Preparation Of Multifunctional Nanodrugs Based On Novel Carbon Dots And Its Application In The Precision Theranostics Of Colorectal Cancer

Objective:Colorectal cancer（CRC）is a malignant tumor that begins in the colorectal mucosa,according to the statistics of the National Cancer Center in Februar y2022,CRC accounted for 40.8%of the 4.064 million cancer cases,second onl y to lung cancer（82.8%）,and CRC accounted for 19.6%of the 2.414 million cancer deaths,ranking fourth.Despite the development of surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy,the overall survival rate for patients with CRC remains low.In order to improve the efficiency of diagnosis and treatment of CRC,more and more researchers begin to focus on nanomaterials.Among them,carbon dots（CDs）has attracted much attention due to its high dispersion rate,long life,high stability,low toxicity,good biocompatibility and photobleaching resistance.In this paper,two multifunctional CDs were prepared and the targeted nanomedicine with multi-modality and multi-means of diagnosis and treatment was constructed to explore its role in the diagnosis and treatment of CRC.The main contents are as follows:1.Fluorescent carbon dots（GCDs）with excellent photothermal properties were synthesized by solvothermal method using citric acid and methotrexate as raw materials and formamide as solvent.Then photosensitizer Ce6 and targeted antibody Cet were connected with G CDs through amide bond to form GCDs-Ce6-Cet nano-drug,and finall y adsorbed on the surface of Mn O ₂ nano-sheet b y physical action to construct Glutathione（GSH）responsive nano-drug Mn O₂@GCDs-Ce6-Cet（MGCC）.GCDs and nano-drugs were characterized and tested in vitro,and their uptake,metabolism,imaging,treatment and biosafet y were evaluated at cell level and in vivo level.2.Red fluorescent carbon dots（Ni/Mn-CDs）with optical absorption and chemical kinetic properties in NIR-II region were synthesized by one-step hydrothermal method using Ethylene diamine tetraacetic acid（EDTA）,nickel chloride,manganese chloride and o-phenylenediamine as raw materials.Then,nano drugs（Ni/Mn-CDs-Cet-SNO,CCS）were synthesized and loaded with thermosensitive Nitric oxide（NO）small molecule donor（SNO）and targeted antibody Cet.Finally,Ni/Mn-CDs and nano-drugs were characterized in structure and tested in vitro,and their uptake,metabolism,imaging,treatment and biosafety were evaluated at cellular and in vivo levels.Method:1.Using citric acid and methotrexate as raw materials and formamide as solvent,GCDs with the best fluorescence and photothermal properties were synthesized by solvothermal method through optimizing the synthesis conditions（reactant ratio,synthesis temperature and synthesis time）,and then Ce6 and Cet were grafted by chemical bonds,and finally bonded to Mn O₂ nanosheets by physical adsorption to obtain the final nanomaterials MGCC.The properties of GCDs and MGCC were characterized and tested by transmission electron microscope（TEM）,atomic force microscope（AFM）,X-ray diffractometer（XRD）,X-ray photoelectron spectrometer（XPS）,Fourier transform infrared spectrometer（FTIR）,ultraviolet-visible spectrophotometer and fluorescence spectrophotometer.The Cellular uptake,the response ability to GSH of MGCC and the active targeting ability of nanomaterials were observed by laser confocal microscopy.The killing effect of nanomaterials on colorectal cancer cells（HCT-116）,breast cancer cell（MCF-7）and Normal esophageal cells（HEEC）was investigated by MTT and Calcein-AM/PI experiments.The model of HCT-116 tumor bearing nude mouse was constructed,and the T1 nuclear magnetic signal of MGCC in vitro and in vivo magnetic resonance imaging（MRI）were studied by small animal nuclear magnetic imaging system.The photothermal imaging（PTI）ability of nanomaterials in vivo was observed by thermal imager.The antitumor effect of nanomaterials at the living level was evaluated by the changes of tumor volume in tumor-bearing nude mice.The biological safety of nanomaterials was evaluated by the changes of body weight,blood biochemical analysis and organ tissue section analysis during treatment.2.Using EDTA,nickel chloride,manganese chloride and o-phenylenediamine as raw materials,four kinds of carbon dots were synthesized by one-step hydrothermal method after combination in different proportions.By testing the visible light absorption spectra and photothermal properties of different CDs,the carbon dots（Ni/Mn-CDs）with excellent photothermal properties in NIR-II region and 1064 nm laser excitation were obtained.Then,the small molecule donor of NO SNO was synthesized from 4-mercaptobutyric acid and tert-butyl nitrite.SNO was characterized by ¹H NMR,UV-visible absorption spectrum and FTIR,and then Cet and SNO were connected with Ni/Mn-CDs by amide bond to synthesize nano-drug CCS.Ni/Mn-CDs and CCS were characterized and tested by TEM,AFM,XRD,XPS,FTIR,UV-visible spectrophotometer and fluorescence spectrophotometer.The Cellular uptake of nano-drugs and the active targeting ability of CCS were observed by laser confocal microscopy,and the killing effect of CCS on HCT-116 cells and Normal colorectal epithelial cells（NCM-460）was investigated by MTT and Calcein-AM/PI experiments.The bearing nude mouse model of HCT-116 tumor was constructed,and the T1 nuclear magnetic signal of CCS in vitro and magnetic resonance imaging（MRI）in vivo were studied by small animal nuclear magnetic imaging system.The metabolism and distribution of nano drugs in vivo were observed by MRI/fluorescence imaging（FLI）/PTI.Next,the anti-tumor effect and biological safety of CCS in vivo were evaluated using the same method as the above part.Results:1.The characterization results showed that the synthesized GCDs had good dispersion and uniform size distribution,the particle size was less than 10 nm.It was an amorphous carbon structure,mainly consisting of C,N and O elements.In vitro test results showed that GCDs had good fluorescence stability and low toxicity.The MGCC was successfully synthesized by absorption spectrum and Zeta potential.The particle size of MGCC was about80 nm.XPS showed that its main elements included C,N,O,Mn.MGCC can produce oxygen（O₂）in the presence of tumor endogenous H₂O₂,which enhances its photodynamic performance.Meanwhile,MGCC also has excellent photothermal and Fenton-like properties.Cell and animal experiments showed that single chemokinetic therapy（CDT）,photothermal therapy（PTT）and photodynamic therapy（PDT）had different anti-cancer effects,but the CDT/PTT/PDT combination treatment had the best effect,and the tumor in the combined treatment group could completely subside after 14 days of treatment.In the small animal MR imaging system,MGCC showed obvious T1 MR signal,and MRI/PTI at the tumor site of mice showed that nanomaterials could actively gather at the tumor site,and reached maximum enrichment at the tumor site 8 h after caudal vein injection.The body weight of mice did not decrease significantly,the blood biochemical indexes were all within the normal range after treatment,and HE staining of major tissues and organs showed no significant pathological changes.The results show that the nanodrug has no hemolysis,excellent anti-tumor effect and good biocompatibility in vivo.2.The characterization results showed that the synthesized Ni/Mn-CDs had good dispersion and uniform size distribution,and the particle size was less than 10 nm.Ni/Mn-CDs had amorphous carbon structure,mainly consisting of C,N,O,Mn and Ni elements.Optical stability experiments show that Ni/Mn-CDs has good fluorescence stability.Nuclear magnetic hydrogen spectroscopy（¹H NMR）proved that SNO was synthesized successfully,and SNO had excellent NO release ability at higher temperature.The final prepared nano drug（CCS）is spherical with a particle size of about 190 nm.XPS characterization shows that it mainly consists of C,N,O,Mn,Ni and S elements.In vitro performance test results show that CCS has excellent photothermal and chemical kinetic properties.CCS can achieve controlled release of NO under 1064 nm laser irradiation,and has the ability to actively target HCT-116 cells.The combination of CDT/PTT/NO gas therapy has the best effect.The small animal MR imaging system observed obvious T1 MR signal of CCS,and the mouse FLI/MRI/PTI multimodal imaging showed that CCS could target to the tumor site in vivo.The mice experiment after 14 days of treatment showed that the tumor was completely subsided in the combined treatment group and no significant changes in the body weight of the mice.After treatment,the blood biochemical indexes were all within the normal range,and HE staining of major tissues and organs showed no obvious pathological changes.The above experiments confirmed that nano drugs have excellent anti-tumor effect and good biocompatibility in vivo.Conclusion:In this paper,two multifunctional carbon dots were synthesized and two new nanomaterials applied in the integration of CRC diagnosis and treatment were constructed.The nanomaterials were all grafted with targeted antibody Cet,which endowed them with active targeting ability.After the nanomaterials MGCC in the first part is targeted to CRC tumor cells,fluorescence recovery is achieved in the microenvironment of high GSH in cancer cells,and the efficacy of nanomaterials PDT is enhanced by the self-oxygen supply of tumor endogenous H₂O₂.PTI and PTT are performed under 808 nm laser irradiation.CDT/PTT/PDT multi-means combined targeted therapy for CRC guided by MRI/PTI dual-mode imaging;In order to obtain a photothermal agent with deeper tissue penetration ability,Ni/Mn-CDs with strong absorption and heat generation ability in the NIR-II window was further developed in the second part of the work.Meanwhile,the heat-sensitive NO small molecule donor SNO was synthesized and combined with Ni/Mn-CDs to achieve controlled release of NO.Combined with the CDT effect of Ni/Mn-CDs and the photothermal properties of Ni/Mn-CDs-Cet-SNO（CCS）,the novel nanomaterials（Ni/Mn-CDs-Cet-SNO,CCS）are prepared after NO gas therapy.CCS can be used for multi-modal targeting of CRC imaging,providing effective support for the diagnosis and treatment of nanomaterials.In vitro and in vivo experiments have verified that CCS can be used in the treatment of CRC by the combination of CDT,PTT and NO gas therapy,indicating that CCS can significantly improve the therapeutic effect of CRC.Two novel nanomaterials have achieved good therapeutic effects on CRC at the cellular level and in vivo level,taking into account the advantages of multi-modal imaging.This paper provides new ideas and new means for the integration of clinical diagnosis and treatment of CRC.