The Construction And Preliminary Evaluation Of Two-stage PH-sensitive Doxorubicin Hydrochloride Loaded Nanoparticles With Dual Drug-loading Strategies

Chemotherapy is the most commonly used treatment for many cancers.However,its success has been limited by several drawbacks,such as low efficacy and toxic side effects.For chemotherapy,how to improve the therapeutic efficacy and reduce the side effects is an urgent problem to be solved.Combination therapy of chemotherapy and gene therapy is another common strategy for improving anti-tumor efficacy of chemotherapy.Co-delivery of drug and gene is the common method to achieve combination therapy.It's important that co-delivery drug and gene in one carrier,because drug and gene could be loaded with suitable dosage and reach the same target cell.In recent years,nano-drug delivery system(NDDS)was widely used in cancer therapy.NDDS could passively reach tumor target through the EPR effect,which make NDDS preferentially accumulate in tumor tissues.Compared with low molecular weight anti-tumor agents,NDDS could change the non-selectivity biodistribution of drug in the whole body and then improve anti-tumor efficacy.At the same time,co-deliveiy of drug and gene in one carrier could be achieved by NDDS.NDDS was also a hot-topic incombination therapy.After accumulated in tumor tissues by EPR effect,controlled drug release should be achieved,so that the effective anti-tumor efficacy could be achieved.Due to the distinct pH gradients among normal tissues(pH 7.4),tumor extracellular environments(about pH 6.5)as well as endo/lysosome(about pH 5.0),it's important to build a two-stage pH-sensitive core-shell NDDS.The design of two-stage pH-sensitive core-shell NDDS was that:In blood circulation and normal tissues,negatively charged shell of NDDS was expected to coated on positively charged core.In order to keep NDDS stable and prolong the circulation time,relieve side effects of drug caused by drug leakage.After NDDS accumulated in tumor tissues via EPR effect,the outer shell could dissociate at the subacid microenvironment and core was exposed to facilitate the tumor cellular uptake.After internalization,drug was pH-sensitively released.The two-stage pH-sensitive strategy of firstly/tumor microenvironment pH-sensitive combining with secondly/intracellular pH-sensitive was beneficial to achieve controlled intracellular drug release,which could improve anti-tumor efficacy and relieve side effects.The high drug-loading capacity was the basic for anti-tumor efficacy.Dual drug loading strategy was a common used method to improve drug-loading capacity.Dual drug loading strategy of physical loading combining with chemical medication was used in order to improve the drug loading capacity and anti-tumor efficacy of NDDS.Based on the design,dual drug-loading strategy combined with two-stage pH-sensitive strategy was used to build a two-stage pH-sensitive nanoparticle with dual drug loading strategy.Firstly,hydrazone linked cationic conjugates poly(ethyleneimine)-C6-succinimidyl 6-hydrazinonicotinate acetone hydrazone-DOX(PEI-C6-SANH-DOX HCl,PEI-C-DOX)was synthesized.Secondly,21-base(CGA)7 oligodeoxynucleotides(CGA-ODNs)was chosen for the carrier of DOX,DOX loaded CGA-ODNs(CGA-ODNs/DOX,OD)was obtained with incubation at room temperature.OD was condensed by PEI-C-DOX to get the complex with dual drug loading strategy PEI-C-DOX/OD(POD).O-car-boxymethyl-chitosan(CMCS),a kind of biodegradable pH-sensitive amphiprotic polyelectrolytes,was coated on POD in order to built CMCS/POD(CPOD).Based on the study mentioned above,one-step strategy was used to bulid the co-delivery nanoparticle.In the present work,plasmid of enhanced green fluorescent protein(pEGFP)was chosen for model gene.The misture of OD and pEGFP was condensed by PEI-C-DOX in order to get the co-delivery complex PEI-C-DOX/OD&pEGFP(PODE).CMCS was coated on PODE to build the co-delivery nanoparticle CMCS/PODE(CPODE).CPODE was obtained the same two-stage pH-sensitive of CPOD,and a good foundation was settled of combination therapy.Main methods and results were as follows:1.Determination of DOX contentFirstly,DOX content was determined by UV spectrophotometry.The results showed that the linearity of DOX in the concentration of 5-35 ?,g/mL was good(r=0.9994),methodological evaluation was conformed the requirement.Subsequently,DOX content was determined by fluorescence spectrophotometry.At pH 7.4,the linearity of DOX in the concentration of 51-1020 ng/mL was good(r=0.9995),methodological evaluation was conformed the requirement.At pH 5.0,the linearity of DOX in the concentration of 50-1000 ng/mL was good(r=0.9993),methodological evaluation was conformed the requirement.2.The construction of two-stage pH-sensitive DOX loaded nanoparticle and the primary evoluation of its physicochemical property.PEI-C-DOX was successful synthesized,and confirmed by 1H-NMR and IR.OD was prepared,and the optimal drug loading ratio was 0.12:1.At 4? for 24 h,DOX was no obvious release,indicating the pretty stability of OD.POD and CPOD were prepared based on the electrostatic interaction.The optimal prescription of POD was w/w 339:64,and the optimal prescription of CPOD was w/w 75:4.Both POD and CPOD displayed a spherical morphology with a narrow distribution.The average size of POD was(140.2 ± 5.2)um,PDI was 0.144±0.018,zeta potentialwas(20.4 ±1.9)mV.The average size of CPOD was(165.0±3.3)nm,PDI was 0.191 ±0.001,zeta potential was-(15.6±0.82)mV.The results of plasma stability confirmed that,in 10%plasma,CPOD could keep stable for 24 h.The results of hemolytic tests confirmed that,the calculated HRs of CPOD were all less than 5%,CPOD had good hemocompatibility under preliminary experimental conditions.3.The evaluation of the two-stage pH-sensitive as well as in vitro and in vivo anti-tumor efficacy of CPODIn vitro evaluation showed that,at tumor extracellular pH,CMCS could dissociate from CPOD.In B16 and HepG2 cell lines,the cellular uptake of CPOD was much higher than that at pH 7.4(p<0.005 in B16 cells,p<0.01 in HepG2 cells).The firstly/tumor microenvironment pH-sensitive was confirmed.In vitro release study revealed that the drug release amount from CPOD at endo/lysosome pH was significantly more than that at pH 7.4(p<0.005).In the nuclear localization test,both in the B16 and HepG2 cell lines,at pH 6.5 and pH 6.0,CPOD could internalization and release DOX,almost all DOX was located in nuclear in 4 h.The secondly/intracellular pH-sensitive was confirmed.The results of cytotoxicity assay confirmed that the cytotoxicity of free DOX was better than CPOD in B16 cell line,while in HepG2 cell line,the cytotoxicity of CPOD was similar to free DOX.Blank nanoparticles showed great safety in the test concentration.Furthermore,in vivo NIRF imaging indicated that CPOD could accumulate in tumor after 1 h,and lasted for 24 h.At 24 h,the fluorescence intensity of tumor in CPOD group was 3.46 times stronger than that of free Cy5 group.In the in vivo anti-tumor test,CPOD exhibited a better anti-tumor efficacy even than double dosage DOX solution,and weight lose caused of mice wasn't revealed.In the histological analysis,no visible tissue lesions of main organs in CPOD group were observed,indicating the preliminary in vivo safety of CPOD.4.The construction of co-delivery DOX and pEGFP nanoparticles with primary evoluationCo-delivery DOX and pEGFP complex PODE was built,and CMCS was coated on PODE to build CPODE.The optimal prescription of PODE was w/w 68:24,and the transfection efficiency was 11.8±0.10%.The optimal prescription of CPODE was w/w 75:8.Both POD and CPOD displayed a spherical morphology with a narrow distribution.The average size of PODE was(77.61 ±5.7)nm,PDI was 0.265±0.019,zeta potential was(4.63±0.33)mV.The average size of CPODE was(169.313.5)nm,PDI was 0.191±0.097,zeta potential was-(12.8±0.61)mV.The results of nuclease resistance test showed that,the ability of CPODE was better than PODE.The results of plasma stability confirmed that,the plasma stability of CPODE was pretty,CPODE could protect pEGFP from degradation.The coating of CPODE could improve the nuclease resistance and plasma stability of CPODE.The procedural release test showed that,DOX was released from CPODE at the beginning,at 12 h,the cumulative release percentage was 51.5%.The release of pEGFP need a longer time more than 12 h.The co-delivery test of CPODE confirmed that,in HepG2 cell line at pH 6.0,the co-delivery efficiency was 29.5%.In conclusion,after loaded in CPOD,the anti-tumor efficacy of DOX was improved with pretty safety.Potential application value was shown for CPOD.The co-delivery DOX and pEGFP were achieved by CPODE.A new idea for co-delivery NDDS in combination therapy was obtained by CPODE.