Construction And Preliminary Study Of Iron Alginate Nano-Drug Delivery System

Tumor is a serious disease threatening human health.Due to lack of specificity,the traditional tumor chemotherapy has great toxicity and side effects on normal tissue.The nano-drug delivery system?NDDS?could increase the accumulation of chemotherapeutic drugs in tumor tissue through the enhanced permeability and retention?EPR?effect,but there are still problems such as low delivery efficiency,incomplete drug release,and inability to achieve deep tumor tissue.Hence,we designed an intelligent drug delivery system with the characteristic of size transformation under tumor micro-environment?TME?.It could improve the tumor-targeting ability of drugs,achieve TME-responsive drug release at the tumor site,and further deliver drugs to the deep region of tumor tissue.This system employed natural polysaccharide sodium alginate?Alg?with good biocompatibility as the matrix material,FeCl₃·6H₂O as the cross-linking agent,doxorubicin?DOX?as the model drug and Cysteine-Arginine-Glycine-Aspartic acid-Lysine?CRGDK?peptide as the target molecule.In this study,Iron-alginate nanogel?FeAlg?was synthesized as a drug carrier by a simple one-step cross-linking method.The factors affecting the particle size and morphology of FeAlg nanogel were optimized.Firstly,the tumor targeting peptide CRGDK was grafted onto Alg skeleton by amide bond?CRGDK-Alg?,to give FeAlg nanocarrier active targeting performance.Then,DOX was efficiently encapsulated in CRGDK-Alg nanogel during Fe³⁺cross-linking to prepare a CRGDK-FeAlg/DOX system.In the mildly acidic and reducing TME,Fe³⁺was reduced to Fe²⁺.CRGDK-FeAlg/DOX nanogel dissociated along with the particle size conversion from large to small.On the one hand,it could realize drug release in response to TME,and on the other hand,it could enhance the penetration effect of nano-preparation in the deep tumor region.Moreover,FeAlg nanoegel could efficiently catalyze H₂O₂ to produce·OH by Fenton reaction in tumor hypoxic environment,achieving local chemodynamic therapy without O₂ mediation.Therefore,CRGDK-FeAlg/DOX system could realize tumor multi-mechanism treatment?ROS and chemotherapy?at the same site.In vitro study showed that CRGDK was successfully grafted onto the Alg skeleton,and the graft rate was 14.68%.Under the simulated TME in vitro,the size of FeAlg reduced from 50 nm to<10 nm.This indicated that FeAlg could achieve size conversion at the tumor site,facilitating the penetration of nano-formulation into deep tumor tissue.CRGDK-FeAlg could efficiently load DOX,and the drug loading efficiency and encapsulation efficiency were 37.56%and 40.12%,respectively.Drug release experiments showed that the cumulative release rate of CRGDK-FeAlg/DOX in the mildly acidic and reducing medium?pH=5.5 and 2 mM Glutathione?GSH??was 81.98%,which was higher than that of the PBS group?49.69%?.This feature indicated CRGDK-FeAlg/DOX nanogel could realize the accurate drug release in tumor cells,enhancing the anti-tumor effect and reducing the adverse reaction of DOX.The results of cell uptake indicated that the uptake amount of CRGDK-FeAlg/DOX by A549 cells with high expression of neuropilin-1?Nrp-1?was 2.87 times than that of FeAlg/DOX group at 1h,due to the high affinity of CRGDK with the Nrp-1 receptor.While in MCF-7 cells with low expression of Nrp-1receptor,the uptake amount showed no significant difference for the different preparations.The results of lysosomal colocalization experiments showed that the lysosomal escape could be effectively achieved after the nano-preparation entering cells,promoting DOX into the nucleus to exert anti-tumor effect.Reactive oxygen species?ROS?test indicated that CRGDK-FeAlg could effectively induce ROS production.The results of cell apoptosis experiment showed that the apoptosis rate of CRGDK-FeAlg/DOX?5?g/mL?at 6 h was as high as 81.9%,which was 1.7 times higher than that of DOX group,indicating CRGDK-FeAlg/DOX could effectively induce apoptosis of A549 tumor cells.The results of Calcein-AM/PI double staining showed that the percentage of cell death was 65.93%when CRGDK-FeAlg/DOX was applied at 0.6?g/mL for 24 h,which was 53.73%higher than that of DOX group.This was because CRGDK-FeAlg/DOX not only had active targeting effect,but also owned sustained release effect.It is beneficial to maintain a high and lasting intracellular drug concentration.Furthermore,the?OH radicals produced by Fenton reaction also enhanced the anti-tumor effect.The results of cell inhibition showed that the inhibition rate of CRGDK-FeAlg/DOX?5?g/mL?for 48 h on A549 cells was75.26%,which was 36%higher than free DOX group.In vitro three-dimensional multi-cell spheres?3DMCS?penetration experiment results showed that CRGDK-FeAlg/DOX could effectively achieve deep tumor penetration.Compared with SiO₂ nanoparticles with constant particle size,the penetration distance of CRGDK-FeAlg/DOX increased about 60?m,which was beneficial for its anti-tumor effect.This was originated from the TME-triggered size conversion effect of FeAlg,thus delivering DOX to deep tumor and achieving the uniform distribution of DOX in the tumor tissue.In vivo,A549 tumor-bearing nude mice were used as animal models.In vivo imaging results showed that CRGDK-FeAlg/DOX enabled efficient accumulation and long-term retention in the tumor site.The results of deep tumor penetration showed that CRGDK-FeAlg could efficiently deliver DOX to the deep region of tumor tiss ue.The results of ROS production at tumor sites showed that CRGDK-FeAlg could effectively induce tumor cells to produce ROS,which was more effective than FeAlg group.Pharmacodynamics experiments showed that CRGDK-FeAlg/DOX could significantly inhibit the tumor proliferation in vivo.The relative tumor volume?V/V₀?in CRGDK-FeAlg/DOX group after treatment was only 1.13,significantly lower than that of blank control group with V/V₀ of 4.79.