¹³¹I-Labeled Controlled Release Nitric Oxide（NO） Nanodelivery System For Combination Tumor Therapy

Objective:Malignant tumor has become a major threat to human health.At present,the treatment of malignant tumors mainly consists of surgery,radiotherapy and chemotherapy.However,all the above treatment methods lack specificity and are easy to cause complications and toxic side effects.Therefore,the development of a new precision treatment has become an urgent problem to be solved.Nitric oxide（NO）,as an important gas signal molecule,plays a key regulatory role in many physiological and pathological processes,has attracted much attention because it can directly kill cancer cells at high concentrations.As we all know,the site of NO release,exposure time and dose are the key factors in cancer treatment.However,the half-life of NO in organisms is relatively short,and it is easy to diffuse and unstable,so it is difficult to achieve high concentration aggregation in space and time to achieve the expected therapeutic effect.In order to solve this problem,researchers have designed and developed many NO donors,but most of the donors are easy to decompose and difficult to store,and there will be premature leakage in practical applications.Therefore,it is urgent to construct a new means to achieve NO donor accumulation and controlled release at the tumor site.With the vigorous development of nanotechnology,people are committed to designing and developing the combination of NO donors and nanocarriers.Due to the high permeability and retention effect（EPR effect）of nanocarriers on solid tumors,it can achieve passive targeting at tumor sites,laying the foundation for the precise and controllable release of NO at the tumor site.In addition,radionuclides have a strong killing effect on abnormal proliferation of tumor cells,and the radiation released in the decay process can directly cause the damage of tumor cell DNA,so as to achieve therapeutic effect.Studies have shown that NO can enhance the sensitivity of tumor cells to radionuclides,providing a new way to solve the problem of radionuclide therapy resistance.Therefore,this study aims to construct a controlled release nitric oxide（NO）nanodelivery system based on radionuclide ¹³¹I labeling,so as to achieve the effect of combining ¹³¹I with NO gas therapy and other methods for tumor treatment.Methods:（1）Through the organic synthesis of NO donor（LN-NO）,using polyethylene glycol modified polydopamine（PDA-PEG）as nano-carrier,LN-NO was loaded on the surface of PDA-PEG byπ-πstacking.Finally,¹³¹I was labeled by chloramine-T method to obtain¹³¹I-LN-NO@PDA-PEG.The successful synthesis of LN-NO was proved by ¹H NMR,¹³C NMR and MS;The LN-NO@PDA-PEG was characterized by UV-Vis,FT-IR,XPS and TEM,and its successful preparation was verified.The labeling rate,radiochemical purity and radiostability of ¹³¹I-LN-NO@PDA-PEG were determined byγcounter and TLC.The controlled release behavior of LN-NO@PDA-PEG triggered by 808 nm laser irradiation was investigated in vitro by Griess reagent,and its photothermal performance was monitored by infrared thermal imager.The uptake of LN-NO@PDA-PEG by He La cells was studied by confocal imaging.NO fluorescence probe DAF-FMDA was used to monitor the production of NO in cells under 808 nm irradiation.The killing effect of¹³¹I-LN-NO@PDA-PEG in cells was further studied by MTT and Calcein-AM/PI staining.The therapeutic effect of ¹³¹I-LN-NO@PDA-PEG in nude mice was proved by comparing the tumor volume and tumor weight,and the biological safety of ¹³¹I-LN-NO@PDA-PEG in nude mice was evaluated according to biochemical indexes,organ tissue sections and changes in the body weight of nude mice.（2）Hyaluronic acid modified manganese dioxide（MnO₂/HA）was used as nanocarrier to enhance its active targeting ability,NO donor（SD-NO）was loaded on the surface of MnO₂/HA by hydrophobic interaction,and ¹³¹I was labeled by chloramine-T method to prepare ¹³¹I-SD-NO@MnO₂/HA.The successful synthesis of SD-NO,the successful preparation of SD-NO@MnO₂/HA and the successful labeling of ¹³¹I-SD-NO@MnO₂/HA were verified by means described in（1）,and the labeling efficiency,radiochemical purity and radiostability of ¹³¹I-SD-NO@MnO₂/HA were investigated.Using Griess reagent method to further explore the controllable NO release performance of SD-NO@MnO₂/HA under ultrasonic.The Fenton-like effect of SD-NO@MnO₂/HA was detected by methylene blue（MB）in vitro.Reactive oxygen species probe DPBF and ONOO^-probe DHR123 were used to study the ability of SD-NO@MnO₂/HA to produce ROS and ONOO^-under ultrasound stimulation in vitro.Confocal imaging was used to study the cell targeting ability of SD-NO@MnO₂/HA,and DAF-FMDA and DHR123 were used to verify the production of intracellular NO and ONOO^-,respectively.MTT assay and cell Calcein-AM/PI staining were used to study the cytotoxicity of ¹³¹I-SD-NO@MnO₂/HA.The tumor-bearing nude mice were photographed and recorded,and the therapeutic effect of ¹³¹I-SD-NO@MnO₂/HA in vivo was evaluated by observing the changes of tumor volume and tumor weight,and the biological safety of¹³¹I-SD-NO@MnO₂/HA in nude mice was evaluated according to the biochemical indexes,organ tissue sections and body weight changes of nude mice after the treatment cycle.Results:（1）Successfully synthesized LN-NO and prepared LN-NO@PDA-PEG and ¹³¹I-LN-NO@PDA-PEG.In vitro experimental results showed that LN-NO@PDA-PEG has high drug loading rate and good in vitro stability,and can achieve NO production and controlled release under 808 nm laser irradiation.The results of in vitro experiments showed that LN-NO@PDA-PEG had a high loading rate and good stability,was able to produce and release NO under 808 nm laser irradiation with good photothermal properties.Confocal imaging results showed that LN-NO@PDA-PEG could be taken up by cells within 8 hours;MTT assay and Calcein-AM/PI staining further verified that ¹³¹I-LN-NO@PDA-PEG had strong killing ability under 808 nm laser irradiation;The results of the treatment in nude mice showed that ¹³¹I-LN-NO@PDA-PEG had good anti-tumor effect after 808 nm laser irradiation;the results of organ sections and biochemical indexes did not show any significant abnormalities,which proved that ¹³¹I-LN-NO@PDA-PEG had good biological safety.（2）SD-NO,SD-NO@MnO₂/HA and ¹³¹I-SD-NO@MnO₂/HA were successful prepared.The experimental results in vitro show that SD-NO@MnO₂/HA has high drug loading rate and stability in vitro.After ¹³¹I labeling,it has high labeling rate,radiochemical purity and radiostability.In addition,SD-NO@MnO₂/HA can achieve the production of NO,ROS and ONOO^-under in vitro ultrasonic stimulation.Confocal imaging results showed that SD-NO@MnO₂/HA can be fully taken up by He La cells within 4 hours,which can trigger the production of NO,ROS and ONOO^-in the cell by SD-NO@MnO₂/HA under ultrasonic stimulation.At the same time,MTT results confirmed that ultrasonic stimulation ¹³¹I-SD-NO@MnO₂/HA had strong cytotoxicity in He La cells,and the results of Calcein-AM/PI staining were basically corresponding to the results of MTT.Compared with the single treatment,the ¹³¹I-SD-NO@MnO₂/HA group showed a more obvious advantage in the treatment of tumor-bearing nude mice,further verifying the significantly improved anti-tumor effect of combination therapy.There were no obvious abnormalities in organ tissue sections and biochemical indexes after the end of the treatment cycle,indicating that ¹³¹I-SD-NO@MnO₂/HA has good biological safety.Conclusion:In this work,a ¹³¹I labeled controlled release NO nanodelivery system was constructed for the study of collaborative tumor therapy,which realized the aggregation of¹³¹I and NO donors at the tumor site and the controlled release of NO in response to stimulation,reducing the toxic and side effects of normal sites.In addition,the combination of NO gas therapy with other treatments overcomes the defects of single therapy and provides a new strategy and basis for combined and efficient therapy.（1）PDA-PEG nanocarriers were used to obtain ¹³¹I-LN-NO@PDA-PEG loaded with LN-NO and ¹³¹I,so as to achieve more efficient aggregation in tumor sites.Under 808nm laser irradiation,PDA-PEG can convert near-infrared light into heat to kill cancer cells and be used for photothermal treatment of tumor.The N-N bond fracture of LN-NO can be further induced by converting near-infrared photons into active electrons to achieve controlled release of NO.In addition,NO can enhance the sensitivity of tumor cells to ¹³¹I,reduce the radiation damage to the normal site,and ultimately achieve the effect of ¹³¹I-LN-NO@PDA-PEG gas/photothermal/radionuclide combination therapy.（2）Compared with near-infrared light,ultrasonic,as a mechanical wave,has the characteristics of deep tissue penetration,low cost and operability.Inspired by the first part,in order to further improve the targeting and anti-tumor performance of the nanodelivery system,SD-NO,which can produce ROS and NO simultaneously under ultrasonic stimulation,was designed and synthesized.At the same time,MnO₂/HA as a nano-carrier can not only realize the active targeting of He La cells with high CD44 receptor expression,but also consume the high concentration of GSH in the tumor microenvironment,thus mediating the Fenton-like reaction and achieving the effect of chemokinetic therapy.SD-NO and ¹³¹I were loaded onto the surface of MnO₂/HA for the synergistic treatment of cervical cancer with NO gas/sonodynamic/chemokinetic/radionuclide targeted under ultrasonic stimulation.