alleviating pain experienced by patients during photodynamic therapy, and simultaneously enhances the efficacy of photodynamic therapy by utilizing LC's ability to inhibit angiogenesis. The nanomedicine has a regular and uniform particle size, exhibits good stability within 7 days, shows excellent accumulation at the tumor site, and demonstrates good photodynamic therapy efficacy. Simultaneous intravenous injection of lidocaine and the photosensitizer to the lesion site not only reduces surgical steps but also allows for targeted lesion treatment, reducing patient anxiety regarding the potential impact of anesthesia on intelligence (especially in children undergoing facial treatment).

[0016] Furthermore, by weight, the formulation includes 1-2 parts of photosensitizer Ce6, 1-2 parts of analgesic drug LC, and 5-10 parts of amphiphilic polymer. This ratio ensures that the amphiphilic polymer has the highest drug loading capacity for both drugs, thereby improving the efficiency of drug use.

[0017] The present invention also discloses a method for preparing the nanomedicines, wherein ECCL and ESCL nanomedicines are prepared by self-assembly of two hydrophobic drugs Ce6 and LC. The preparation method is simple and easy to prepare.

[0018] The present invention also discloses the application of the nanomedicines. It has been verified that the nanomedicines ECCL and ESCL can effectively inhibit nerve impulses generated during photodynamic therapy and effectively relieve pain generated during photodynamic therapy. Attached Figure Description

[0019] Figure 1 A physical image used to test the electrophysiological signals of the sciatic nerve in mice; Figure 2 Transmission electron microscope image; Figure 2 a and 2c are nanomedicines ECC and ESC containing only the photosensitizer Ce6; Figure 2 b and 2d are transmission electron micrographs of the nanomedicines ECCL and ESCL prepared in this invention; Figure 3 This is a dynamic light scattering diagram of nanomedicines; Figure 4 This is a graph showing the particle size change of nanomedicines over 7 days. Figure 5 The toxicity of nanomedicines to melanoma cells (B16F10) under dark conditions; Figure 6Figure 1 shows the ROS generated under illumination after intracellular endocytosis of nanomedicines or bare drugs (scale bar = 100 μm) under a fluorescence microscope. Figure 2 shows the fluorescence microscopy image of ROS generated under dark conditions after intracellular Ce6 endocytosis, Figure 3 shows the fluorescence microscopy image of ROS generated under dark conditions after intracellular endocytosis of nanomedicine ESCL, Figure 4 shows the fluorescence microscopy image of ROS generated after intracellular Ce6 endocytosis and illumination, and Figure 5 shows the fluorescence microscopy image of ROS generated after intracellular endocytosis of nanomedicine ESCL and illumination. Figure 7 To assess cell activity under different light exposure times after endocytosis of nanomedicines; Figure 8 The effect of nanomedicine on duct formation inhibition (scale bar=500 μm); Figure 9 Comparison of relative catheter lengths calculated from nanomedicines ECC, ECCL, ESC, and ESCL after inhibiting angiogenesis; Figure 10 The changes in sciatic nerve signal in mice during photodynamic therapy; Figure 11 The number of posture changes in mice during photodynamic therapy; Figure 12 The change in the relative volume of tumors in mice after photodynamic therapy; Figure 13 The change in survival rate of mice after receiving photodynamic therapy. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of the present invention, and not all of them.

[0021] The components described and illustrated in the accompanying drawings and embodiments of this invention can be arranged and designed in various different configurations. Therefore, the detailed description of the embodiments of the invention provided in the following drawings is not intended to limit the scope of the claimed invention, but merely to illustrate one selected embodiment of the invention. All other embodiments obtained by those skilled in the art based on the accompanying drawings and embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0022] This invention utilizes commonly used amphiphilic polymers—polyethylene glycol-polycaprolactone (PEG-PCL) and ROS-responsive amphiphilic polymers—polyethylene glycol-polypropylene sulfuride (PEG-PPS)—as drug carriers for Ce6 and LC, respectively, to prepare nanomedicines (ECCL and ESCL). Both drugs are simultaneously delivered to the disease site via intravenous injection and the unique high-permeability long retention (EPR) effect of nanomicelles. During photodynamic therapy at the disease site, the photosensitizer Ce6 generates reactive oxygen species, providing cytotoxicity; the analgesic LC blocks sodium ion influx, alleviating pain experienced by the patient during photodynamic therapy. Simultaneously, the analgesic LC's ability to inhibit angiogenesis enhances the efficacy of photodynamic therapy (e.g., for port-wine stains, tumors, etc.).

[0023] The preparation methods of ECCL and ESCL are as follows: Weigh 1 mg of Ce6, 2 mg of LC, and 10 mg of the amphiphilic polymer, and dissolve them in 1 mL of dimethyl sulfoxide (DMSO); after mixing the solution evenly by stirring, seal it in a dialysis bag (MWCO=2000), and dialyze it in ultrapure water for three days, changing the ultrapure water every 8 hours during the period; after dialysis, make up the volume of the solution in the dialysis bag to 10 mL, filter it with a 0.45 µm filter, collect the prepared nanomedicine, and store it in a refrigerator at 4℃ for later use.

[0024] To demonstrate the analgesic and photodynamic therapy-enhancing effects of LC, nanomedicines (ECC and ESC) encapsulating only the photosensitizer Ce6 were prepared as controls. The preparation method was the same as that for ECCL, except that LC was not added during the preparation of the DMSO solution.

[0025] In clinical practice, the primary method for measuring pain in patients is the questioning method, which requires the patient's active cooperation. Before developing new clinical treatments, it is necessary to validate their effectiveness using experimental animal models. Clearly, this method of measuring pain is not suitable for experimental animal models. Currently, experiments mainly assess the degree of pain suffered by mice through pain-related behaviors and facial expressions. However, these methods require the mice to be in a free state, making it impossible to assess the pain experienced by mice under deep anesthesia undergoing photodynamic therapy. A novel experimental method has been developed to detect the degree of pain experienced by mice during treatment. The specific process is as follows: Mice were deeply anesthetized with 0.3% sodium pentobarbital (0.1-0.2 mL / 10g), and hair was extracted from the left hind limb. The hair was cut at the extraction site using sterilized surgical scissors, and the sciatic nerve on the left posterior side of the mouse was carefully dissected using hemostatic forceps. The mouse's limbs were bound with rubber bands, which were then fixed to a mouse board. A hook electrode was placed on the dissected sciatic nerve, and a grounding electrode was clamped onto the nearby skin tissue. The electrode was connected to a bio-acquisition and analysis system (with the following parameters: sampling rate: 100 kHz, range: 5 mV, time constant: 200 ms, low-pass filter: 100 Hz). A photosensitizer was injected into the mouse's left hind leg, and the area was irradiated with laser light. Figure 1 A physical image used to test the electrophysiological signals of the sciatic nerve in mice.

[0026] This invention uses amphiphilic polymers as drug carriers to prepare ECCL and ESCL nanomedicines by self-assembly of two hydrophobic drugs, Ce6 and LC. The nanomedicines exhibit regular and uniform particle size morphology, good stability within 7 days, excellent enrichment effects at tumor sites, and good photodynamic therapy efficacy.

[0027] like Figure 2 As shown, the nanomedicine prepared by this invention exhibits a uniform spherical shape under a transmission electron microscope.

[0028] Figure 3 The dynamic light scattering detection results of the nanomedicine show that its average particle sizes are 87.78 nm (ECC), 86.86 nm (ECCL), 59.91 nm (ESC), and 57.39 nm (ESCL), and it exhibits uniform dispersion. Furthermore, as shown... Figure 4 As shown, its particle size did not change significantly within 7 days, proving that it has good stability.

[0029] Figure 5 The toxicity of nanomedicines to melanoma cells (B16F10) under dark conditions was compared, showing that the photosensitizer toxicity significantly decreased after being encapsulated by a drug carrier. Subsequently, as... Figure 6 As shown, the ability of bare Ce6 and Ce6 encapsulated by a drug carrier to generate ROS in vivo after light exposure was compared. Figure 6 Figure a shows a fluorescence microscopy image of ROS generated under dark conditions after cytotoxicity of Ce6. Figure 6 Figure b shows a fluorescence microscopy image of ROS generated under dark conditions after endocytosis of the nanomedicine ESCL. Figure 6 Figure c shows a fluorescence microscopy image of ROS generated after light stimulation following Ce6 endocytosis in cells. Figure 6Figure d shows a fluorescence microscopy image of ROS generated after light stimulation following endocytosis of the nanodrug ESCL. It can be seen that Ce6 can generate more ROS after being encapsulated by the drug carrier PEG-PPS, proving that it has a stronger ability to kill cells.

[0030] like Figure 7 As shown, the phototoxicity of the nanomedicines after intracellular endocytosis was then tested at different light exposure times. It can be seen that under the same concentration of photosensitizer, both nanomedicines (ECCL and ESCL) have a good killing effect on B16F10 cells.

[0031] In addition, the inhibitory effect of nanomedicines on angiogenesis was tested. Figure 8 The Control group was the group that used culture medium for catheter imaging experiments, and its relative catheter length was calculated. Figure 9 It is evident that the nanomedicines ECCL and ESCL have a good inhibitory effect on angiogenesis.

[0032] During photodynamic therapy in mice, sciatic nerve signals were observed. Figure 10 It is evident that the nanomedicines ECCL and ESCL can effectively inhibit nerve impulses generated during photodynamic therapy. Moreover, compared with ECC and ESC nanomedicines, the number of postural changes in mice injected with ECCL and ESCL nanomedicines during photodynamic therapy was significantly reduced. Figure 11 This demonstrates that ECCL and ESCL nanomedicines effectively alleviate pain during photodynamic therapy.

[0033] Finally, the antitumor efficacy of nanomedicine was investigated in a mouse model of melanoma. In this study, the tumor volume was 50 mm². 3 Tumor-bearing mice were randomly divided into three groups: (1) saline (NS) group; (2) ECC group; (3) ECCL group; (4) ESC group; and (5) ESCL group. First, nanomedicine or NS was injected into the mice via tail vein injection (Ce6 dose was 5 mg / kg). Photodynamic therapy was then administered to the tumor sites at 12 h and 24 h, with the energy density of the 660 nm laser during treatment being 288 J / cm². 2 The targeting mechanism is that, by utilizing the high permeability and retention effect (EPR effect) unique to tumor sites, nanoparticles with a size of 100-200 nm can be enriched in tumor sites.

[0034] During the treatment process, changes in tumor size and mouse survival rates were measured and statistically analyzed. For example... Figure 12As shown, compared with nanomedicines without LC encapsulation, the nanomedicines ECCL and ESCL have more significant anti-tumor effects. This is because the angiogenesis inhibition effect of lidocaine enhances the efficacy of photodynamic therapy.

[0035] like Figure 13 As shown, mice treated with ESCL nanomedicine had the most stable survival rate after photodynamic therapy, indicating that ESCL-treated mice have better safety.

[0036] In summary, the nanomedicines ECCL and ESCL prepared in this invention can effectively relieve pain during photodynamic therapy and have a good photodynamic therapeutic effect.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A nanomedicine, characterized in that, It includes a drug carrier and an active ingredient. The drug carrier uses an amphiphilic polymer, and the active ingredient includes the photosensitizer Ce6 and the analgesic drug LC. The photosensitizer Ce6 is used to generate reactive oxygen species, providing cytotoxicity. The analgesic drug LC is used to block sodium ion influx, relieve pain, and inhibit angiogenesis. The product comprises, by weight, 1-2 parts of photosensitizer Ce6, 1-2 parts of analgesic drug LC, and 5-10 parts of amphiphilic polymer; wherein the photosensitizer Ce6 and the analgesic drug LC are encapsulated in the amphiphilic polymer. The amphiphilic polymer used is polyethylene glycol-polypropylene sulfurization; The nanomedicine appears as a uniform sphere with a regular morphology under a transmission electron microscope.

2. The method for preparing the nanomedicine according to claim 1, characterized in that, Includes the following steps: (1) Weigh Ce6, analgesic drug LC and amphiphilic polymer, dissolve them in solvent to obtain a mixed solution; (2) Stir the mixed solution until homogeneous, and then dialyze it in ultrapure water; (3) The solution obtained by dialysis was diluted to a certain volume and filtered to obtain nanomedicine.

3. The method for preparing nanomedicine according to claim 2, characterized in that, In step (1), dimethyl sulfoxide is used as the solvent.

4. The method for preparing nanomedicine according to claim 2, characterized in that, In step (2), the dialysis process is as follows: the mixed solution is sealed in a dialysis bag and dialyzed in ultrapure water for several days, during which the ultrapure water is replaced every 8 hours; In step (3), the nanomedicine is refrigerated after it is obtained.

5. The application of the nanomedicine according to claim 1 in the preparation of tumor-targeting drugs, characterized in that, The tumor is melanoma.

6. The application according to claim 5, characterized in that, The nanomedicine is used to relieve pain during photodynamic therapy.

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