The Synthsis Of New Type Of Bortezomib With Tumor-targeted And PH-responsive Behavior And Its Application In Bone Tumor Therapy

ObjectiveUbiquitin-Proteasome System(UPS)is one of the ways of degradation of intracellular proteins.Excessive activities of this system may accelerate the occurrence of diseases which includes malignant tumors.One of the critical reasons is that proteasomes play great roles in cellular growth,propagation,difference as well as apoptosis.Thus,the inhibitors of proteasomes could retard these proceedings by blocking the degradation of intracellular proteins.Bortezomib(BTZ),an inhibitor of UPS,has been used extensively in the treatment of tumors such as multiple myeloma and mantle cell lymphoma in recent years.However,its bad ability of enhanced permeation retention effect(EPR)makes it a low level of concentration in lesions,followed by relatively strong side-effects to normal tissues.Therefore,more and more researches concentrated their focus on nanoparticles such as hydrogel,nano-sized mesoporous silica dioxide,poly lactic-co-glycolic acid(PLGA)and so on to establish the drug-controlled release systems.Nevertheless,these systems did not have the ability of targeted delivery of BTZ,and the unsatisfying drug concentration in the tumor location for effective treatment.This not only declined the rate of drug delivery,but also resulted in great adverse effects for the body and organs.Herein,we aim at preparing a pH-responsive polymer which realizes the ability of targeted delivery for BTZ.The novel type of BTZ can be delivered to tumor locations with the help of RGD and releases BTZ at slight acidic tumor microenvironments with the hope of improving the therapeutic effects and reduce the side effects at the same time.MethodsOn the basis of G5 PAMAM dendrimer(G5 PAMAM),we synthesized a new vector modified with cRGD and polyethylene glycol(PEG),then connected with catechol groups and named it as cRGD-PEG-PAMAM-Cat.The biocompatibility,target ability and the anti-tumor activity of the novel vector were further studied.The compound cRGD-PEG-PAMAM-Cat/BTZ was produced through the reaction of catechol and borate.With bag filter method and 1H NMR spectroscopy,the vitro drug release of cRGD-PEG-PAMAM-Cat/BTZ was measured first under different pH conditions.The cellular uptake of cRGD-PEG-PAMAM-Cat/BTZ was detected with the help offluorescent pyrene borate.To pinpoint the cytotoxicity of cRGD-PEG-PAMAM-Cat/BTZ in vitro,the method of 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di–phenytetrazoliumromide raexperiment(MTT)was performed.Finally,tibia metastasis model of breast cancer was built up in nude mice,then were divided into four groups according to the tumor fluorescent: the cRGD-PEG-PAMAM-Cat/BTZ group(RGD+);the PEG-PAMAMCat/BTZ group(RGD-);the free BTZ group(BTZ)and the phosphate buffer solution group(PBS).After the sectionlization,all the four groups were treated through tail intravenous injection with a dose of 0.5 mg/Kg/time in one mouse,followed by regular measurement of body weight,tumor perimeter.As soon as the finish of treatment,the bioluminescence imaging was made again.In addition,the tibia X-ray and Micro-CT of the lesion side were conducted respectively to explore the anticancer activity of the new BTZ type.Results(?)Synthesis of Targeted and pH-responsive polymer and the new form BTZThe new vector,which was modified with cRGD,PEG and catechol group,was successfully synthesized.On average,the 1H NMR showed that the compound was connected with 36 PEGs,2 cRGDs and 30 catechol groups for each vector.At last,the cRGD-PEG-PAMAM-Cat/BTZ was acquired through the reaction of catechol group and borate to form a borate ester bond.(?)In vitro release,cellular uptake and inhibition to tumor cells of cRGD-PEG-PAMAM-Cat/BTZThe bag filter method and ~1H NMR spectroscopy results indicated that release of BTZ from cRGD-PEG-PAMAM-Cat/BTZ less than 5% at pH 7.4,while the release of BTZ speeded up as the decline of pH values.Moreover,the cellular uptake experiment of the new vector showed that the pure pyrene boric acid could hardly penetrate the cells,but that with cRGD-PEG-PAMAM-Cat did well.The cytotoxicity of cRGD-PEG-PAMAM-Cat/BTZ was measured using MTT method.Great toxicity of cRGD-PEG-PAMAM-Cat/BTZ to breast cancer cells had been detected.However,no obvious toxic effect was observed in PEG-PAMAM-Cat/BTZ that without cRGD.(?)Therapeutic effect evaluation of cRGD-PEG-PAMAMCat/BTZ in tibia metastasis models of breast cancer in nude miceThe body weight of all the nude mice did not change significantly during the treatment experiment.The results of the relative fluorescence intensity of the tumors(before and after the treatment)showed a great significance(P<0.05)between RGD+group and PBS group as well as RGD-group.Though significance was not acquired between RGD+ group and BTZ group,the relative fluorescence intensity in RGD+ group was about half of that in BTZ group.The change in perimeter of tumor showed great obvious difference between them with a significance of P<0.05.Both the X-ray and the Micro-CT imaging of tibias revealed the least bone destruction in RGD+ group compared with others.The bone volume and the Structure linear density(Tb.N)were further calculated with a significance of P<0.05,as well as the difference of tumor weight between RGD+ group and other groups.ConclusionsIn summary,we present a new drug vector for boronic acid containing anticancer drugs such as BTZ with good biocompatibility.With the connection of cRGD,the vector acquires the ability of targeting and penetration,which establishes the targeted-delivery of drugs.The catechol groups conjugated on the dendrimer playe a critical role in binding and releasing the anticancer drug through the formation of borate ester bond,which showed good stability in physiological environment while could be activated by the slight acidity microenvironment with the result of drug release from the prepared formulation.Thus,the local drug concentration improved with the result of better therapeutic efficacy.All in all,this study realized the targeted delivery and pH-responsive release of drugs,which provides a promising therapeutic option for the future treatment of cancers.