| Background and objective:Breast cancer(BC)is the most common malignant tumor in women today and has a serious impact on women's life and health.At present,the treatment concept of BC has achieved remarkable results in surgery,chemotherapy(CT),targeted therapy,radiotherapy(RT),endocrinotherapy,and immunotherapy.The multidisciplinary diagnosis and treatment model for breast cancer has been popularized,and BC has become a curable disease.Chemotherapy(CT)still has an indispensable and essential position in the comprehensive treatment of BC;however,the serious side effects and multidrug resistance caused by CT are still serious problems that threaten the lives and health of BC's patients.In practice,BC is based on combined chemotherapy,with the purpose of achieving dose reduction,enhancement,and reduction of drug resistance.Currently,taxane chemotherapeutics are widely used in the treatment of BC,such as nanoparticle albumin-bound paclitaxel(Nab-PTX).Nab-PTX was approved for the treatment of metastatic BC by FBA in 2005 and is also used in the application of neoadjuvant and postoperative adjuvant chemotherapy for BC.In particular,Nab-PTX makes the hydrophobic paclitaxel easily soluble in water and safer in administration.Meanwhile,the albumin on the surface of the nanoparticles is targeted to bind with SPARC over-expressed by cancer cells,which further increases the accumulation of Nab-PTX in tumor tissues.However,taxane drugs have a weak ability to kill cancer stem cells(CSCs)that cause BC recurrence,metastasis,and drug resistance.CSCs refer to a special subgroups of tumor cells with self-renewal and tumor initiation capabilities in tumor tissue.Also,the presence of CSCs is the internal reasons for resistance to CT drugs,new tumor-targeted drugs,and radiotherapy,so that CSCs are able to survive in the standard cancer treatment and start tumor recurrence and metastasis.Studies revealed that breast cancer stem cells(BCSCs)are found in BC tissues,and BCSCs are the main cause of BC recurrence,metastasis,and drug resistance.Salinomycin(Sal)has a strong ability to inhibit BCSCs and the efficacy of Sal is 100 times stronger compared to paclitaxel.Specifically,Sal not only kills CSCs in mice,inhibits mice from producing new tumor cells,but also restricts the growth of existing tumors.At the same time,PTX combined with Sal has a synergistic effect in the treatment of BC,which can improve the treatment effect.In addition,these two chemotherapeutic drugs have different mechanisms of action,and their combined use can prevent the emergence of multidrug resistance.However,Sal and its sodium salt are also insoluble in water,and these toxic and side effects on the body's nerves and muscles are the main problems limiting its clinical application.Using medical multifunctional nanomaterials as carriers,a research hotspot,is one of the directions to overcome its clinical application problems.Among multifunctional nanomaterial carriers,layered double hydroxide(LDH)nanoparticles(NPs)composed of anionic clay have a unique two-dimensional spatial structure.Due to the considerable advantages of LDH in biomedical applications,it has attracted attention from many biomedical and composites experts in recent decades.First,LDH can easily and uniformly incorporate various combinations of divalent and trivalent metal cations(including Mn(II)or Cu(II))into the layered structure as a high-performance magnetic resonance imaging contrast agent for the cancer diagnosis;secondly,LDH can effectively load and deliver anionic chemical drugs,photosensitizers and therapeutic genes in vivo and in vitro;thirdly,after intravenous injection,LDH lamellar nanoparticles can be effectively enriched in tumor tissues and released drugs and promote the phagocytosis of cancer cells;fourthly,LDH is easily degraded into biosafe ions and molecules after it exerts a therapeutic function at the diseased site,reducing the side effects that remain in the body for a long time;fifthly,LDH is usually positively charged under physiological conditions and can be used at any time adhere to the cell membrane,greatly promote the cells to absorb the loaded therapeutic drugs through the endocytic escape pathway.At present,LDH as a nano-medicine carrier also has some shortcomings.For example,LDH is positively charged under physiological conditions.Although it shows high affinity to the cancer cell membrane,it is easy to interact with plasma proteins and be quickly eliminated,and it is easy to cause the entry of positively charged NPs into normal cells causes adverse reactions,which is not conducive to the enrichment of LDH at the tumor site.The nanoparticle carrier modified with a negatively charged group can effectively reduce the adsorption of plasma proteins and the clearance rate of the mononuclear phagocytic system,extend the circulation time of the nancparticles in the blood,and increase the enrichment in the tumor site.However,modifying the negatively charged coating on the surface of the LDH will greatly weaken the binding ability of the nanocarrier and the tumor cell membrane,which is not conducive to the uptake of the drug-carrying nanoparticles by the cancer cells,resulting in low therapeutic efficacy.In order to solve this problem,our research group used BSA-coated nanoparticles formed by LDH,which can reduce the aggregation of LDH particles.BSA coating reduces the adsorption of serum proteins,prolongs the circulation time,and enhances the enrichment of tumor tissue.BSA coating is effective for cancer.Cell phagocytosis has little effect.Moreover,it can depolymerize in a solution with pH 6.8 or more acidic to release the positively charged LDH in the composite nanoparticles,so that the composite nanoparticles are protected by the negatively charged bovine serum albumin(BSA)coating in the blood circulation and are enriched by the EPR effect in the tumor microenvironment,and under the weak acid condition of the tumor microenvironment,the BSA on the surface of NPs depolymerizes and falls off,exposing the positively charged LDH,and promoting tumor cells to swallow the drugs carried by LDH,increasing the efficacy;At the same time,after LDH enters the tumor,it adsorbs the proteins in the tumor microenvironment and slightly aggregates,which is more conducive to increase the enrichment at the tumor site.Our hypothesis:We plan to use multifunctional LDH to co-carry Sal and Nab-PTX and modify LDH with albumin to extend the circulation time of the negatively charged Sal/LDH@Nab-PTX NPs system in the blood and enhance its accumulation in tumor tissues.The strategy of cellular uptake makes it more conducive to the implementation of an effective mode of BC treatment based on nanomaterials combined chemotherapy.This research consists of three parts:The first part is the preparation and identification of multifunctional nanocarriers co-delivery Sal and Nab-PTX;the second part is the multifunctional nanocarrier co-carrying Sal and Nab-PTX inhibit breast cancer 4T1 cells in vitro;the third part is the study of multifunctional nanocarriers carrying Sal and Nab-PTX for targeted inhibition of BC and metastatic cancer in vivo.Part ? Preparation and identification of multifunctional nanocarriers co-delivery salinomycin and nanoparticle albumin-bound paclitaxelObjective:Through the preparation and optimization of multifunctional nano-carrier co-loaded salinomycin and nanoparticle albumin-bound paclitaxel,a method for detecting the content of chemotherapeutic drugs was established and the preparation was characterized and identified,and create a foundation for further experimental research in vitro and in vivo.Methods:1.The detection wavenumber of Sal was determined by the UV spectrophotometer test,the intra-day and inter-day precision of Sal were investigated,and the standard curve for the determination of Sal content was drawn.Synthesize LDH nanoparticles(LDH NPs)and LDH nanoparticles(Sal/LDH NPs)successfully loaded with Sal by high temperature coprecipitation ion exchange method and coprecipitation ion exchange method respectively,and characterize and identify the synthesized Sal/LDH NPs and the content of Sal in Sal/LDH NPs was detected and calculated corresponding encapsulation rate and upload rate.2.Using LDH NPs and Sal/LDH NPs as carriers respectively,through electrostatic interaction,bovine serum albumin(BSA)is carefully adhered to the surface of the corresponding NPs to wrap and construct LDH@BSA NPs and Sal/LDH@BSA NPs,to characterize and identify the synthesized LDH@BSA NPs and Sal/LDH@BSA NPs,At the same time,the pH-responsive Sal/LDH@BSA NPs release situation is studied.3.Using LDH NPs and Sal/LDH NPs as carriers respectively through electrostatic interaction,the mixture of BSA and Nab-PTX(the mass ratio of the two is 90:10 to 99:1)is carefully adhered to the corresponding LDH@Nab-PTX and Sal/LDH@Nab-PTX NPs were constructed by coating the surface of NPs,and the synthesized LDH@Nab-PTX NPs and Sal/LDH@Nab-PTX NPs were characterized and identified.Results:1.The detection wavelength of Sal by uv spectrometer is 226 nm.The intra-day precision and intra-day precision RSD%values of Sal content determined by this method are both less than 2%,and the precision is good,which could meet the requirements of this study.The average quality of LDH was 8.35 ±0.45 mg/mL,and the optimal Sal/LDH NPs with high upload rate was 98.69%and 48.66%,respectively.The standard curve is drawn for the determination of Sal content,which maintain a good linear relationship(R2=1)within the concentration range study.2.LDH,Sal/LDH,LDH@BSA,Sal/LDH@BSA,LDH@Nab-PTX and Sal/LDH@Nab-PTX NPs are characterized respectively.The average particle size ranges from 75.4 nm of LDH NPs to 131.3 nm of LDH@BSA NPs;from 92.8 nm of Sal/LDH NPs to 122.8 nm of Sal/LDH@BSA NPs;from 143.2 nm of LDH@Nab-PTX NPs to 155.5 nm of Sal/LDH@Nab-PTX NPs.The above particles size is uniform,the distribution is uniform,and the PDI value of the NPs are about 0.1.Analyze the mass ratio of LDH:BSA by combining the NPs particle size change and PDI value after reconstitution of the lyophilized powder,and the ratio of 1:10 is preferably used as the test formulation.3.The surface charge of the particles shows:After positively charged Sal/LDH NPs are coated with BSA,Sal/LDH@BSA NPs,LDH@Nab-PTX NPs and Sal/LDH@Nab-PTX NPs are all negatively charged,indicating that the potential is determined by 45.5 mV for Sal/LDH NPs to-22.4 mV for Sal/LDH@BSA NPs,-17.2 mV for LDH@Nab-PTX NPs,and-23.0 mV for Sal/LDH@Nab-PTX NPs.4.In the stability experiment,Sal/LDH@Nab-PTX NPs have good stability within 24 hours in deionized water,PBS and medium containing 10%FBS at room temperature.5.The results of release experiments show that the release behavior of the two drugs is good,and the release in the pH 6.0 buffer is significant compared with the pH 7.4 buffer,the difference is as high as 63%in vitro.6.When the mass ratio of Sal to LDH is 0.1:1,0.5:1 and 1:1,the particle size of Sal/LDH NPs does not change much.7.The x-ray diffraction(XRD)pattern analysis of the lyophilized powder of Sal/LDH NPs produced by the high-temperature co-precipitation ion exchange method shows characteristic reflections corresponding to the planes(003)and(006)of the LDH,indicating that the interlayer spacing of LDH and Sal/LDH nanoparticles is similar,which means that the layered structure of LDH crystals is not affected when Sal is loaded and intercalated.This also indicates that Sal molecules are randomly inserted into the middle layer of LDH,resulting in a wider XRD peak of Sal/LDH.8.The FT-IR spectra of LDH,Sal/LDH,LDH@Nab-PTX and Sal/LDH@Nab-PTX samples show that LDH NPs show a wide band at 3479 cm-1,corresponding to the H2O molecules absorbed in LDH NPs and OH stretching of the OH group(the absorption band at 3479 cm-1 indicates the stretching vibration of physically adsorbed water molecules in active hydroxyl or brucite layers in LDH).It is speculated that the bands at 1632 cm-1 and 1367 cm-1 are attributed to H2O shear vibration and contaminated CO32-stretching vibration respectively.The M-O vibration and M-O-M bending(M=Mg or Al)in the main layer of LDH NPs resulted in a strong adsorption peak in the range of 510-800 cm-1.These typical bands also appear in all samples containing LDH.It is worth noting that three peaks at 2960 cm-1,1655 cm-1 and 1544 cm-1 of BSA are clearly observed in the LDH@Nab-PTX nanohybrid.These peaks are related to CH,C=O and the stretching vibration of the NH bond is related.However,since the Nab-PTX content on LDH nanoparticles is very small(Nab-PTX accounts for 0.89 wt%,PTX accounts for 0.089 wt%),no obvious PTX bands are seen in the FT-IR spectrum of LDH@Nab-PTX.The LDH phase is also present in the Sal/LDH sample,indicating the properties similar to the LDH material.Since Sal exists in the LDH layer in the form of ions,we neutralized the prepared Sal-Na samples with NaOH and compared them in the FT-IR spectrum.The strong peak of Sal-Na's characteristic band at 2964 cm-1 was preliminarily identified as the stretching vibration of-CH3.This kind of vibration is also evident in the Sal(1)/LDH and Sal(0.5)/LDH nano hybrids.This shows that Sal was successfully embedded in the middle of the LDH layer.It is worth noting that the two strong peaks of the characteristic band of Sal at 1715 cm-1 and 1565 cm-1 are preliminarily identified as C=O(-O)symmetrical and asymmetrical stretching vibrations.This vibration occurs in the Sal(1)/LDH nanohybrid at 1712 cm-1 and 1569 cm-1 is clearly visible.It was confirmed that the negatively charged Sal was successfully loaded in the middle of the LDH layer.9.The TEM images of LDH,Sal/LDH@BSA and Sal/LDH@Nab-PTX in complete medium and the TEM image of Sal/LDH@Nab-PTX in deionized water stained with phosphotungstic acid,confirm the two-dimensional morphology of LDH,Sal/LDH@BSA,Nab-PTX and Sal/LDH@Nab-PTX and the coating layer of BSA.Conclusion:The Sal/LDH@Nab-PTX nanocarrier system that can load both Sal and Nab-PTX chemotherapeutics is accurately prepared,and the upload amount can be accurately controlled.Part ? Multifunctional nanocarriers co-delivery salinomycin and nanoparticle albumin-bound paclitaxel system to inhibit breast cancer 4T1 cells in vitroObjective:To compare the inhibitory effects of salinomycin and nanoparticle albumin-bound paclitaxel system carried by multifunctional nanocarriers on breast cancer 4T1 cells through cell experiments in vitro.Methods:1.Using confocal scanning microscope(CLSM)to study the cellular uptake of FITC/LDH@Nab-PTX NPs by 4T1 breast cancer cells.2.Use cell counting kit-8(CCK-8)to detect different concentrations of Sal and Nab-PTX liquids,Sal/LDH@BSA NPs liquids,LDH@Nab-PTX NPs liquids and the effect of Sal/LDH@Nab-PTX NPs liquid on the growth of breast cancer 4T1 cells,and the experimental study was compared to evaluate its toxicity to breast cancer 4T1 cells.Results:1.The most 4T1 cells can effectively endocytose FITC/LDH@BSA NPs after 2 hours of incubation,and as the incubation time increases,more NPs are gradually absorbed by the cells;CLSM images confirm the green fluorescence signal of FITC/LDH@BSA was observed inside the cell after 4 hours of culture,and it was mainly located in the cytoplasm.2.When the concentration of LDH NPs is as high as 400 ?g/mL,the viability of breast cancer 4T1 cells treated with LDH@BSA NPs is still higher than 90%,indicating that the nanocarrier we designed has good biocompatibility.3.Comparison of the cell viability of 4T1 cells treated with Sal at 100 ? for 1 hour and Sal/LDH at room temperature for 1 hour,and the cell viability of 4T1 cells treated with Sal/LDH at 100? for 1 hour and Sal/LDH at room temperature show:Sal after high temperature treatment does not affect the cell viability of inhibiting 4T1 cells.In the cytotoxicity experiment under single-drug treatment,Sal/LDH@BSA NPs,Nab-PTX NPs and LDH@Nab-PTX NPs compared with Sal/LDH@BSA NPs and LDH@Nab-PTX NPs.As the dose increased,the survival rate of cells in all groups gradually decreased.4.Sal and LDH@Nab-PTX NPs have lower 50%inhibitory concentrations(IC50),6.94 and 4.62 ?g/mL,respectively,and the 50%inhibitory concentrations(IC50)of Sal/LDH@BSA NPs and Nab-PTX NPs,they are 20.66 and 9.79 ?g/mL respectively.5.Choose the load ratio of Sal and Nab-PTX to be 1:1 for further combination therapy(mainly considering that PTX accounts for only 10 wt%of Nab-PTX,that is,Sal:PTX=10:1).Sal/LDH@Nab-PTX NPs treatment for 24 h reduced the viability of 4T1 cells in a dose-dependent manner.The cell survival rate of Sal(0.47-7.5?g/mL)and NDR-PTX NPs(0.47-7.5?g/mL)at the same dose and concentration is 44.8-16.6%.Significantly lower than Nab-PTX NPs,or Sal/LDH@BSA NPs,or LDH@Nab-PTX NPs alone,and Sal/LDH@Nab-PTX showed significant synergy(combination index from 1.74-1.98).Conclusion:1.LDH NPs carrier has good biocompatibility.2.Sal/LDH@Nab-PTX NPs can be used as an ideal nanocarrier to deliver anti-tumor drugs to the cytoplasm to induce cytotoxicity.3.The use of the same low dose of Sal/LDH@Nab-PTX NPs combined treatment of breast cancer 4T1 cells has a significant synergistic effect.The Sal/LDH@Nab-PTX NPs co-carrying Sal and Nab-PTX for breast cancer can overcome the limitations of single-drug therapy,reduce side effects and improve treatment effects.Part ?:Multifunctional nanocarriers co-delivery salinomycin and nanoparticle albumin-bound paclitaxel for targeted inhibition of breast cancer and metastatic cancer in vivoObjective:To compare the anti-tumor efficiency of salinomycin and nanoparticle albumin-bound paclitaxel carried by multifunctional nano-carriers in inhibiting breast cancer and metastatic cancer in vivo through experimental research on anti-tumor efficiency.Methods:1.A tumor model of breast cancer 4T1-Luc cells in tumor-bearing mice was established,and Balb/C mice were divided into the corresponding control group and drug treatment group.By observing the general situation and measuring the changes of body weight and tumor volume of mice.2.When the tumor grows to 50-100 mm3,divide the mice into groups for drug treatment under different conditions,observe and measure the general condition,body weight,tumor size changes,and important organs and lymph node tissue tumor metastasis for comparison and analyze.3.Through pathological immunohistochemical analysis of important organs,evaluate the combined treatment effect of Sal/LDH@Nab-PTX NPs in vivo.Results:1.In terms of orthotopic breast tumor,the tumor in the control group(PBS)grew rapidly and reached 1472.5±280.1 mm3 on the 28th day after the first vaccination.Compared with the control group,the low-dose(3 mg/kg)Sal/LDH@BSA NPs group had no statistically significant inhibitory effect on tumors suppression(882.7±650.2 mm3,p=0.19);the free Sal and Nab-PTX mixture group has a very significant inhibitory effect on tumor growth(72.9±82.7 mm3,p<0.01);Sal/LDH@Nab-PTX NPs group showed a very significant inhibitory effect on tumor growth(30.8±43.9 mm3,p<0.01),LDH@Nab-PTX NPs group showed a significant inhibitory effect on tumor growth(310.4±448 mm3,p<0.01).Compared with the Sal/LDH@BSA NPs group,Sal/LDH@Nab-PTX NPs group and the free Sal and Nab-PTX mixture group have a significant inhibitory effect on tumor growth(p<0.05);LDH@Nab-PTX NPs group showed no difference in the inhibition of tumor growth(p=0.16).Compared with the free Sal and Nab-PTX mixture group and the LDH@Nab-PTX NPs group,the Sal/LDH@Nab-PTX NPs group had no difference in tumor growth inhibition(p=0.90).There is no significant difference in the weight change of the mice.2.The tumor growth of the Sal/LDH@Nab-PTX NPs group was significantly inhibited.On the 28th day,the tumor tissues of 3 mice almost completely disappeared,and the tumor tissues of the other 2 mice were also very small(30.8±43.9 mm3).3.After treatment,tumor sections is stained with hematoxylin and eosin(H?E)and analyzed with terminal deoxynucleotide transferase dUTP marker(TUNEL).Many tumor cells in the Sal/LDH@Nab-PTX NPs group lost their membrane integrity,and their nuclear density was significantly reduced.TUNEL staining of tumor tissue also showed that the Sal/LDH@Nab-PTX NPs treatment group significantly induced tumor cell apoptosis and inhibited its proliferation in vivo.4.Comparison of the fluorescence values of the lungs and liver organs in each group showed:on the 28th day after the first vaccination,the fluorescence value of lung tissue in the PBS group reached?3.75×105,and the fluorescence value of liver tissue reached?2.93×105;Sal/LDH@BSA NPs group which the fluorescence value of lung tissue reached?1.07×106,and the fluorescence value of liver tissue was not measured;the tissue fluorescence value of the free Sal and Nab-PTX mixture group reached?5.07×105,and the fluorescence value of liver tissue was not measured;Sal/LDH@Nab-PTX NPs showed a significant inhibitory effect on tumor growth of 1.61×105,and the fluorescence value of liver tissue was not detected;LDH@Nab-PTX NPs showed a significant inhibitory effect on tumor growth of 1.07×106,and the fluorescence value of liver tissue was not detected.5.Histopathological analysis of hematoxylin and eosin(H?E)staining showed that there were obvious tumor nodules in the lung tissues of the PBS group,tumor cells were found in the lung tissues of the LDH@Nab-PTX group,and no obvious tumors cells were found in the lung tissues of the other three groups.Except for axillary lymph node enlargement with tumor cells in PBS group,no significantly enlarged lymph nodes are found in the other 4 groups.No significant abnormalities are observed in other major organs(heart,liver,spleen and kidney)in each group,further demonstrating the excellent biosafety of LDH nanoparticles.Conclusion:1.Sal/LDH@Nab-PTX NPs have a significant inhibitory effect on tumor growth,and can use LDH nanoparticles to effectively deliver to tumor tissues,promote cell uptake and release drugs in the acidic microenvironment of tumors.2.Sal/LDH@Nab-PTX NPs can inhibit the growth of tumor metastasis,and can reduce lung and liver tumor metastasis.The research firstly used the high-temperature co-precipitation ion exchange method to successfully insert Sal,which inhibits BCSCs,into the middle of the Mg3Al-LDH layer,and screened out Sal/LDH NPs with high upload rates;negatively charged Nab-PTX and BSA on the surface.The positively charged Sal/LDH@NPs adhere to the surface of LDH nanoparticles through electrostatic interactions between Nab-PTX and BSA;after identification,a pH-responsive Sal/LDH@Nab-PTX NPs carrier system was successfully constructed.Secondly,it was once again verified that the LDH NPs carrier has good biocompatibility through in vivo animal experiments and in vitro cell experiments.Sal/LDH@Nab-PTX NPs can be used as an ideal nanocarrier to deliver antitumor drugs to the cytoplasm to induce tumor cell apoptosis;use the same low dose of Sal/LDH@Nab-PTX NPs to treat breast cancer 4T1 cells,has significant mutual synergy.The Sal/LDH@Nab-PTX NPs co-carrying Sal and Nab-PTX for breast cancer can overcome the limitations of single-drug therapy,reduce side effects and improve treatment effects.In addition,the use of tumor models of tumor-bearing mouse breast cancer 4T1-Luc cells for drug treatment studies found that Sal/LDH@Nab-PTX NPs have a significant inhibitory effect on breast tumor growth,due to the effective delivery of LDH nanoparticles to tumor tissues,promote cell uptake and release drugs in the acidic microenvironment of tumors.At the same time,it was found that Sal/LDH@Nab-PTX NPs can effectively reduce lung and liver tumor metastasis.In summary,we use multifunctional LDH nanoparticles to co-carry Sal and Nab-PTX,modify LDH with albumin,and successfully constructed Sal/LDH@Nab-PTX NPs,which can enhance their accumulation and cellular uptake in tumor tissues.It provides an experimental basis for the implementation of an effective mode of breast cancer treatment based on nanomaterials combined chemotherapy. |
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