CN118453902B - A nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer and its preparation method and application - Google Patents
A nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer and its preparation method and application Download PDFInfo
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Abstract
本发明提供了一种用于三阴性乳腺癌的双靶向光动力治疗的纳米递送材料及其制备方法和应用。通过将具有三阴性乳腺癌靶向能力的Dasatinib衍生物修饰于聚环糊精表面并与内质网靶向光敏剂混合组装，所构建的新型双靶向纳米材料可精准靶向识别肿瘤细胞，增加光敏剂在肿瘤部位的富集，且能够进一步实现在内质网中的蓄积，产生的ROS可直接作用于内质网，引起更强的内质网应激，进而显著提高靶向光动力的抗肿瘤效果。本发明制备的新型双靶向功能纳米载药体系解决了传统光敏剂靶向性差，生物溶解性以及治疗效果有限的问题，为实现三阴性乳腺癌的靶向光动力治疗提供了一种创新性策略。
The present invention provides a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, and a preparation method and application thereof. By modifying a Dasatinib derivative with triple-negative breast cancer targeting ability on the surface of a polycyclodextrin and mixing and assembling it with an endoplasmic reticulum-targeted photosensitizer, the constructed novel dual-targeted nanomaterial can accurately target and identify tumor cells, increase the enrichment of photosensitizers at the tumor site, and can further achieve accumulation in the endoplasmic reticulum. The generated ROS can directly act on the endoplasmic reticulum, causing stronger endoplasmic reticulum stress, thereby significantly improving the anti-tumor effect of targeted photodynamic therapy. The novel dual-targeted functional nano-drug delivery system prepared by the present invention solves the problems of poor targeting, biological solubility and limited therapeutic effect of traditional photosensitizers, and provides an innovative strategy for achieving targeted photodynamic therapy of triple-negative breast cancer.
Description
Technical Field
The invention belongs to the field of novel targeted nano-drugs, and in particular relates to a nano-delivery material for double-targeted photodynamic therapy of triple-negative breast cancer, and a preparation method and application thereof.
Background
Triple-negative breast cancer (TNBC) is a subtype of breast cancer and has the characteristics of strong invasiveness, easy metastasis and extremely poor prognosis, so that the survival rate of patients is low. Because the Estrogen Receptor (ER), the Progestogen Receptor (PR) and the proto-oncogene Her-2 on the TNBC tumor surface are negative, few anticancer drugs can enter tumor cells, and the treatment effect is greatly reduced. For this reason, increasing the dose of anticancer drugs is often used to achieve the desired therapeutic effect. Unfortunately, this inevitably increases the toxic side effects of the drug as well as tumor resistance, which in turn reduces the therapeutic options for TNBC. At present, chemotherapy, immunotherapy, surgery, radiotherapy and the like are often used to treat TNBC clinically according to the different stages of TNBC. However, the above treatments cause serious side effects such as wound infection, bone marrow suppression, hepatotoxicity, skin and heart damage. Furthermore, resistance to chemotherapy and radiation therapy, metastasis and recurrence also frequently occur in TNBC. Thus, the search for targeted drugs that can effectively treat TNBC to improve the therapeutic efficacy of TNBC is currently a major challenge.
The tyrosine kinase inhibitor has the characteristics of high selectivity and few side effects, can be used as a competitive inhibitor for combining Adenosine Triphosphate (ATP) and tyrosine kinase, can also be used as a tyrosine analogue, blocks the activity of the tyrosine kinase, inhibits cell proliferation, has a wide target, such as BCR-ABL kinase, SRC family kinase (SRC, LCK, YES, FYN) and the like, and is currently used as a first-line medicament for clinically treating tumors. Furthermore, several studies have shown that SRC protein levels are significantly higher in TNBC than in other breast cancer subtypes. And Dasatinib is used as a tyrosine kinase inhibitor, can accurately target SRC family proteins (including SRC, LCK, FYN and the like), and provides a feasible scheme for realizing TNBC targeted treatment. However, the currently existing Dasatinib-targeted TNBC treatment is still applied to preclinical studies in the form of chemotherapy or combination chemotherapy, which also inevitably causes problems of drug resistance and adverse reactions of patients. Therefore, dasatinib combined with other tumor treatment modes is hopeful to become a brand-new tumor treatment strategy for targeted treatment of TNBC.
Photodynamic therapy (PDT) has become a novel cancer treatment modality due to its unique advantages of minimal invasive, high efficiency, and no significant resistance. However, the conventional photosensitizers such as phthalocyanine and porphyrin have the problems of insufficient targeting of tumor tissues, poor biological solubility and the like, so that the PDT has limited therapeutic effect. With the development and application of nanomaterial technology, a strategy of constructing a nano delivery system as a photosensitizer carrier is undoubtedly provided with a new platform for the development of PDT, and the selective enrichment of photosensitizer at tumor sites is increased. Unfortunately, this strategy still faces two obstacles, 1) poor biological solubility of the nanocarrier and premature leakage of the photosensitizer causing stronger toxic side effects, 2) limited radius of action (< 0.02 μm) due to short ROS half-life (< 0.04 μm), which is undoubtedly limited in the vicinity of the site of generation, resulting in limited PDT treatment efficacy. Therefore, in view of these problems, there is a need to develop a soluble nano-delivery vehicle with tumor cell and subcellular organelle targeting function to achieve the purpose of effectively improving photodynamic anti-tumor effect.
Disclosure of Invention
The invention aims to provide a nano delivery material for double-targeting photodynamic therapy of triple-negative breast cancer and a preparation method thereof. Firstly, covalently coupling p-Toluenesulfonamide ligand with endoplasmic reticulum targeting function and photosensitizer Ce 6 to obtain photosensitizer CET with endoplasmic reticulum positioning capability, then, modifying Dasatinib derivative on the surface of polycyclodextrin material by means of interaction of host and guest to obtain nano delivery material Da-CD with TNBC targeting capability, and finally, mixing and assembling photosensitizer CET and Da-CD to obtain nano material Da-CD@CET with tumor cell targeting and endoplasmic reticulum targeting function. The high loading rate and high biocompatibility of the polycyclodextrin can reduce biotoxicity, in addition, the Dasatinib derivative loaded on the surface of the polycyclodextrin can accurately target and identify tumor cells, so that the photosensitizer is promoted to be more enriched at a tumor part, the endoplasmic reticulum of the material package targets the photosensitizer CET, more photosensitizers can be further accumulated in the endoplasmic reticulum, the generated ROS can directly act on the endoplasmic reticulum, stronger endoplasmic reticulum stress is caused, and the effect of targeted photodynamic therapy TNBC is further remarkably improved. The dual-targeting functional nano drug-carrying system prepared by the invention has higher biocompatibility and obvious targeting of tumor cells and endoplasmic reticulum, shows negligible toxic and side effects, obviously improves the photodynamic anti-tumor effect, and provides an innovative strategy for realizing the targeted photodynamic therapy of TNBC. Meanwhile, the invention also provides the nano delivery material for the double-targeting photodynamic therapy of the triple negative breast cancer, which is obtained by the method, and application of the nano delivery material.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The invention discloses a preparation method of a nano delivery material for double-targeting photodynamic therapy of triple-negative breast cancer, which comprises the following steps:
(1) Firstly, accurately weighing beta-CD, dissolving in 15wt% NaOH aqueous solution, and stirring at normal temperature. Then, toluene was added dropwise to the mixture and stirring was continued, and epichlorohydrin as a crosslinking agent was slowly added dropwise thereto to continue the reaction. After the reaction, the crude product was subjected to a pre-precipitation treatment with isopropanol, the precipitate was transferred to a suitable amount of water and neutralized with dilute hydrochloric acid. It was dialyzed through a dialysis bag to thoroughly remove monomers and oligomers, and finally lyophilized in vacuo to give the white solid product Poly- β -CD.
(2) Accurately weighed Dasatinib and succinic anhydride are added into anhydrous DMF, the mixture is stirred uniformly, and accurately weighed triethylamine is slowly added into the mixed reaction drop by drop. Finally, nitrogen is introduced for protection, and stirring is carried out at room temperature for continuous reaction. After the reaction, the reaction system was rotary evaporated in vacuo to remove DMF, and the mixture was purified by column chromatography. Finally, mixing and evaporating the collected organic solvent to obtain a white solid compound Da-COOH;
(3) Accurately weighing Da-COOH obtained in the step (2), adding an anhydrous DMF solution to stir and mix uniformly, and then adding accurately weighed NHS and EDCI into the mixed reaction solution to react in an ice-water bath. Then, the accurately weighed amantadine was added to the mixed solution, and the reaction was stirred at room temperature. After the reaction, the reaction solution is transferred into a mixed solution of acetone and water to form a precipitate, and the precipitate is filtered in vacuum. Vacuum drying to obtain white solid compound Da-Ad;
(4) And (3) dissolving the accurately weighed ethylenediamine in the anhydrous dichloromethane solution, stirring in an ice bath, dissolving the weighed p-toluenesulfonyl chloride in the anhydrous dichloromethane solution, slowly dripping the solution into a reaction system, continuing to react at normal temperature, and protecting by N 2. After the reaction is finished, purifying the mixture by adopting a column chromatography method, and finally obtaining a pale yellow solid compound ET;
(5) And (3) dissolving the Ce 6, the HOBT and the EDCI which are accurately weighed into anhydrous DMF solution, carrying out light-shielding reaction at room temperature, then adding the ET obtained in the step (4) for continuous reaction, and protecting N 2. After the reaction is finished, purifying the mixture by adopting a column chromatography method to finally obtain a brownish-black solid compound CET;
(6) And (3) dissolving the Poly-beta-CD obtained in the step (1) and the CET obtained in the step (5) in a mixed solution of DMSO and water, and carrying out light-shielding reaction at room temperature for 2 hours. The Da-Ad obtained in the step (3) is dissolved in a mixed solution of DMSO and water, and then slowly added into the reaction system in a dropwise manner. The reaction was continued at room temperature in the dark. After the reaction, the reaction mixture was transferred to a dialysis bag for dialysis. Finally, the reaction solution is freeze-dried to obtain brown-black solid powder Da-CD@CETNPs.
Further, the dialysis conditions in step (1) were dialysis in dialysis bags of mw=8000 for 24 hours, with water being exchanged every 3 hours.
Further, in the step (3), the dosage ratio of the mixed solution of acetone and water is 1:4.
Further, the mixed solution of DMSO and water in the step (6) is used in a ratio of 1:3. Dialysis conditions were dialysis for 24h in dialysis bags with mw=3000, with water being exchanged every 3 h.
It is an object of a second aspect of the present invention to disclose a nano-delivery material for dual targeting photodynamic therapy of triple negative breast cancer, the material being prepared by the above method.
The invention aims at disclosing the application of the nano delivery material for the double-targeting photodynamic therapy of triple negative breast cancer in a double-targeting functional nano drug carrying system.
The invention has the remarkable advantages that:
(1) The polycyclodextrin type nano delivery carrier has good biocompatibility and stability under physiological conditions, has the characteristic of responding to tumor microenvironment disintegration and drug release, can remarkably reduce toxic and side effects and adverse reactions caused by premature leakage and poor solubility of nano drugs in the transportation process, and can be used as an ideal nano drug carrier.
(2) The nano delivery material modified by the small molecule targeting drug Dasatinib derivative shows remarkable targeting recognition capability on triple negative breast cancer cells, and provides a triple negative breast cancer targeting treatment strategy with great prospect.
(3) The nano medicine carrying system has excellent stability and homogeneity, and is favorable to use and long term preservation.
(4) The preparation method is simple, has less side reaction, easily obtained raw materials and low cost, and is favorable for realizing industrial production.
Drawings
FIG. 1 is a graph showing transmission electron microscopy and particle size distribution of different nano-drug delivery systems;
FIG. 2 is a graph showing the stability and particle size variation of different nanodrug delivery systems in deionized water and culture medium;
FIG. 3 is a tumor cell targeting graph of flow cytometry characterization of Da-CD@CETNPs;
FIG. 4 shows an endoplasmic reticulum targeting ability map of Da-CD@CETNPs for laser confocal microscopy;
FIG. 5 shows the inhibition ability of cell activity of different drug groups tested by MTT method;
FIG. 6 is a graph showing inhibition ability of different drug groups on tumor models of triple negative breast cancer mice;
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The preparation method of the nano delivery material for double-targeting photodynamic therapy of triple-negative breast cancer provided by the invention comprises the following steps:
(1) First, 10.0g of β -CD was weighed accurately and dissolved in 15mLNaOH (15 WT%) of an aqueous solution, followed by stirring at room temperature for 2 hours. Subsequently, 2mL of toluene was added dropwise to the mixture and stirring was continued for 2 hours, and 3.8mL of epichlorohydrin as a crosslinking agent was slowly added dropwise thereto and reacted for 3 hours. After the reaction, the crude product was subjected to a preliminary precipitation treatment with 200mL of isopropanol, and the precipitate was transferred to a suitable amount of water and neutralized with dilute hydrochloric acid. Finally, it was dialyzed through a dialysis bag of mw=8000 for 2 days, with water being exchanged every 3 hours to thoroughly remove monomers and oligomers, and finally freeze-dried in vacuo to give the white solid product Poly- β -CD.
(2) Accurately weighed Dasatinib (1.60 g,3.27 mmol) and succinic anhydride (0.37 g,3.70 mmol) were placed in a 100mL double-necked flask. Then, 15mL of anhydrous DMF was added to the double-necked flask, stirred well, and accurately weighed triethylamine (0.70 g,6.90 mmol) was slowly added dropwise to the mixture. Finally, nitrogen is introduced for protection, and stirring is carried out at room temperature for continuous reaction for 48 hours. After the reaction, the reaction system was subjected to rotary evaporation under vacuum to remove DMF, and the mixture was purified by column chromatography (CH 2Cl2/CH3 OH 30:1 v/v). Finally, the collected organic solvents are mixed and evaporated to obtain 1.73g of white solid compound Da-COOH with the yield of 90％.1H NMR(400MHz,DMSO-d6)δ＝11.45(s,1H),9.89(s,1H),8.23(s,1H),7.38(d,J＝7.4Hz,1H),7.27(dd,J＝10.6,7.5Hz,2H),6.06(s,1H),4.15(t,J＝5.7Hz,2H),3.50(s,4H),2.58(t,J＝5.7Hz,2H),2.49(s,8H),2.40(s,3H),2.23(s,3H).13C NMR(101MHz,DMSO-d6)δ＝172.60,165.63,162.93,160.41,157.43,141.34,139.30,134.03,132.93,129.48,128.62,127.47,126.18,83.12,61.97,58.95,56.42,53.18,52.86,44.04,29.29,26.05,18.79.HRMS(ESI)：m/z calculated for C26H30ClN7O5S[M+H]+:588.1790;found:588.1781.
(3) Da-COOH (0.9 g,2.0 mmol) obtained in the step (2) was accurately weighed and placed in a 50mL round bottom flask, 30mL of anhydrous DMF solution was added to stir and mix well, then accurately weighed NHS (0.3 g,2.4 mmol) and EDCI (0.5 g,2.4 mmol) were added to the mixed reaction solution, and reacted in an ice-water bath for 1h. Subsequently, an accurately weighed amount of amantadine (0.3 g,2.1 mmol) was added to the mixture, and the reaction was stirred at room temperature for 12 hours. After the reaction, the reaction mixture was transferred to a mixture of 300mL of acetone and water (1:4) to form a precipitate, which was vacuum filtered. Finally, drying in vacuo at 50℃for 24h gave 0.72g of compound Da-Ad as a white solid in yield 65％.1H NMR(400MHz,DMSO-d6)δ＝10.21(s,1H),8.79(s,1H),8.46(s,1H),7.35(d,J＝7.4Hz,1H),7.32(dd,J＝10.6,7.5Hz,2H),5.68(s,1H),4.35(t,J＝5.7Hz,2H),3.62(s,4H),3.44(s,4H),2.97(t,J＝5.7Hz,2H),2.52(s,4H),2.40(s,3H),2.23(s,3H),2.21(s,2H),1.98(s,4H),1.87(s,3H),1.76(s,6H).13C NMR(101MHz,DMSO-d6)δ＝173.60,172.70,169.23,165.43,161.31,158.43,143.58,138.25,135.63,130.73,128.93,127.78,127.47,126.18,121.52,79.32,62.15,57.65,56.36,52.08,51.26,47.89,43.94,41.16,36.48,32.12,29.29,26.05,24.65,18.79,16.58.HRMS(ESI)：m/z calculated for C36H45ClN8O4S[M+H]+:721.3257;found:721.3296.
(4) Accurately weighed ethylenediamine (1.5 g,25 mmol) was dissolved in 50mL of anhydrous dichloromethane solution, stirred in an ice bath, then weighed p-toluenesulfonyl chloride (0.95 g,5 mmol) was dissolved in 5mL of anhydrous dichloromethane solution, then slowly added dropwise to the reaction system, and the normal temperature reaction was continued for 24h, with N 2 for protection. After the reaction was completed, the mixture was purified by column chromatography (CH 2Cl2/CH3 OH 30:1 v/v) to finally obtain 0.75g of a pale yellow solid compound ET in yield 72％.1H NMR(400MHz,DMSO-d6)δ＝7.66(s,2H),7.39(s,2H),3.37(s,2H),2.76(t,J＝5.7Hz,2H),2.43(s,3H).13C NMR(101MHz,DMSO-d6)δ＝137.86,137.25,129.78,129.43,128.31,127.28,44.13,39.28,27.39.HRMS(ESI)：m/z calculated for C9H14N2O2S[M+H]+:214.0846;found:214.0913.
(5) Accurately weighed Ce 6 (48.0 mg,0.08 mmol), HOBT (16.0 mg,0.12 mmol) and EDCI (23.0 mg,0.12 mmol) are dissolved in 3mL of anhydrous DMF solution, reacted at room temperature in the absence of light for 30min, then ET (21.4 mg,0.10 mmol) obtained in step (4) is added, and the reaction is continued for 24h, N 2 protects. After the reaction was completed, the mixture was purified by column chromatography (CH 2Cl2/CH3 OH 30:1 v/v) to finally obtain 18.3mg of a tan solid compound CET in a yield of 29％.1H NMR(400MHz,DMSO-d6)δ＝7.86(s,2H),7.54(s,2H),6.65(s,1H),6.46(d,J＝6.4Hz,1H),5.32(dd,J＝10.6,7.5Hz,2H),5.16(s,1H),4.35(t,J＝5.7Hz,2H),3.66(s,2H),3.37(s,2H),2.92(t,J＝5.7Hz,2H),2.44(s,2H),2.40(s,3H),2.35(s,3H),2.16(s,6H),2.05(s,2H),2.01(s,1H),1.55(s,2H),0.87(s,3H),0.76(s,3H).13C NMR(101MHz,DMSO-d6)δ＝172.24,171.46,169.58,164.79,150.28,148.65,147.58,146.72,145.75,144.58,142.15,139.68,138.48,137.47,136.63,134.56,131.84,130.53,129.92,129.23,128.78,128.37,125.69,123.38,119.32,113.15,97.68,96.56,95.28,54.36,42.49,38.64,38.18,34.58,30.15,28.89,21.65,19.75,17.65,15.69,12.69,11.58,10.79.HRMS(ESI)：m/z calculated for C43H48N6O7S[M+H]+:792.3248;found:792.3286.
(6) Poly-beta-CD (21.0 mg) obtained in the step (1) and CET (2.0 mg) obtained in the step (5) were dissolved in a mixed solution of DMSO and water (1:3), and reacted at room temperature in a dark place for 2 hours. Subsequently, 0.5mg of Da-Ad obtained in the step (3) was dissolved in a mixture of DMSO and water (1:3), and then slowly added dropwise to the reaction system. The reaction was continued for 12h at room temperature in the dark. After the reaction was completed, the reaction mixture was transferred to a dialysis bag with mw=3000 for dialysis for 24 hours, with water being changed every 3 hours. Finally, the reaction solution is freeze-dried to obtain brown-black solid powder Da-CD@CETNPs.
Nanomaterial characterization (see figure 1)
The morphology of Da-CD@CET NPs was characterized using a transmission electron microscope (Transmission Electron Microscope, TEM) at ambient temperature. Next, hydrodynamic dimensions of Da-CD NPs and Da-CD@CETNPs in aqueous solution were determined by Malvern Nano-ZS ZEN 3600. TEM images show that both nano materials show spherical structures, and the sizes are about 60nm respectively. The hydrodynamic diameters and stability of the Da-CD NPs and Da-CD@CET NPs were then further confirmed by Dynamic Light Scattering (DLS), the average hydrodynamic diameters being about 61nm, respectively, which is nearly identical to TEM data, and the polydispersity index being 0.23 and 0.22, respectively, indicating that the preparation of Da-CD@CET NPs does not change the original morphology of Da-CD NPs, and also exhibits smaller particle size, more favorable for achieving endocytosis of tumor cells.
Characterization of Material stability (see FIG. 2)
The stability of the material is always the precondition and key of the application, so that the stability of various nano materials is researched. 2mg of Da-CD NPs, CD@CETNPs and Da-CD@CETNPs solid powder were dissolved in 3mL of deionized water, and the solution was observed. Da-CD NPs, cd@cet NPs, da-cd@cet NPs after 7 days in deionized water showed no significant turbidity and precipitation, consistent with the initial state of the solution on the first day, and all nanomaterials exhibited less PDI and remained essentially unchanged for 7 days. Meanwhile, the average hydrodynamic diameter is stable without obvious size change after being treated in deionized water for 7 days, and the size of the target drug Da-CD@CET NPs in the deionized water and the culture medium hardly changes within 7 days, which shows that the nanometer materials based on the poly beta-CD have long-term stability and uniformity. The ultraviolet-visible spectrophotometry and the calculation of Drug Loading Capacity (DLC) and Drug Loading Efficiency (DLE) of CET according to the drug standard curve are 5.02+ -0.13% and 79.90+ -0.35%, respectively, which also show that Da-CD NPs have good drug loading capacity.
Characterization of cell targeting (see FIG. 3)
(1) Firstly, selecting 4T1 cells with good growth state, removing old culture medium, adopting sterile PBS to wash for three times, then adopting 0.25% pancreatin to make digestion treatment, adding fresh culture medium to make heavy suspension after centrifugation, counting cell suspension by means of cell counter, diluting cell suspension to 2.0X10 5 cells/mL, and adding 1mL into confocal dish. Finally, all confocal dishes were incubated overnight in a 37 ℃ 5% co 2 cell incubator.
(2) Adding medicine, namely removing the culture medium in the laser confocal dish after the cells are completely adhered, then adopting sterile PBS to wash for three times, finally adding 1mL of fresh culture medium of Da-CD@CET NPs or CD@CETNPs with the concentration of 20 mug/mL into the laser confocal dish, and placing into a cell incubator with the temperature of 37 ℃ and the concentration of 5% CO 2 for incubation for 12 hours.
(3) And (3) after the incubation is finished, removing all the drug-containing culture mediums in the laser confocal dish, cleaning the culture mediums with sterile PBS for three times, and adding 1mL of sterile PBS to wait for photographing. Meanwhile, 0.5mL of 0.25% pancreatin was added to the other washed copolymer Jiao Min for digestion treatment, and 1mL of sterile PBS was added for resuspension after centrifugation. Finally, the fluorescence test of the intracellular drugs (excitation wavelength: 633nm, emission wavelength: 650-750 nm) was performed by using a laser confocal electron microscope and a flow cytometer, respectively. All cell blank groups in the experiment were incubated with the drug-free medium and other procedures were consistent with the experimental group.
The fluorescence intensity of intracellular Da-CD@CET NPs was detected by means of a confocal laser microscope and a flow cytometer. Da-CD@CET NPs exhibit a stronger fluorescent signal intensity than Da-CD@CET NPs, about 3 times thereof, indicating that Da-CD@CETNPs exhibit a stronger cell-targeted uptake ability. This indicates Dashatinib introduction greatly enhances specific recognition of 4T1 cells by nanomaterials, thereby facilitating uptake by cells. Competitive studies were targeted to further reveal whether the enhanced uptake of Da-cd@cet NPs in 4T1 cells is mediated by Dashatinib. Different concentrations of free Dashatinib (0, 5, 10 and 20 nM) were pre-incubated with 4T1 cells for 12h before incubation with Da-CD@CET NPs, which resulted in a certain amount of SRC protein being occupied and thus reduced in number. The results of both confocal laser microscopy and flow cytometry showed that with increasing concentration of free Dashatinib, the intracellular fluorescence intensity of Da-cd@cetnps also decreased, suggesting that more Da-cd@cetnps may accumulate in 4T1 cells by SRC receptor mediated endocytosis due to the presence of targeting unit Dashatinib. Therefore, all cell uptake experimental results show that Dashatinib modified nano-carriers can remarkably improve the targeting capability of Da-CD@CETNPs to SRC protein over-expressed triple negative breast cancer cells.
Endoplasmic reticulum targeting characterization (see figure 4)
(1) Spreading dish, firstly, selecting 4T1 cells with good growth state, removing old culture medium, adopting sterile PBS to wash for three times, then adopting 0.25% pancreatin to make digestion treatment, adding fresh culture medium to make heavy suspension after centrifugation, counting cell suspension by means of cell counter, diluting cell suspension to 1.0X10 4 cells/mL, and adding 1mL into confocal dish. Finally, all confocal dishes were incubated overnight in a 37 ℃ 5% co 2 cell incubator.
(2) Adding medicines, namely removing the culture medium in the laser confocal dish after the cells are completely adhered, then adopting sterile PBS to wash for three times, finally adding 1mL of fresh culture medium with concentration of 20 mug/mL Ce6, CET or Da-CD@CET NPs into the laser confocal dish, and placing the culture medium into a cell incubator with the temperature of 37 ℃ and the concentration of 5% CO 2 for incubation for 12 hours.
(3) Staining was performed by first removing 2. Mu.L of each of the stock solutions of endoplasmic reticulum green probes at a concentration of 1mM, and mixing with a new medium to finally give a probe solution at a concentration of 2. Mu.M. The drug-containing medium in the laser confocal dish was then removed, washed three times with sterile PBS and the cells were resuspended in 1mL of medium containing the probe dye and transferred to the corresponding laser confocal dish, respectively. Finally, the cells were incubated in a 37℃5% CO 2 cell incubator for 20min.
(4) And (3) photographing, namely after the co-incubation is finished, centrifuging and removing the corresponding probe dye in the dish, and centrifugally washing for three times by adopting sterile PBS (phosphate buffered saline), so as to remove the redundant probe dye and prevent the photographing background from being disturbed. Then a small amount of PBS was added to resuspend the cells and the cells were transferred to a new confocal dish before photographing. Finally, the cells were photographed using a confocal laser microscope equipped with a 405nm or 488nm multi-argon laser and a 633nm diode laser. The excitation and emission wavelength parameters of the photosensitizer compound are Ex/Em=633/650-750 nm, and the excitation and emission wavelength parameters of the endoplasmic reticulum probe dye are Ex/Em=488/510-570 nm.
The red fluorescence of Ce6 and CET and the green fluorescence of the endoplasmic reticulum probe were observed using laser confocal. All red fluorescence of free Ce6 has little overlap with the green fluorescence signal representing the endoplasmic reticulum. Further qualitative analysis of the line scan spectra of fluorescence intensities indicated that Ce6 group and endoplasmic reticulum dye had significant separation signals (fig. 4 b). By introducing p-toluenesulfonamide, the free CET group is found that a red fluorescent signal and an endoplasmic reticulum dye show obvious fluorescent overlap after 12h incubation, a yellow fluorescent spot is shown in the endoplasmic reticulum, and the line scanning spectrogram qualitative analysis of the fluorescent intensity also shows that the CET group and the endoplasmic reticulum dye have partial overlapping signals, so that the free CET has shown visible endoplasmic reticulum targeting capability. Whereas in the Da-cd@cet NPs group, compared to free CET, the endoplasmic reticulum exhibited intense yellow fluorescent spots, and at the same time, the line scan profile qualitative analysis of fluorescence intensity also exhibited a high overlap of CET and endoplasmic reticulum dye. Therefore, these data strongly demonstrate that Da-cd@cet NPs can significantly enhance the enrichment of photosensitizers in the endoplasmic reticulum, achieving the goal of targeting the endoplasmic reticulum.
Characterization of photodynamic activity ex vivo (see FIG. 5)
The cytotoxicity exhibited by photosensitizers during photodynamic therapy is one of the main research principles for evaluating its properties. The MTT method is an experimental method widely used at present to evaluate the ability of drugs to induce apoptosis. MTT (3- (4, 5) -dimethylthiahiazo (-z-y 1) -3, 5-di-phenytetrazoliumromide) acts as a yellow colored dye, and is known under the trade name thiazole blue. The principle of MTT detection can be summarized as that the mitochondria of living cells contain a reducing succinate dehydrogenase, which can reduce yellow MTT to insoluble blue-violet crystalline material formazan (Formazan) and deposit in the cells. In contrast, mitochondria in dead cells do not contain such a reducing succinate dehydrogenase, and therefore MTT cannot be reduced, and formazan having blue-violet crystals cannot be produced. Related experiments show that dimethyl sulfoxide (DMSO) organic solvent can dissolve formazan deposited in cells, and an enzyme-labeled instrument is adopted to measure the light absorption value (OD value) of the formazan at the wavelength of 490nm or 570nm, so that the corresponding living cell number is indirectly obtained. The formula for calculating cell viability (I%) is as follows:
I%=[(A-A0)/(As-A0)]×100%
Wherein A, A 0 and As are OD values of the cytosolic group, the blank solvent control group and the cytosolic solvent control group, respectively. In the experimental process, the logarithmic value of the corresponding concentration of the drug is taken as an abscissa, the cell survival rate (%) is taken as an ordinate, and experimental data are expressed by Mean + -SD of three independent experiments. Finally, the experimental data are plotted by GRAPHPAD PRISM 6.0.0 software to obtain the relationship curve between cytotoxicity and drug dosage
Experimental method
(1) Plating, namely selecting 4T1 cells with good growth state, removing old culture medium, and cleaning three times by adopting sterile PBS. Subsequently, 1mL pancreatin (0.25% EDTA) was added for digestion and centrifugation. Cell resuspension was performed using fresh cell culture medium, counted by a cell counter, and its density was diluted to 8.0X10 4 cells/mL. 100 μl of diluted different cell suspensions were then added to each well of the 96-well plate, and the different drug concentration groups and all control groups were set to 6 multiplex wells to reduce experimental errors. Finally, all 96-well plates were placed in a 37 ℃ 5% co 2 cell incubator for adherent culture.
(2) Dosing-first, old medium in 96-well plates was removed and each well was washed three times with sterile PBS. Then, the drugs formulated by fresh cell culture medium were sequentially added to different 96-well plates and incubated in a 37 ℃ 5% co 2 cell incubator for 12h.
(3) Illumination after incubation was completed, the old medium containing the drug was removed from the 96-well plate and washed three times with sterile PBS to completely remove the non-ingested drug. Then, 100. Mu.L of new cell culture medium was added to each well, and the 96-well plate for phototoxicity test was placed under an LED laser plate (λ=670nm, 4mW/cm 2,1.2J/cm2) and irradiated for 5min. Finally, the cells were incubated in a 37℃5% CO 2 cell incubator for 24h. The dark toxicity experiment process is free from illumination treatment, and other operation steps are consistent with the light toxicity experiment and are carried out in a light-proof environment.
(4) After 24h incubation, 96-well plates of both cells were removed from the incubator and 10. Mu.L of the pre-formulated MTT solution was added to each well, followed by continuous incubation in the cell incubator for 4h. Then, the supernatant from the 96-well plate was removed and 100 μl of DMSO solvent was added to each well and placed in a 37 ℃ shaker for 30min to allow for adequate solubilization of formazan that may be generated. Finally, the OD of the 96-well plate at 570nm was measured by an enzyme-labeled instrument.
From the experimental data, no significant cytotoxicity was observed for 4T1 cells in the absence of laser irradiation for all drug groups, indicating negligible dark toxicity for the different drug groups. However, under the condition of 5min irradiation under an LED laser plate (λ=670nm, 4mw/cm 2,1.2J/cm2), the photo cytotoxicity of the Da-cd@cet NPs group was significantly higher than that of the single-targeted CET, cd@cetnps group and blank control group, indicating that the Da-cd@cetnps with dual targeting function exhibited more significant killing ability on 4T1 cells in a smaller concentration range, thereby achieving stronger anti-tumor cell proliferation ability.
In vivo photodynamic activity characterization (see FIG. 6)
200 Mu L of physiological saline containing 4T1 cells (2X 10 6 cells) is inoculated into the right hind limb of a mouse subcutaneously to establish a tumor model, and the growth condition of the tumor is closely observed. After 5 days, after tumors grew to a size of 50-100mm 3 (tumor volume was calculated using the equation of length x width 2/2), all mice were randomly divided into five groups (Control, ce6, CET, cd@cetnps, da-cd@cetnps), five per group (n=5) for the experiment.
Before intravenous drug administration, the mice were weighed and tumor size was measured, then each mouse was respectively intravenous injected with a different drug (100 ug/mL,200 μl) and physiological saline solution in groups, after 12 hours of administration, the tumor area was irradiated with a 670nm laser for 10 minutes, and the tumor volume was monitored every other day. The growth status of mice and their tumor size indicate that the single targeted material group, including the free Ce6, CET and cd@cet NPs group, only partially inhibited tumor growth, whereas the Da-cd@cet NPs group almost completely inhibited tumor growth, which fully suggests that the dual targeted nanomaterial photosensitizer improved the tumor inhibition ability of PDT. Meanwhile, da-CD NPs group tumors grew rapidly, similar to control group. From the relative tumor growth curves plotted in the measurement data, it is evident that the Da-CD@CET NPs group can almost completely inhibit the growth of tumors. In addition, the weight of resected tumor after 14 days gave the same tumor inhibiting results. All experimental results show that Da-CD@CET NPs with double targeting functions can accurately deliver photosensitizers to triple-negative breast cancer cells and further deliver the photosensitizers to an endoplasmic reticulum, so that the targeted photodynamic therapy of triple-negative breast cancer is realized, and the obvious in-vivo tumor inhibition effect is shown.
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