The Study On Intratumoral Injection Hydrogels Of Doxorubicin-loaded Gold Nanocages Gated With Temperature Sensitive Polymers For Enhancing Synergistic Effect Of Hyperthermia And Chemotherapy

Cancer is one of the leading causes of human mortality.Though chemotherapy is one of the most commonly used anticancer treatment in clinic,clinical practice shows that there are only limited antitumor efficacy and serious toxicity for systemic chemotherapy via intravenous administration,owing to lack of selective accumulation in tumor tissue for most of drugs.In the last two decades,a variety of approaches with different antitumor mechanism have been combined for cancer therapy.Among them,hyperthermia has received great attention for its multiple antitumor effect.It was found that a synergistic effect between hyperthermia and chemotherapy was achieved under relative low temperature(39 ~ 43 oC),However,how to achieve the precise synchronization of hyperthermia and chemotherapy,and the impact of this precise synchronization on the therapeutic effect and its mechanism still need to be further explored.Aiming to precisely synergistic effect of thermo-chemotherapy,a hydrogel drug carrier of gold nanocages modified with two temperature sensitive polymers was constructed for achieving near-infrared-triggered on-demand release,and precise synchronization between hyperthermia and chemotherapy.The main contents and results are as follows:(1)The preparation and characterization of temperature/pH sensitive polymers.N-isopropyl acrylamide(NIPAM)and tert-butyl acrylate(tBA)were chosen as temperature sensitive monomer and pH sensitive monomer precursors,respectively.Two types of block copolymers pNIPAM100-b-ptBAm(PNtB100-b-m,m=50,100)and three types of random copolymer pNIPAM400-co-ptBAn(PNtB400-co-n,n=6,8,10)were synthesized by atom transfer radical polymerization.The molecular weight of PNtB100-b-100 and PNtB100-b-50 are 51.3 and 37.5 kDa,respectively,in agreement with the theoretical value calculated from feeding ratio.The monomer ratios of the two block copolymers,which confirmed by the 1H-NMR,were 1:1 and 2:1,respectively.PNA100-b-50 and PNA100-b-100 were obtained with completed hydrolysis,and their lower critical solution temperatures(LCSTs)were measured as 36.5 oC and 37 oC,respectively.By controlling the hydrolysis parameters of PNt B100-b-100,four PNAx polymer with different degrees of hydrolysis(PNA30,PNA50,PNA75,PNA100)were obtained.The nanoparticles with hydrophobic core and temperature sensitive pNIPAM shell were formed via the self-assemble of PNAx in different pH solution.Moreover,the molecular weight of three random copolymers measured by GPC was 41.6,46.1 and 45.6 kDa,respectively.The corresponding temperature/pH-sensitive PNA400-co-n polymers were obtained by the hydrolysis of PNt B400-co-n polymers with trifluoroacetic acid,and their LCSTs were easily adjusted via pH.Specifically,PNA400-co-8 was used for the following studies about photothermal-triggered on-demand release,since its LCST(39.9 oC)was between body temperature(37 oC)and hyperthermia temperature(43 oC).(2)The studies about co-assembling behaivor of Dox-induced temperature/pHsensitive block copolymer PNAx(D-PNAx)hydrogels for the evaluation local antitumor efficacy through intratumoral injection.Weakly basic antitumor drug,doxorubicin(Dox),was used for co-assembling with weakly acidic PNAx polymers via acid-base neutralization.Specifically,D-PNA100 self-assembled nanogels showed the best stability(zeta potential-32mV),the highest drug loading(41.2%)and long term drug release characteristics.The sol-gel transition results confirmed that D-PNAx nanogels and the corresponding PNAx polymer form gel under the physiological temperature.Pharmacodynamic experiments show that D-PNA100 nanogels can effectively inhibit tumor growth.In comparison with free Dox(1.13 ± 0.04 times at 9 days)and PNA100(2.11 ± 0.34 times),D-PNA100 showed best antitumor effect,that is,the tumor treated by them grew to only 0.77 ± 0.13 times of original tumor volume in the experiment period.All mice in D-PNA100 group survived during the experiment period,indicating the good biological safety of as-synthesized nanogels.In contrast,80% of the mice died in the free Dox group.The intratumoral retention result showed that after 10 days treatment,41% drug was still left in the D-PNA100 nanogels group,while only 20% drug remained in the free Dox group.These results indicated that in local antitumor therapy,injectable hydrogels of D-PNA100 has many advantages as minimal invasion,high drug loading amount,sustained release,long-term and high antitumor efficacy and good biocompatibility.(3)The preparation and characterization of Dox-GNC@PNA-hls.Firstly,silver nanocubes(SNCs)with good dispersibilities were synthesized by polyalcohol reduction method.Their sizes were 45 ± 5 nm.Using SNCs as template,GNCs whose absorbtion peak localed at 800 nm were prepared by electrochemical replacement reaction.The results showed that the particle size was 45 ± 5.0 nm,the hydrodynamic diameter was about 100 nm,and the zeta potential was-7.7 mV.Elemental analysis showed that GNCs were gold and silver alloy nanoparticles.PNA400-co-8 was screened as a temperature sensitive gatekeeper(high temperature ligand,PNA-h),and used to surface modification of GNCs with high photothermal conversion efficacy.Further,PNA100-b-50 was modified subsequently onto GNC@PNA-h,as low temperature ligand(PNA-l).The resultant GNC@PNA-hls with dual modification of PNA-h and PNA-l,has 9.0 %(PNA-h)and 25 %(PNA-l)of weight fractions by thermogravimetric analysis(TGA).TEM image showed GNCs surface is covered with a polymer layer with 3 nm thickness.The characterization of energy dispersive spectrometer(EDS)confirmed the polymer had been successfully modified on the surface of GNCs.The hydrodynamic diameters of GNCs increased from 100 nm to 130 nm after surface modification of PNA.Dox was loaded in GNCs by ammonium sulfate loading approach,and the drug loading was as high as 8.0 %.In vitro drug releasing profiles indicated that Dox-GNC@PNA-hls exhibited infrared(NIR)-triggered on-demand releasing behaviors,which has been affected greatly by laser power,radiation time and frequency and medium pH.Concentrated dispersion of Dox-GNC@PNA-hls has excellent sol-gel phase transition behavior,and could be used in intratumoral injection as their gellating temperature was 34 ℃.Their storage modulus(G’)reached to 1870 Pa,an increase of 2000 times than that before gellation.(4)Evaluation of antitumor effects of Dox-GNC@PNA-hlsin vitro and in vivo.MTT experiment results show that Dox-GNC@PNA-hls can effectively kill the tumour cell under the 808 nm laser irradiation,and demonstrate that hyperthermia and chemotherapy synchronous treatment has stronger tumor cell killing ability than sequential treatment.And the pharmacodynamics experiment showed that Dox-GNC@PNA-hls group showed better antitumor effect compared with the free Dox group and the GNCs photothermal group.The precise synchronization of animal experiments show that Dox-GNC@PNA-hls can achieve synchronous photothermal drug release under illumination,showed higher Dox content in tumor tissue.What’s more,we found that Dox-GNC@PNA-hls could realize on-demand durg release to achieve the precise coordination of hyperthermia and chemotherapy,so as to enhance the efficacy of tumor therapy effectively.In this thesis,a hydrogel drug carrier of gold nanocages modified with two temperature sensitive polymers was constructed.Which achieved the precise coordination of hyperthermia and chemotherapy for long-term repeated under near infrared laser irradiation,and puts forward some new ideas of precision synergy for tumor therapy.