A Self-guidance Biological Hybrid Drug Delivery System Driven By Anaerobes To Inhibit The Proliferation And Metastasis Of Colon Cancer

In recent years,the incidence and mortality of cancer have increased significantly worldwide,among which colon cancer ranks third in incidence and second in mortality.and colon cancer has the typical characteristics of high metastasis and high diffusion,such as causing liver metastasis,lung metastasis,etc.Surgical therapy causes large trauma,prone to postoperative complications and has limited therapeutic effects in cases where cancer cells have metastasized.Traditional chemotherapy drugs can freely pass through the vascular walls of normal tissues and tumor tissues,with poor targeting and strong toxicity.Although emerging nano-drugs can accumulate in tumor sites through enhanced permeability and retention（EPR）effect,most of them remain in the periphery of tumor blood vessels due to the obstruction of tumor matrix and the penetration into the deep part of the tumor is very weak.Therefore,it is of great significance to design preparations which can achieve precise targeting,deep penetration and controlled release of drugs at tumor sites to effectively inhibit tumor growth and metastasis.This topic used sodium alginate（Alg）as the matrix material,Fe³⁺as the cross-linking agent,doxorubicin（DOX）as the therapeutic drug,and the Bifidobacterium infantis（BI）transgenic with Endostatin（ES）gene as the biological carrier.Finally,a biological hybrid drug delivery system BI-ES-FeAlg/DOX driven by anaerobic bacteria was designed,which realized the synergy of genetic engineering technology and chemotherapy to inhibit the growth and metastasis of colon cancer.In this experiment,firstly,anaerobic bacteria BI was cultured under suitable conditions,and the plasmid of p GEX-4T-1-Endostatin was transferred into BI by electroporation to obtain engineering bacteria BI-ES.Then,we used the one-step cross-linking method to synthesize iron-alginate（FeAlg）nanogel on the surface of BI-ES and loaded DOX to obtain BI-ES-FeAlg/DOX.When BI-ES-FeAlg/DOX entered the body,due to the hypoxia tropism of the anaerobic bacteria BI-ES,the preparations could accurately target the tumor site and enter the deep hypoxic area.Under the weakly acidic and highly reducing conditions of the tumor microenvironment（TME）,Fe³⁺was reduced to Fe²⁺.Consequently,the FeAlg nanogel disintegrated and DOX was released to kill tumor cells.At the same time,BI-ES growed in the hypoxic zone and expressed ES protein.ES protein could down-regulate bFGF and cooperate with DOX to down-regulate the expression of VEGF,inhibiting tumor growth and metastasis by blocking tumor angiogenesis.In addition,Fe²⁺could react with H₂O₂ to produce a large amount of·OH,which could further induce tumor cell death.The related characterization results showed that FeAlg was successfully synthesized on the surface of BI-ES,with a thickness of about 70-80 nm.BI-ES-FeAlg could effectively load DOX,with a drug loading rate of 22.19%and encapsulation rate of 8.99%.The growth rate of each cultures had no obvious abnormality,indicating that the previous operations did not affect the activity of BI.And under simulated TME conditions,the cumulative release rate of BI-ES-FeAlg/DOX reached 67.72%after 72h,while it was only 6.83%in the PBS group.It proved that BI-ES-FeAlg/DOX could be effectively released in the tumor environment but remained relatively stable in the blood circulation.The results of in vitro bacterial invasion assay,uptake assay and deep penetration assay showed that CT-26 colon cancer cells could quickly and efficiently absorb BI-Es-FeAlg/DOX and carry DOX to the center of tumor globules,which proved that BI-ES had excellent ability to invade tumor cells and target hypoxia,and could deliver drugs to the deep hypoxia area.BI-ES-FeAlg/DOX had a significant anti-tumor effect,the inhibition rate of BI-ES-FeAlg/DOX（10μg/m L）could reach 89.15%at 48 h.At the same time,the reactive oxygen experiment indicated that FeAlg could produce a large amount of·OH in tumor cells,which further enhanced the anti-tumor effect.As for the anti-metastasis effect,Western blot,wound-healing experiments and Transwell experiments all showed that by down-regulating the expression of bFGF and VEGF,BI-ES-FeAlg/DOX could effectively block the formation of tumor blood vessels,thus inhibiting tumor metastasis.In the AOM/DSS mouse model and the CT-26 tumor-bearing model,BI-ES-FeAlg/DOX showed well tumor targeting and deep penetration capabilities,which helped it to fully exert its anti-tumor effect.Besides,Fe²⁺could react with the H₂O₂ in tumor to generate a large amount of·OH,which strengthened the inhibitory effect and promoted the death of tumor cells.Pharmacodynamics results showed that BI-ES-FeAlg/DOX could effectively alleviate the pathological symptoms related to colon cancer and had significant anti-tumor efficiency.The tumor inhibition rate of CT-26tumor-bearing mice reached 82.12±3.08%,which could significantly inhibit tumor volume and had good biosafety.In CT-26 lung metastasis model,BI-ES-FeAlg/DOX also showed excellent anti-metastasis effect.The lung condition was improved,and the number of metastases was significantly reduced.The histological section showed that the lung tissue morphology was close to normal,and the alveolar structure remained intact.There was no obvious abnormality in body weight and the biological safety was good.ELISA analysis results showed that the expression levels of bFGF and VEGF in BI-ES-FeAlg/DOX group were decreased,which was consistent with the Western blot results.