Study On Cisplatin-loaded Nanoparticles Against Osteosarcoma

Osteosarcoma is the most common bone malignancy in children and adolescents. Owingto the high rate of systemic spread, the cure method is rare with surgical treatment alone. Infact, the5-year survival rate of patients is no more than10%~20%with amputation treatmentalone. With the application of adjuvant chemotherapy, the current5-year survival rate ofpatients has currently markedly increased to approximately60%~70%for metastaticosteosarcoma.Cis-diamminedichloroplatinum (II)(CDDP, cisplatin or cisplatinum) is a frequently usedanticancer drug in adjuvant chemotherapy for the treatment of osteosarcoma. It exertsantitumor effects by binding to deoxyribonucleic acid (DNA) and bringing about theformation of intrastrand and interstrand cross-links which triggers the apoptosis of cancercells. The therapeutic activity of cisplatin is proportional to the dosage, however thedose-dependent nephrotoxicity and neurotoxicity have limited its use as a treatment forosteosarcoma. In addition, in vitro studies have shown cisplatin to have a direct effect ongrowth plate chondrocytes that results in decreased growth and final height in animals. Sinceosteosarcoma usually occurs in children and teenagers, these side effects would have anegative impact on the quality of life of patients. Therefore, the strategy that can reduce theside effects without loss of anticancer efficacy of CDDP against osteosarcoma is highlydesired.Polymeric nanocarriers can dramatically improve the pharmacokinetics andbiodistribution of cisplatin, thus the enhanced anticancer efficacy and reduced side-effects canbe obtained. Since most solid tumors have elevated vascular permeability and usually lackeffective lymphatic drainage, nanomaterials (Diameter:20-200nm), such as liposomes,nanoparticles, and macromolecular drugs, tend to accumulate in tumor tissue much more thanin normal tissues, this is referred to as the enhanced permeability and retention (EPR) effect.As compared with free CDDP, PEGylated CDDP-loaded polymeric nanoparticles usuallyhave remarkably prolonged blood circulation time. This leads to that the CDDP-loadedpolymeric nanoparticles have more opportunity to target tumor tissue through the EPR effect,suggesting the enhanced anticancer efficacy and reduced side-effects.In addition, the penetration of nanoparticles into tumor tissue is usually poor. The tumor penetration depth of block copolymer micelles (BCMs) is dependent on the particle size.BCMs with diameters of25nm (BCM-25) diffused further away from the blood vessels(Dmean=42±9μm) following extravasation, compared to BCMs with diameters of60nm(BCM-60) which mainly remained in the perivascular regions (Dmean=23±4μm). Because ofthe poor penetration of nanoparticles into tumor tissue, there is a low contact probability forsome nanoparticle to reach a majority of target cells within the tumor. As a result, thetherapeutic efficacy of many anticancer nanomedicines is limited. But an internalizing RGD(iRGD) was found be able to selectively enhance the vascular and tissue permeability oftumors overexpressing αv integrins and neuropilin-1. Therefore the coadministration of iRGDmay have some possibility to enhance the anticancer efficacy of drug-loaded nanoparticlesagainst osteosarcoma.Herein, we present the treatment of osteosarcoma with the CDDP/PLG-g-mPEGnanoparticles alone or with iRGD. MNNG/Hos is used as the osteosarcoma cancer model.The CDDP delivery system is evaluated in vitro and in vivo in detail. The coadministration ofiRGD is expected to enhance the permeability of tumor vascular and tissue, consequentlyenhancing the efficacy of the CDDP-loaded nanoparticles for the treatment of osteosarcoma.（1）Preparation of PLG-g-mPEG and cisplatin-loaded nanoparticles. Poly (L-glutamicacid) was prepared by the ring-opening polymerization of BLG-NCA and deprotection.PLG-g-mPEG was then prepared by the esterification of poly (L-glutamic acid) withpolyethylene glycol monomethyl ether. CDDP/PLG-g-mPEG nanoparticles (CDDP-NPs) wasproduced by the reaction of PLG-g-mPEG with CDDP for72hrs in aqueous solution. Thedrug-loading content was up to26wt%. The drug-loading efficiency was up to more than95%, which indicates that PLG-g-mPEG can efficiently encapsulate cisplatin（2）In vitro evaluation of CDDP-NPs. The diameter of CDDP-NPs is43.4+1.3nmthat is in the range of20-200nm, which implies the CDDP-NPs may have EPR effect. Thezeta potential of CDDP-NPs is-13.6+0.6mV. The slight negative charge can effectivelyreduce the absorption of protein and liver distribution, and prolong the circulation time ofCDDP-NPs. The in vitro drug release behavior of CDDP-NPs was investigated. It was foundthat burst release did not appear for the CDDP-NPs. The drug release rate is increased withthe increase of chloride ion concentration and the decrease of solution pH value. In particular,the drug release rate in the blood environment is far below that in the lysosomal environment,which is good for the tumor specific release of CDDP. PLG-g-mPEG material has no toxicity on human osteosarcoma cell line MNNG/Hos. In contrast, CDDP-NPs has obvious inhibitoryeffect on the proliferation of MNNG/Hos cells, the IC50of12.2μg/mL (on the basis of CDDP)is3times of cisplatin (IC50value of4mg/L), which can be explained by the slow releaserate of CDDP-NPs.（3）In vivo evaluation of CDDP-NPs for the treatment of osteosarcoma. Comparedwith the original free CDDP, the CDDP-NPs have greatly extended blood circulation time.This is because the surface of CDDP-NPs is negatively charged and protected by hydrophilicpolyethylene glycol, effectively reducing the interaction with serum protein. In addition, thesize of CDDP-NPs is appropriate to effectively reduce the renal filtration, therebymaintaining a high plasma concentration of drug. The long blood circulation is conducive forthe passive targeting of nanoparticles in tumor by EPR effect. The average body weight ofmice in the CDDP groups declined sharply, and some mice died. These indicated that CDDPhas systemic acute toxicity. In contrast, the average body weight of mice in the CDDP-NPsgroup increased after treatments, indicating lower systemic toxic side effect. Regard to therate of tumor inhibition, the CDDP-NPs showed equal to or slightly higher efficacy for thetreatment of MNNG/HOS osteosarcoma. Through the combined use of iRGD, the anticancereffect of CDDP-NPs was further enhanced, while the systemic toxicity and side effects didnot increase. The tumor necrosis of different treatments was compared using hematoxylineosin staining (H&E staining). The CDDP-NPs+iRGD group showed larger necrosis areathan CDDP, CDDP-NPs and CDDP+iRGD groups, which is consistent with results from thegrowth inhibition studies. Therefore, CDDP-NPs, especially the CDDP-NPs+iRGD, havegreat potential for the treatment of osteosarcoma.