Eniginering Of A Cisplatin/Yeast Capsule Oral Delivery System For Targeted Treatment Of Cancer

Cisplatin(cis-diamminedichloroplatinum(II),CDDP)was first approved by the U.S.Food and Drug Administration(FDA)for the treatment of testicular cancer and bladder cancer in 1978.Then it has been widely used in clinical practice.As a broad spectrum anticancer drug,cisplatin exerts anticancer effects via multiple mechanisms,yet its most prominent mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of mitochondrial apoptosis.Cisplatin has a remarkable effect for varieties of solid tumors,including head,lung,colorectal,breast,ovarian cancer etc.Normally at the beginning of the cisplatin-based treatment of solid tumor,the tumor growth is obviously inhibited and even be significantly decreased.The condition of patients is also stable.As the treatment continues,finally the majority of patients develop resistance to cisplatin therapy(Especially in the patients bear colorectal,lung and prostate cancer).Moreover,a large part of tumors which are initially sensitive to cisplatin develop drug resistance eventually(Especially in ovarian tumor model).Cisplatin is intravenously given to patients in the clinical treatment of tumor,considering of the nephrotoxicity of cisplatin,it is necessary for the patient to accept hydration and diuresis simultaneously when the dose come up to 80~120mg/m2 during the long-term hospitalization.Since the resistance and nephrotoxicity limit the application of cisplatin in tumor treatment to a large degree,studies on the strategy of cisplatin-based tumor treatment mainly focused on two aspects: Reversal of cisplatin resistance and reduce the nephrotoxicity.Compared with other delivery systems,oral delivery with better patients’ compliance and security is the most widely-used.Clinical statistics shows that about 80% of the drug works via oral delivery system.During the research of oral drug delivery system,we found that yeast fungus and other microbes were confirmed to infect plenty of macrophages through gastrointestinal in the body.So it is promising to achieve the tumor targeted drug delivery by the monocyte/macrophage recruitment effect on the tumor site.Under such background,in this study we put forward the construction of cisplatin/yeast capsule(Pre CDDP/YC)oral targeted drug delivery system.We synthetic positively charged cisplatin prodrug(Pre CDDP)at first,then build the delivery system through electrostatic interaction between Pre CDDP and YC.After successfully construct of Pre CDDP/YC delivery system,we evaluate its safety and antitumor effect.Achieving the antineoplastic effect of cisplatin derived drugs by oral delivery,reduce the side effects of intravenous drip and improve the compliance of patients with no long-term hospitalization.Method1.Preparation of yeast capsuleTreating certain weight of Baker’s yeast,with Na OH and HCl respectively,and then extract with isopropyl alcohol and acetone to remove soluble components,vacuum drying at 20-25°C.2.Optimal preparation conditions of yeast capsuleTo study the influence of YC loading efficiency dealing with different alkali/acid processing time,we fixed alkali processing time constantly,change the acid treatment time to get the optimal acid treatment time.In the same way,we fixed acid processing time constantly,change the alkali treatment time to get the optimal alkali treatment time.3.Preparation of Pre CDDPCDDP active pharmaceutical ingredients(API)were dispersed into suspension in the water,then add Ag NO3 crystals.The mixture is stirred and incubated for the whole night.The solution of Pre CDDP is vacuum freeze dried to get the(Pre CDDP)-cis-[Pt(NH3)2(H2O)2](NO3)2 freeze-dried samples.4.Preparation and characterization of Pre CDDP/YCThe suspension of YC mixed with Pre CDDP solution were co-incubated at 37°C for certain time.Remove the supernatant after centrifugation,precipitation is vacuum dried to get Pre CDDP/YC.Then using Transmission electron microscopy(TEM)and Scanning electron microscopy(SEM)on the morphology characterization.5.In vitro release experimentsWe simulated the condition of gastrointestinal environment by using p H 1.2 HCl solution and p H 7.4 PBS solution.Certain amount of Pre CDDP/YC was suspended in the HCl and PBS solution,samples of different time points were determination by atomic absorption spectrometry(AAS),then calculating the accumulation of drug release percentage,draw the corresponding drug release curve.6.The influence of the biological activity of macrophagesMix the concentration gradient Pre CDDP solution with Raw264.7 macrophage cell.After a certain time,the cell viability was quantified by MTT to evaluate the influence of the biological activity of macrophages.7.Evaluation of in vitro antitumor effectHuman breast cancer MCF-7 and MCF-7 CDDP-resistance cell line(MCF-7/ADR),human hepatocellular carcinoma Hep G2,human cervical cancer Hela and human lung cancer A549 cells were treated with the medium containing concentration gradient Pre CDDP/YC for 24 h and 48 h.The cell viability was quantified by CCK8 kit.8.Evaluation of in vivo antitumor effectA549 cells were subcutaneous inoculated under left axillary of the nude mice.When the average volume of xenograft tumors reached 2 cm3,the mice were randomly divided into five groups-Control,Pre CDDP/YC oral 6 mg/kg,Pre CDDP/YC oral 2 mg/kg,and CDDP iv 6mg/kg.After 15 days,all mice were sacrificed,then collected blood samples,main organs and tumors for further analysis.9.Chronic toxicity evaluationMale Balb/c mice were randomly divided into five groups,including Control,Pre CDDP/YC oral 6 mg/kg,Pre CDDP/YC oral 2 mg/kg,CDDP oral 6 mg/kg and CDDP iv 6 mg/kg.After 7 times administration(21 days totally),evaluating the toxicity of Pre CDDP/YC.Results1.Successfully prepare YC mainly composed of β-1,3-glucan in the outer wall after treat with alkali,acid and organic solvent.The size of YC symmetrical distributing in 2.5-3 μm,Zeta potential is approximate-10 m V.The morphological characterization of YC also showed that most of the yeast fungus cell contents have been removed.2.The loading efficiency of positively charged nanoparticles is highest when yeast was treated with alkali for 1.5 h and acid for 0.5 h.3.We successfully construct the Pre CDDP/YC delivery system through electrostatic effect in the process of co-incubation,our research suggests that to the optimal preparation conditions of incubation time is 1 h,and p H 7 ultrapure water is the optimal solvent of Pre CDDP.The morphological characterization of Pre CDDP/YC also showed that after the load of Pre CDDP,contents of YC are significantly increased,and concave-convex evenly distributed on the surface of YC.4.The release curve in vitro showed that Pre CDDP/YC can slowly and uniformly release in gastrointestinal tract environment,and has a good stability.5.Pre CDDP/YC delivery systems had no significant effect on the proliferation activity of macrophage cell.6.The results of In vitro antitumor experiment show that Pre CDDP/YC has different degree of cytotoxicity to several types of tumor cells,it can effectively kill cancer cells,even the cell has a resistance to cisplatin.The cytotoxicity is dose-dependent and time-dependent.7.We successfully establish A549 lung cancer xenograft tumors on nude mouse.After a period of treatment,the results showed that both Pre CDDP/YC oral and cisplatin intravenous can inhibit the growth of tumor,but Pre CDDP/YC therapy do not cause weight decreased obviously which CDDP iv group shows a obvious downward trend.At the same time hematological parameters,liver and kidney function show no significant effect.In addition in vivo imaging of the experimental results also showed that Cy7.5/YC targeted focus on the tumor site,this is helpful to improve tumor local drug concentration and therapeutic effect.8.Compared with the clinical administration of cisplatin,Pre CDDP/YC has no significant impact on body weight,organ index,liver and kidney function and hematological parameters of Balb/c mice.HE staining also proved Pre CDDP/YC cause no significant pathologic change to organs of mice,CDDP cause a severe kidney and spleen pathologic damage in stark contrast.The results show that Pre CDDP/YC has a good safety given by oral.Conclusions1.We successfully prepared a negative-charged microcapsule YC which has a good loading efficiency of positive-charged nanoparticles through electrostatic effect.The delivery system of positive-charged cisplatin prodrug and YC can raise the accumulation of drug by tumor site monocyte/macrophage recruitment effect to achieve targeted delivery.2.Pre CDDP/YC oral drug delivery system has good security in the tumor treatment compared with traditional CDDP intravenous therapy with counterpart antitumor effect,lower side effects,targeted effect at the same time.In a word,our study is based on the theory of some fungi and other microbes can infect large number of macrophages through the gastrointestinal tract,so we put forward the idea to build the CDDP/YC oral drug delivery system.We first synthesis positive-charged cisplatin prodrug(Pre CDDP),then construct Pre CDDP/YC delivery system through electrostatic effect.Traditional way of platinum drugs normally administrated by intravenous injection,Pre CDDP/YC has a good antitumor effect and greatly improve some of the side effects caused by intravenous injection,and the preparation method is simple and efficient,so it has promising application prospect.