Cancer Stem Cell Treatment With Nano Drug Delivery System And The Mechanism Investigation

Based on recent researches,the cancer stem cells(CSCs)in tumor may be responsible for cancer initiation,progression,resistance,recurrence,and metastasis.They spend most of their time in non-dividing G0 cell cycle state,being insensitive to many chemical drugs which functioned on cell division.They showed characteristics resembling normal stem cells.When stimulated by surrounding environment,they could differentiate and transform to new tumor cells to promote tumor growth and tumor relapse.Besides,the strong tumorigenic ability of CSCs was believed to be the main course for tumor metastasis.Lots of researches had pay attention to targeting CSCs to eliminate tumor and prevent its recurrence.In this research,we focused on nanocarrier-based therapeutic agents to specifically and effectively target CSCs.The main content of the thesis is as follows:The serum-free suspension culture method was adopted to obtain breast cancer stem cell(breast CSCs)from MCF-7 cell line.The single MCF-7 cell formed a compact spheroid structure(mammospheres)with a well-defined circular shape and a diameter of 200μm.The CSCs surface marker CD44+/CD24-,the transcription and expression level of stem genes(Nanog,OCT4 and SOX2)as well as drug resistance protein ABCG2 were detected and it proved that the mammospheres were enriched of cancer stem cells.The glycolipid-like stearic acid-g-chitosan oligosaccharide(CSOSA)showed strong cellular uptake and deep penetration on mammoshperes,delivering drug doxorubicin(DOX)into its core.Mammosphere suppression effect was measured by acid phosphatase(APH)assay.The drug loaded CSOSA(CSOSA/DOX)showed enhanced mammosphere suppression effect than DOX·HCl with a decrease of IC50 value from 2.64 μg/mL to 1.07 μg/mL(*p<0.05).Mammosphere tumor model and MCF-7 tumor model were separately established under the mammary gland of nude mice.We demonstrated that mammosphere cell induced-tumors had a more complicated architectural pattern and cell components,remodeled the mammary tissue,recapitulated the primary tumor.Mammosphere tumor model and multi-cycle repetitive administration method were adopted for better simulating clinical therapy regimen.In the 1st treatment cycle,DOX·HCl showed stronger anti-tumor efficacy than CSOSA/DOX.From the 2nd treatment cycle,tumors treated by DOX·HCl kept growing,while tumors treated by CSOSA/DOX showed little expansion.At the end of the tri-cycle treatment,CSOSA/DOX micelles showed stronger anti-tumor effect than DOX·HCl(**p<0.01).CSOSA/DOX could kill both CSCs and non-CSCs without CSCs enrichment.Only non-CSCs were killed by DOX·HCl and the percentage increased from 10.95%to 69.36%.CSOSA/DOX also promoted the decay of microenvironment by decreasing the number of cancer-associated fibroblasts,thus return facilitated anti-tumor effect.The MCF-7 tumor model was adopted to explore the changes of drug resistance caused by multi-cycle repetitive administration.We found CSOSA/DOX showed stronger anti-tumor efficacy than DOX·HCl after tri-cycle treatment.Tumors treated by DOX·HCl showed obvious increase expression of P-glycoprotein,while in tumors treated by CSOSA/DOX,the P-glycoprotein level was equal to that of the untreated group.Two stimulating methods,pulsatile treatment and continuous treatment,were used on in vitro cell line to simulate the in vivo process.Drug sensitivity of cells stimulated by DOX·HCl decreased,whose IC50 values increased to 0.80 ±0.03 and 0.88±0.07 μg/mL.Drug sensitivity of cells stimulated by CSOSA/DOX changed little and in consistent with the in vivo results,almost no increase of P-glycoprotein was seen in cells stimulated by DOX encapsulated in nanocarrier.The appearance of drug resistance was related to genetic changes.The mRNA levels of mdrl gene in DOX·HCl stimulated cells were thousands folds of that in parent sensitive cell.In contrast,for those treated by DOX encapsulated in nanocarrier,they stayed the same as the original sensitive cell.The CSOSA/DOX didn’t induce mdrl gene transcription.Lipid nanoparticles which had excellent biocompatibility and strong loading capacity for drugs with poor solubility were widely applied in anti-tumor therapy.In this study,anti-tumor drug oxaliplatin(OXA)was combined with CSCs specific drug salinomycin(SAL)to treat hepatoma.The A54-PEG-ODA conjugate was synthesized by conjugating the A54 peptide through PEG chains to octadecylamine(ODA)in the presence of EDC and NHS.The fatty chain of the A54-PEG-ODA conjugate was inserted to the solid lipid to prepare A54 peptide modified solid lipid nanoparticle(A54-PEG-SLN).To promote entrapment of oxaliplatin in A54-PEG-SLN,soybean phospholipid was used to form a complex with oxaliplatin.The drug encapsulation efficiency and drug loading were 65.3±1.9%and 3.46±0.12%(at a feed ratio of 5%).The results of cellular uptake indicated that after modified by A54 peptide,internalization of A54-PEG-SLN was accelerated in model cell line BEL-7402.It also showed specific selective uptake in BEL-7402 cells when incubated with other two cells,HepG2 and L-02 cells,together.The aptamer A15 could target CD 133 molecular,CSCs surface marker,with high high affinity.The free amino group on A15 reacted with the other free amino group from NH2-PEG-SLN/SAL nanoparticle in the presence of DSC to synthesis A15 modified solid lipid nanoparticle A15-PEG-SLN/SAL.Poorly soluble drug SAL was effectively loaded in the A15-PEG-SLN/SAL nanoparticle.Its drug encapsulation efficiency and drug loading were 84.42±0.55%and 7.77±0.28%(at a feed ratio of 10%).In vitro,the IC50 values of OXA and A54-PEG-SLN/OXA on BEL-7402 cell were 8.11±1.50 and 16.0±1.20 μg/mL.The decreased cytotoxicity may be related to the delayed release.No obvious CSCs cytotoxicity was seen after SAL treatment,while the A15-PEG-SLN/SAL formulation displayed high cytotoxicity on CD133 positive cancer stem cells with an IC50 value of 0.69±0.02μg/mL.Considering the insensitivity of BEL-7402 cell to SAL,sequential administration was applied to perform the in vivo anti-tumor therapy.OXA formulations were injected two treatment cycles before SAL formulation,when sensitive BEL-7402 cells were killed and the drug resistant CSCs were exposed.The proportion of CSCs increased from 0.1%to 3.0%.A54 peptide modification enhanced the anti-tumor effect of SLN formulations which was comparable with free OXA.What’s more,after sequential administration of SAL,the A54-PEG-SLN/OXA and A15-PEG-SLN/SAL combined therapy regimen showed strongest tumor suppression effect.In conclusion,the glycolipid-like nano-drug delivery system showed favourable anti-tumor effect during multi-cycle treatment.The CSOSA/DOX could kill CSCs and inhibite tumor recurrence.The combined therapy of A54-PEG-SLN/OXA and A15-PEG-SLN/SAL showed synergistic effect and exhibited great therapeutic potential on hepatoma.