Design,Preparation And Application Of Glucose Oxidase-based Novel Intelligent Nanoreactors In Targeted Therapy Of Breast Cancer

As the "number one killer" of women’s health,the morbidity and mortality of breast cancer are rising year by year.It surpassed lung cancer for the first time in 2020 and became the most common cancer in the world,seriously harming human health.At the same time,its complex pathogenesis makes the unsatisfactory effect of traditional anti-breast cancer therapies(such as chemotherapy,surgery,radiotherapy),and it is difficult to meet the increasing breast cancer patients’ demand for efficient and safe therapeutic programs,so urgently need to develop new treatments.Glucose oxidase(GOx),as a biocatalyst,can effectively catalyze the oxidation of intratumoral glucose into gluconic acid and toxic hydrogen peroxide(H2O2),which can not only inhibit tumor proliferation through "new" starvation therapy,but also regulate the tumor microenvironment(such as increasing acidity and improving the level of H2O2).Therefore,GOx can be strategically combined with traditional anti-breast cancer therapies(such as chemotherapy)to make up for the shortcomings of traditional treatments(such as reduced sensitivity),develop "super-additive"multi-modal synergistic therapeutic programs,and provide new options for breast cancer patients.However,GOx is a protein with poor stability and short half-life in vivo,which greatly limits its application in tumor therapy.At the same time,traditional chemotherapy drugs lack tumor selectivity and targeting,have side effects and are easily eliminated by the body.Therefore,it is necessary to develop a suitable drug delivery system to solve the above problems and effectively deliver them to the breast cancer site.Due to its excellent biocompatibility,good in vivo pharmacokinetic behavior and high drug loading capacity,liposomes have received extensive attention in drug delivery,and some liposomal nano-drugs(such as Myocet)have been approved for market.However,traditional liposomes have poor targeting ability for breast cancer,which seriously affects the therapeutic effect of drugs.Based on the therapeutic principle of "targeted tumor suppression,non-toxic anti-tumor" and the weak acidic of tumor microenvironment,this paper designed and synthesized a variety of novel intelligent liposome ligands with excellent breast cancer targeting and tumor microenvironment response abilities for the first time.Then,with liposome as the basic carrier and based on the unique catalytic properties of GOx,two kinds of novel intelligent liposomal nanoreactors with clinical transformation potential were exploited.By effectively regulating the tumor microenvironment,the liposomal nanoreactors were complementary with chemotherapy,so as to achieve accurate,intelligent,safe and efficient treatment for breast cancer.In addition,this paper for the first time ingeniously combined the nanoreactors’ ability of regulating the tumor microenvironment(such as increasing acidity and in-situ production of NO)with the intelligent breast cancer targeting of liposomes,achieving self-promoting deep penetration and retention in solid tumors,maximizing the delivery of drugs to the core region of breast tumors and exert their effects.The research contents and main conclusions of this paper are as follows:The first part of the work:In view of the poor targeting ability of traditional liposomes for breast cancer,this part selected biotin molecule which can specifically recognize the sodiumdependent multivitamin transporter(SMVT)as the breast cancer active targeting group of liposome.After being modified by polyethylene glycol(PEG)long chain,it was linked to the cholesteric moiety through an acid-sensitive hydrazone bond(Hz).Using biotin,cholesterol,etc.as raw materials,a series of novel smart liposome ligands Bio-PEGn-Hz-Chol(n=2000,3350,5000)with different PEG chain lengths,breast cancer active targeting and tumor microenvironment response capabilities were designed and synthesized through 11-step reactions.In addition,liposome ligand DSPE-PEG2000-R8 with deep penetrating ability of solid tumor was designed and synthesized by coupling R8 peptide with DSPE-PEG2000-Mal through Michael addition reaction.Subsequently,through particle size potential and cell uptake experiments,the ability of Bio-PEGn-Hz-Chol ligands with different PEG chain lengths to conceal the R8 peptide was evaluated.Finally,Bio-PEG3350-Hz-Chol and DSPE-PEG2000-R8 were used to prepare an intelligent liposome(Bio-R8-Lip)that has breast cancer active and passive targeting,tumor microenvironment response,and solid tumor deep penetration capabilities.Then,a novel intelligent liposomal nanoreactor GOx-Bio-R8-Lip was exploited by encapsulating GOx with this liposome.Through sensitizing chemotherapy(PTX-Bio-R8-Lip),synergistic breast cancer treatment was achieved.After entered the body,under the synergistic effect of the EPR effect and biotinmediated active targeting,Bio-R8-Lip specifically accumulated at the breast tumor site.The acid-sensitive hydrazone bond gradually hydrolyzed in the weakly acidic environment of tumor,exposing R8 peptide,promoting the penetration of liposomes in the tumor tissues,thus delivering GOx and paclitaxel(PTX)to breast cancer cells accurately and efficiently.Subsequently,GOx could oxidize intratumoral glucose into gluconic acid and H2O2 rapidly,improve the acidity and H2O2 content of the tumor microenvironment,accelerate the breakage of hydrazone bond and the exposure of R8 peptide on the liposome surface,further increase the penetration ability of liposomes in the deep breast tumor site,and amplify the efficacy of chemotherapy,and effectively treat breast cancer through synergistic chemo/starvation/oxidation therapy.We evaluated the ability of GOx-Bio-R8-Lip to regulate the breast tumor microenvironment through in vivo and in vitro catalysis experiments.And a series of in vitro and in vivo breast cancer targeting and pharmacodynamics evaluation of the co-administration group(PTX-Bio-R8-Lip&GOx-Bio-R8-Lip)were performed with the single administration group(PTX-Bio-R8-Lip or GOx-Bio-R8-Lip)as control.The results of living imaging,excised tumor tissue imaging and immunofluorescence staining of tumor sections showed that PTX-Bio-R8-Lip&GOx-Bio-R8-Lip had the strongest breast cancer targeting ability at all time points after administration,and could penetrate into the deeper tissues of the tumor.In vivo efficacy test results showed that PTX-Bio-R8-Lip&GOx-Bio-R8-Lip had the best anti-breast cancer effect,even if the dose of paclitaxel reduced to 1 mg/kg,the tumor inhibition rate(TIR)was still 93.8%.In addition,the preliminary safety evaluation results showed that PTX-Bio-R8-Lip&GOx-Bio-R8-Lip did not cause lesions in the major organs,and the liposomal materials had no obvious cytotoxicity,which met the demand of"targeted tumor suppression,non-toxic anti-tumor".The second part of the work:Breast cancer(especially triple-negative breast cancer)is highly aggressive,prone to distant metastasis of tumors,and lacks effective treatments.According to statistics,more than 90%of breast cancer patients die from tumor metastasis.In this part,we further optimized the active targeting group and acid-sensitive bond on the basis of the first part,designed and synthesized an integrated multifunctional intelligent liposome ligand(Bio3-PEG3350-BIm-Chol)which has stronger breast cancer active targeting ability,higher tumor microenvironment responsiveness,and the ability to deeply penetrate tumors and escape lysosomes,successfully realizing the maximum function of a single ligand.Then,the liposome(Bio3-BIm-Lip)modified by the above ligand was used to simultaneously encapsulate GOx and Larginine to construct a novel multifunctional intelligent liposomal nanoreactor(GOx+L-Arg-Bio3-BIm-Lip).And for the first time,it was combined with chemotherapy(PTX-Bio3-BIm-Lip)to effectively inhibit the proliferation and metastasis of breast cancer through the multi-modal synergistic effect of chemo/starvation/oxidation/NO gas therapy.The specific content is as follows:In order to further enhance the targeting ability of liposomes,this part modified the branched biotin structure to the terminal of PEG3350 for the first time,and linked to the cholesteric moiety through an acid-sensitive hydrazone bond.Using biotin,diethanolamine,etc.as raw materials,a new class of branched liposome ligands(Bio2-PEG3350-Hz-Chol and Bio3-PEG3350-Hz-Chol)containing different biotin densities were designed and synthesized through 21-step and 25-step reactions,respectively.Then through preliminary targeting experiments,we selected the threebranched biotin structure(Bio3)as the breast cancer active targeting group of liposome ligand.Subsequently,we continued to optimize the acid-sensitive bond and used the imine bond as the acid-sensitive bond of the liposome ligand for the first time.Using biotin,4-formylbenzoic acid,etc.as raw materials,a series of novel liposome ligands(Bio3-PEG3350-AIm-Chol,Bio3-PEG3350-PIm-Chol and Bio3PEG3350-BIm-Chol)containing different types of imine bond were designed and synthesized via 23-step,26-step and 25-step reactions,respectively.Through in vitro bond breaking experiment,we finally selected the benzyl imine bond(BIm),which is more responsive to tumor microenvironment than hydrazone bond,as the acid sensitive bond of liposome ligands.Compared with Bio-R8-Lip,the liposome(Bio3-BIm-Lip)modified by Bio3PEG3350-BIm-Chol can be more concentrated in the breast cancer site due to the synergism of EPR effect and three-branched biotin mediated stronger breast cancer active targeting.In addition,the benzyl imine bond(BIm)can be hydrolyzed rapidly in the tumor microenvironment,so that the Bio3-PEG3350 fragment,which has been fully used,can quickly fall off from the liposome surface.Then,the amino group resulting from the hydrolysis of the imine bond is protonized,and the surface charge can be reversed while the particle size of the liposome is reduced,thus achieving efficient tumor tissue penetration.After the liposomal nanoreactor GOx+L-Arg-Bio3-BIm-Lip successfully entered breast cancer cells through the above effects,it could automatically trigger a GOx-centered cascade reaction without external conditions.Firstly,GOx catalyzed the oxidation of intratumoral glucose into gluconic acid and toxic H2O2,which could trigger starvation and oxidative therapy;Secondly,the large amount of H2O2 generated in situ reacted with L-Arg to produce high concentration of NO gas,which could inhibit the growth,invasion and metastasis of breast cancer;Thirdly,the increase of tumor microenvironmental acidity could also promote the breakage of imine bond and the protonation of amino group on the surface of liposome.And the vascular effect produced by NO could enhance EPR effect and the deep delivery of liposomes.These effects further promoted the efficient penetration and retention of liposomes in the deep tissues of breast tumors,and significantly improve the therapeutic effect of breast cancer.As in the first part of the work,we carried out a series of in vivo and in vitro breast cancer targeting and pharmacodynamics evaluation of the co-administration group(GOx+L-Arg-Bio3-BIm-Lip&PTX-Bio3-BIm-Lip).In addition,systematic catalytic experiments,tumor blood oxygen saturation experiments,and classical cell invasion and lung metastasis experiments were conducted to investigate the ability of nanoreactor(GOx+L-Arg-Bio3-BIm-Lip)in producing NO,modulating breast tumor microenvironment and inhibiting breast cancer cell invasion and metastasis.Catalytic experiments showed that GOx+L-Arg-Bio3-BIm-Lip could produce sufficient H2O2 and NO gas continuously and efficiently,and significantly reduce the pH value of the solution and the cell living environment.The results of photoacoustic imaging(blood oxygen saturation)showed that NO produced by GOx+L-Arg-Bio3BIm-Lip could effectively increase the permeability of blood vessels,enhance blood perfusion in the deep part of the breast cancer,improve hypoxia,and promote GOxmediated glucose oxidation.The results of living imaging,excised tumor tissue imaging,tumor spheroids uptake and immunofluorescence staining of tumor sections showed that the co-administration group had the strongest breast cancer targeting ability and deep tumor penetration ability.The results of in vivo and in vitro pharmacodynamic experiments showed that GOx+L-Arg-Bio3-BIm-Lip&PTXBio3-BIm-Lip had the strongest kill capability on breast cancer cells and exhibited good therapeutic effect in tumor-bearing mice.Even if the dosage of paclitaxel reduced to 1 mg/kg,some mice were completely cured(tumor disappeared).The results of wound healing,invasion and lung metastasis assay showed that GOx+LArg-Bio3-BIm-Lip could effectively inhibit the in vitro migration and invasion of breast cancer cells(4T1 and MDA-MB-231)and the in vivo lung metastasis,and GOx+L-Arg-Bio3-BIm-Lip&PTX-Bio3-BIm-Lip had the best effect.To sum up,in this paper,a variety of bran-new intelligent liposome ligands were designed and synthesized for the first time by means of chemistry,and two novel intelligent liposomal nanoreactors were successfully developed based on GOx.By effectively regulating the tumor microenvironment,a series of positive chain effects were produced,making up for the deficiency of traditional chemotherapy,and realizing precise,secure and efficient "super-additive" multi-modal synergistic treatment of breast cancer.Compared with traditional therapies,these two novel combined treatment strategies we designed have significant anti-tumor effects without obvious toxicity and side effects,which can provide new ideas and inspirations for breast cancer therapy and have broad application prospects.