| BackgroundCisplatin,the first generation of platinum-based antitumor drugs,has shown effective antitumor activity and has been widely used to treat many solid tumors in the clinic.However,cisplatin also brings about serious side effects in the routine clinic use.Therefore,it is of great significance for the treatment of cisplatin with structural modification or pharmaceutical engineering to improve the antitumor activity and reduce its side effect.Current delivery systems reported through encapsulating the cisplatin have common issues of low drug loading and encapsulation efficiency because of the strong hydrophilicity of cisplatin.Polyethyleneimine(PEI)is a kind of cationic polymer with a large number of amino groups,which could be modified easily to achieve high drug loading and entrapment.Moreover,the unique "proton sponge effect" of PEI is conducive to the escape of nano-delivery system from lysosomes and the release of the drugs into the cytoplasm,thus avoiding the degradation of drugs by lysosomes and improving the therapeutic effect.Glutathione,an important antioxidant in biological system,has a great difference in the concentration between the intra and extra of tumor cells.The concentration of glutathione in the cytoplasm is 500-1000 times of that in the blood circulation or extracellular environment.Previous studies have shown that the disulfide bond could remain stable in the blood circulation and extracellular environment and can be rapidly cleaved in the cytoplasm.Therefore,the polymers conjugated with redox-responsive disulfide bond can realize the rapid release of drugs in the cytoplasm where the GSH exists with high concentration.It is an ideal design to construct the delivery system that can aggregate at tumor site and release drugs through stimuliresponsive manner,in order to improve the therapeutic effect and reduce the serious side effects of cisplatin.To transport the chemtherapeutic drug cisplatin to the tumor site and realize the stimuli-responsive drug release,branched polyethyleneimine(BPEI)with large number of amino groups was used as the backbone,and cystamine containing disulfide bond was chosen as the conjugate linker,to construct the redox-responsive polymeric prodrug BPEI-SS-Pt with high drug loading of cisplatin.Based on BPEI-SS-Pt,two series of nano-delivery systems were further designed.The first design is to construct an active tumor-targeted and redox-responsive nano-delivery system by electrostatic adsorption using the prodrug of BPEI-SS-Pt and hyaluronic acid(HA)which can bind to the receptor CD44 highly expressed on the tumor cells.This nano-delivery system could further achieve the active tumor-targeted therapy on the basis of inherent passive tumor-targeted profiles of the nanoparticles via EPR effect.Compared with traditional chemtherapeutic drug cisplatin,the nano-delivery system could realize the active tumor-targeted delivery and redox-responsive drug release,thus improving the therapeutic effect of cisplatin and reducing the side effects as well.The second design is to construct another nano-delivery system using BPEI-SS-Pt as the core and cross-linked by the shell of dialdehyde modified PEG2000(PEG-Di Alde).PEG-Di Alde could conjugate with the amino groups of BPEI via Schiff base to constitute tumor microenvironment and redox-sensitive cascade stimuli responsive nano-delivery system.Compared with cisplatin,this nano-delivery system could detach the PEG shell in the tumor microenvironment and stepwise redox-responsive release drug in the tumor cells,which could avoid the phagocytosis and elimination of macrophages in the blood circulation,ensure the drug uptake and release by tumor cells in high efficacy,and realize the effective antitumor activity.Methods1.Using cisplatin,silver nitrate,cystamine,succinic anhydride and BPEI 25 K as the starting materials,the cisplatin polymeric prodrug BPEI-SS-Pt containing disulfide bond was synthesized.The structures of the synthetic intermediates and polymeric prodrug were confirmed by 1H NMR and IR spectra and the platinum content of the prodrug was detected by ICP-MS.2.HA was combined with BPEI-SS-Pt by electrostatic adsorption to construct the active tumor-targeted and redox-responsive nano-delivery system HA-(BPEI-SS-Pt).The particle size,PDI and zeta potential of the nanoparticles were characterized by Delsa? Nano C Particle Analyzer,and the morphology of the nanoparticles was characterized by the TEM and SEM.The drug loading was detected by ICP-MS and the elemental composition of the nanoparticles was analyzed by the energy spectrum analysis(EDS)with the field emission scanning electron microscope.HA-(BPEI-SS-Pt)nanoparticles were stored in the refrigerator at 4℃ and the change of particle size was measured by DLS analyzer to study the stability of the nano-delivery system.The active tumor-targeted and redox-responsive release behavior of the drug delivery system HA-(BPEI-SS-Pt)in vitro was investigated by simulating the intracellular(10 m M GSH)and extracellular(10 μM GSH)drug releasing condition.The cumulative release of the drug was calculated by detecting the Pt released in the media by ICP-MS to verify the redox drug release behavior of HA-(BPEI-SS-Pt).MTT method was used to study the antitumor activity of HA-(BPEI-SS-Pt)in vitro.In order to investigate the active tumor-targeted effect of HA-(BPEI-SS-Pt),the competitive growth inhibition was measured by MTT method,the quantity of competitive uptake was measured by ICP-MS and the competitive uptake behavior was also observed by TEM and CLSM.The uptake of fluorescence labeled HA-(BPEI-SS-Pt)nanoparticles by A549 cells was observed by CLSM after incubating with different inhibitors of endocytosis(chlorpromazine,amiloride and genistein)at different concentration.BALB/c nude mice bearing A549 subcutaneous tumor were constructed to investigate the antitumor activity of HA-(BPEI-SS-Pt)in vivo.HE staining,fluorescent TUNEL and immunohistochemistry Caspase 3,Ki67 were used to test the apoptosis and the proliferation of tumor cells.The organs of the mice in each group were stained with HE,and the toxic and side effects were analyzed.3.Dialdehyde modified PEG2000(PEG-Di Alde)synthesized by esterification was crosslinked with BPEI-SS-Pt to construct the tumor microenvironment and redox-sensitive cascade stimuli responsive nano-delivery system PEG-(BPEI-SS-Pt).The particle size,PDI and zeta potential of PEG-(BPEI-SS-Pt)were measured by DLS,to investigate the influence of crosslinking time on the formation of the nano-delivery system.The morphology of the nanoparticles was observed by TEM and SEM,and the drug loading of PEG-(BPEI-SS-Pt)was determined by ICP-MS.PEG-(BPEI-SS-Pt)nanoparticles were stored in the refrigerator at 4℃ and the particle size was measured by DLS analyzer to study the stability of the nano-delivery system.The particle size,zeta potential and the morphological change of PEG-(BPEI-SS-Pt)were observed at pH 7.4 and 6.5 to investigate the pH sensitivity in tumor microenvironment.The tumor microenvironment sensitive characteristics of PEG-(BPEI-SS-Pt)were also investigated by 1H NMR at pH 7.4 and 6.5 to observe the pH responsive detachment of the PEG shell.The cumulative drug release behavior of PEG-(BPEI-SS-Pt)through tumor microenvironment and redox-sensitive response was conducted by adjusting pH and GSH concentration to simulate drug release behavior in vivo,and the Pt content was determined by ICP-MS.The antitumor activity of PEG-(BPEI-SS-Pt)in vitro was conducted by MTT method at pH 7.4 and 6.5 respectively.In order to investigate the tumor microenvironment sensitivity of PEG-(BPEI-SS-Pt)nano-delivery system,fluorescence labeled PEG-(BPEI-SS-Pt)was constructed and the tumor cellular uptake of nanoparticles at different pH was observed by CLSM.3D tumor spheroid was constructed and the penetration behavior of the nanoparticles was investigated at different pH on the tumor cell spheres.In order to study the stealth effect of the PEG,the nanoparticles were co-incubated at different pH with the RAW cells and the cellular uptake was observed by CLSM.The cellular uptake of PEG-(BPEI-SS-Pt)nanoparticles was also observed by CLSM after incubating with different inhibitors of endocytosis(chlorpromazine,amiloride and genistein)at different concentration.BALB/c nude mice bearing A549 subcutaneous tumor were constructed to investigate the antitumor activity of PEG-(BPEI-SS-Pt)nano-delivery system in vivo.HE staining,fluorescent TUNEL and immunohistochemistry Caspase 3,Ki67 were used to investigate the apoptosis and the proliferation of tumor cells.The organs of the mice in each group were stained with HE,and the toxic and side effects of nano-delivery system were investigated.ResultsConstruction and antitumor activity evaluation of active tumor-targeted and redox-responsive nano-delivery system1.According to the results of 1H NMR and IR,both the synthesized cisplatin complex and cisplatin polymeric prodrug are the targeted products.The drug loading of cisplatin in BPEI-SS-Pt was 32.66 ± 0.06%.HA and BPEI-SS-Pt could form well-dispersed nanoparticles with the diameters of 150-220 nm at the mass ratio of 1 : 4 and 4 : 1,but could not form smaller size nanoparticles at the mass ratio of 1 : 1.When the mass ratio was more than 1 : 1,the nanoparticle was negatively charged.With the increase of HA content,zeta potential decreased gradually because HA was negatively charged.The drug entrapment efficiency of HA-(BPEI-SS-Pt)-1/4 and HA-(BPEI-SS-Pt)-4/1 was 95.50 ± 0.71% and 90.00 ± 2.83% respectively,and the drug loading was 29.23 ± 0.18% and 7.86 ± 0.15% respectively.The results of storage stability showed that HA-(BPEI-SS-Pt)-1/4 and HA-(BPEI-SS-Pt)-4/1 nanoparticles could be kept stable after being stored at 4℃ for 28 d with minimal size changes.The results of SEM and TEM showed that HA-(BPEI-SS-Pt)-1/4 and HA-(BPEI-SS-Pt)-4/1 nanoparticles were spherical and uniform.Energy spectrum analysis of field emission scanning electron microscopy showed that there are C,N,O,S and Pt elements in the nano-delivery system of HA-(BPEI-SS-Pt)-1/4 and HA-(BPEI-SS-Pt)-4/1,indicating that the nano-delivery system was composed of HA and BPEI-SS-Pt,which was consistent with the design of the delivery system.2.The antitumor activity results showed that HA-(BPEI-SS-Pt)-1/4 and HA-(BPEI-SS-Pt)-4/1 could achieve effective tumoricidal activity in vitro.Considering the particle size,drug loading and MTT results in vitro,the HA-(BPEI-SS-Pt)-1/4 nano-delivery system was selected for the following study.The results of in vitro drug release showed that HA-(BPEI-SS-Pt)-1/4 could release the drug rapidly in high concentration of GSH,and the cumulative release of the drug was high,which behaved in the redox-responsive drug release behavior.In vitro competitive inhibition test showed that the addition of HA could inhibit the endocytosis of nanoparticles into the cells,and weaken the antitumor effect of HA-(BPEI-SS-Pt)-1/4.The in vitro competitive uptake showed that the content of Pt in the cells measured by ICP-MS and the cellular uptake observed by TEM was much lower when HA was added.The results of CLSM showed that the uptake of fluorescence labeled HA-(BPEI-SS-Pt)-1/4 was consistent with those detected by ICP-MS and TEM,indicating the active tumor-targeted effect of this nano-delivery system.The changes of cellular uptake with the addition of different inhibitors of endocytosis showed that the uptake pathway of HA-(BPEI-SS-Pt)-1/4 nanoparticles by A549 cells was mainly through caveolae and clathrin mediated pathway,observed by CLSM.3.The antitumor effect of HA-(BPEI-SS-Pt)-1/4 in vivo showed that HA-(BPEI-SS-Pt)-1/4 and cisplatin could achieve great antitumor effect.TUNEL apoptosis experiment,Caspase 3 and Ki67 immunohistochemistry experiments showed that HA-(BPEI-SS-Pt)-1/4 could cause more apoptosis of tumor cells in tumor tissue,inhibit the proliferation of tumor cells,and have better antitumor effect than cisplatin.Compared with the free drug cisplatin,the HE staining of the organs showed that there was almost no side effect of HA-(BPEI-SS-Pt)-1/4 and this nano-delivery system could effectively reduce the side effects of cisplatin.Tumor microenvironment and redox-sensitive cascade stimuli-responsive nano-delivery system for effective cancer therapy1.The dialdehyde modified PEG(PEG-Di Alde)was synthesized and the structure was characterized by 1H NMR and IR spectra.After the different crosslinking time,including 24 h,48 h and 72 h,the dual responsive nano-delivery system PEG-(BPEI-SS-Pt)was constructed and the particle size,PDI and zeta potential results showed that the crosslinking time of 72 h could constitute the nanoparticles with quite small size about 135 nm,narrow diameter distribution(PDI < 0.3)and the zeta potential was positive.The entrapment efficiency and drug loading of cisplatin in PEG-(BPEI-SS-Pt)detected by ICP-MS were 73.96% and 21.74%,respectively.The morphology of PEG crosslinked nanoparticles was spherical with small particle size and uniform dispersion.The results of storage stability test showed that PEG-(BPEI-SS-Pt)could be kept stable after being stored at 4℃ for 70 d and the particle size of nanoparticles changed relatively small.2.The pH sensitivity results of PEG-(BPEI-SS-Pt)showed that compared with the media at pH 7.4,the nanoparticles at pH 6.5 increased both in size and zeta potential.TEM results showed that at pH 7.4,the nanoparticles were in an independent spherical shape and relatively stable.However,when it comes to pH 6.5,the nanoparticle was irregular in shape,aggregated and had poor stability.In addition,the results of 1H NMR showed that PEG-(BPEI-SS-Pt)had no aldehyde peak at pH 7.4 because of the formation of Schiff base,but aldehyde peak appeared at pH 6.5,which indicated that PEG-(BPEI-SS-Pt)was tumor microenvironment responsive and the PEG shell could be effectively detached under the acidic tumor microenvironment.The results of in vitro drug release study showed that under the condition of pH 7.4,10 μM GSH and pH 6.5,10 μM GSH,the disulfide bond of PEG-(BPEI-SS-Pt)did not respond significantly to the low GSH concentration(10 μM)and the drug release increased very slowly.However,under the condition of pH 7.4,10 m M GSH,the drug release of PEG-(BPEI-SS-Pt)increased rapidly,which indicated that the disulfide bond could respond to the high concentration of GSH and achieve effective redox-responsive drug release.3.The results of MTT assay showed that there was no significant difference in IC50 value between cisplatin at pH 7.4 and 6.5,while PEG-(BPEI-SS-Pt)had much lower IC50 value at pH 6.5(tumor microenvironment),compared with pH 7.4,indicating that the detachment of PEG shell could help the nanoparticles perform better antitumor activity.In vitro cellular uptake experiments showed that PEG shell of PEG-(BPEI-SS-Pt)could stealth the nanoparticles and avoid the uptake by macrophages,which would reduce the rapid clearance of the nanoparticles in the blood circulation and improve the antitumor activity in vivo.The uptake of nanoparticles by A549 cells under different pH showed that the cellular uptake of PEG-(BPEI-SS-Pt)nanoparticles by A549 tumor cells was much more at pH 6.5 than that of pH 7.4.The results of 3D tumor spheroids penetration experiment in vitro showed that nanoparticles could have better tumor penetration ability under the acidic tumor microenvironment and would penetrate the deep tumor tissue,which indicated that the pH sensitive detachment of PEG shell could facilitate the effective delivery and antitumor activity of the nanoparticles.The changes of cellular uptake with the addition of different inhibitors of endocytosis,showed that the uptake pathway of PEG-(BPEI-SS-Pt)nanoparticles by A549 cells was mainly through caveolae and clathrin mediated pathway,observed by CLSM.In vivo antitumor experiments of PEG-(BPEI-SS-Pt)showed that PEG-(BPEI-SS-Pt)could achieve better antitumor effect than cisplatin.TUNEL apoptosis experiment and Caspase 3,Ki67 immunohistochemistry experiments showed that PEG-(BPEI-SS-Pt)could effectively induce apoptosis in the tumor tissue,inhibit the proliferation of tumor cells,and have better antitumor effect.Compared with the free drug,the HE staining of tissues and organs showed that the nanoparticles had little side effects and could reduce the toxic and side effects of cisplatin.ConclusionsIn this paper,based on the backbone of BPEI,BPEI-SS-Pt,a high cisplatin-loaded polymeric prodrug with redox-responsive behavior was synthesized and the structure was confirmed by 1H NMR and IR.By using BPEI-SS-Pt,two kinds of nano-delivery systems,active tumor-targeted and redox-responsive nano-delivery system HA-(BPEI-SS-Pt)and tumor microenvironment and redox-sensitive cascade stimuli responsive nano-delivery system PEG-(BPEI-SS-Pt),were designed and constructed.The antitumor results of two kinds of nano-delivery systems in vitro and in vivo showed that HA-(BPEI-SS-Pt)nano-delivery could realize the active tumor-targeted drug delivery and redox-responsive drug release,improving the antitumor activity and reducing the harm to other organs caused by cisplatin.PEG-(BPEI-SS-Pt)nano-delivery system could detach the PEG stealth shell in the tumor microenvironment and redox-responsive release the drug in the tumor cells,ensuring the effective drug uptake and stimuli drug release at the tumor site and achieving the tumoricidal effects without serious side effects.The construction and evaluation of two kinds of nano-delivery systems provided the theoretical basis and experimental support for the improvement of cisplatin,and showed certain research and application prospects in the clinic. |
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