| Background and Objective:Cancer is seriously threatening the life and health of all human beings.As the number of new cases and deaths of cancer ranked first country in the world,China’s tumor prevention and control situation is very grim.Chemotherapy is one of the most important means of treating cancer.The most critical issue at present is how to make chemotherapeutic agents specifically concentrate in the tumor site and maintain them for a longer period of time to avoid toxic side effects on non-target sites and their premature degradation,metabolism,elimination,as well as reduce the risk of cancer cells developing resistance to drugs caused by their systemic distribution,thereby achieving the purpose of improving curative efficacy and reducing toxicity.According to the consistency of administration site and action site,chemotherapy can be divided into two categories: local chemotherapy(consistent)and systemic chemotherapy(inconsistent).The former is suitable for superficial solid tumors such as skin cancer,oral cancer and deep entities that can be treated by interventional therapy.The latter is suitable for non-solid tumors,deep entities that cannot be treated by interventional therapy,and solid tumors accompanied by numerous metastatic lesions.For local chemotherapy,since the administration route itself has achieved the purpose of "specifically concentrating chemotherapeutic agents in the tumor site",the main problem is how to maintain the high concentration of drugs in the tumor site for a longer period of time,followed by how to make the administration of drug easier and have better patient compliance.For systemic chemotherapy,the primary problem is how to make chemotherapeutic agents specifically concentrate in the tumor site,and then how to maintain the high concentration of drugs in the tumor site for a longer period of time,in order to improve curative efficacy,the most bioavailable intravenous injection(100%)is still the most commonly used route of drug administration.Hydrogel is a dispersion system with water as the dispersion medium,with natural or synthetic high-molecular polymers as the dispersed phase,and the polymer chains crosslinking under certain conditions to form a three-dimensional network structure filled with water in the voids.Under certain conditions,it maintains a certain shape without flowing freely,and allows substances that can be highly dispersed in water to diffuse from it or penetrate into it,besides,its load space is large enough and its bioadhesion is good enough,which are well suited to slowly release drugs as a drug reservoir after loading drugs and applied locally to the site of the lesions in order to improve curative efficacy and reduce toxicity.In the past,due to the limitation of excessive viscosity,traditional hydrogels tended to be implanted into human body through open surgery.In recent years,the development of injectable hydrogel materials has made it possible to implant hydrogels into human body by injection which is much less traumatic,and their tissue gap fillibility and loaded-drugs dispersion uniformity are more excellent than traditional hydrogels.There is a wide variety of injectable hydrogels,but they all have the common feature of rapid conversion from liquid sol to semi-solid gel at the administration site.Among them,the phase transition of injectable thermosensitive hydrogels comes from their response to temperature difference between the environment in vitro and the administration site in vivo.Since the factor that cause the phase transition already exists in the administration site thus do not need to be introduced from outside,not only is the injection operation simpler,but also avoids the possibility of introduced factors adversely affecting the body and the loaded therapeutic agents,plus controlling the temperature of in vitro environment is relatively easy and human body temperature is relatively constant,which make the gelation time and performance in all aspects of hydrogels relatively stable,so they become the earliest also the most in-depth and extensively studied injectable hydrogels.In this project,an injectable thermosensitive hydrogel is used as the carrier to deliver a chemotheraputic drug aiming at solving the problems faced by local chemotherapy.At present,the strategies of specifically concentrating systemically administrated chemotherapeutic drugs in the tumor site can be divided into five aspects: The first is passive targeting,namely utilizing the enhanced permeability and retention effect(EPR)of solid tumors;The second is active targeting,namely utilizing the specific combination between specific or highly expressed antigens or receptors on the surface of cancer cells and their corresponding antibodies or ligands;The third is long circulation,namely PEGylating delivery system to prevent it from being destroyed by biological factors in the circulatory system before reaching the tumor site;The fourth is site-specific drug release,namely utilizing the delivery systems that can respond to the difference between the internal and external environments of tumors to release drug acceleratedly only at the tumor site due to physicochemical properties changes,according to the factors that initiate the changes in physicochemical properties,it can be divided into p H-sensitive,thermosensitive,reduction-sensitive,enzyme-sensitive;The fifth is magnetic targeting,namely adding an external magnetic field to the tumor site after combined administration of drugs and magnetic nanomaterials,using the magnetic field to concentrate the magnetic particles to the tumor site.The above five strategies are not opposed and conflicting,but can be synergistic and complementary,but it is not difficult to see that except magnetic targeting,other aspects are greatly affected by factors of human body,the complexity and individual differences of the body determine the poor controllability and stability of these strategies.The magnetic targeting is hardly affected by factors of human body,and studies have shown that the delivery systems concentrated to the tumor site by magnetic targeting can still be concentrated here for a long time after the external magnetic field is withdrawn.However,magnetic targeting has its limitations,such as not suitable for non-solid tumors and scattered metastatic lesions.Cancer is an extremely complex disease,and its occurrence and development involve multiple signaling pathways,so it is often difficult for a single chemotherapy to completely suppress the development of tumors,cancer cells may develop drug resistance or even return and metastasize with the progress of chemotherapy.Studies have shown that some new cancer treatments emerging in recent years,such as photothermal therapy,magnetic hyperthermia therapy,reactive oxygen-based therapy,gene therapy,immunotherapy,etc.,can be used in combination with chemotherapy to produce powerful anti-tumor effects,or even eliminate tumors completely.In addition to magnetic targeting effect,the iron oxide nanoparticles can also generate heat under the action of an external alternating magnetic field or near-infrared light to produce a thermotherapy effect.The difference between the heat tolerance limit of tumor cells and normal cells can be used to achieve the purpose of killing tumor cells without damaging normal tissues.Furthermore,iron oxide nanoparticles can induce pro-inflammatory M1 macrophage polarization in tumor tissues,iron ions and hydrogen peroxides released by M1 macrophages can produce highly cytotoxic hydroxyl radicals(OH·)via the Fenton reaction,they can also be used to improve the resolution of tumor magnetic resonance imaging(MRI).Therefore,after the systemic administration of a chemotherapeutic drug and iron oxide nanoparticles co-loaded particle system,the drug can be concentrated to the tumor site through magnetic targeting,the combination of multiple therapies(photothermal + reactive oxygen)and chemotherapy as well as the integration of tumor diagnosis and treatment can also be realized.Among various particle structures,nanogels have attracted much attention as a promising nanocarrier due to its character of combining hydrogel properties(such as hydrophilicity,flexibility,versatility,and high water absorption)with nanoscale size.It is a swollen nanoscale network composed of hydrophilic or amphiphilic polymer chains,its mutually cross-linked high-porosity network structure provides sufficient space or binding sites for the loading of substances.Moreover,the composition of nanogel is controllable,which is beneficial to the control of its physical or chemical properties(such as the chargeability and hydrophobicity of the side groups).The above features make it an ideal carrier for delivering therapeutic agents.In this project,nanogel is selected as the carrier for the combined delivery of chemotherapeutic drug and iron oxide nanoparticles aiming at solving the problems faced by systemic chemotherapy and single chemotherapy.Addressing the problems,"how to maintain the high concentration of drugs in the tumor site for a longer period of time" and "how to make the administration of drug easier and have better patient compliance",faced by local chemotherapy,the first chapter of this article constructs a chemotherapeutic drug-hydrogel complex therapeutic system that can be administered by local injection and form a drug reservoir at the tumor site to slowly release the drug.Addressing the problems,"how to make chemotherapeutic agents specifically concentrate in the tumor site" and "how to maintain the high concentration of drugs in the tumor site for a longer period of time",faced by systemic chemotherapy,as well as "how to overcome drug resistance,recurrence and metastasis of tumors" faced by single chemotherapy,the second chapter of this article constructs a chemotherapeutic drug and magnetic nanoparticles co-loaded nanogel therapeutic system that can be administered by intravenous injection and realizes both magnetic targeting and a combination of multiple therapies(photothermal + reactive oxygen)and chemotherapy,as well as the integration of tumor diagnosis and treatment.Methods:In the first part of this study,we used the poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide)(PDLLA-PEG-PDLLA,abbreviated as PLEL)triblock copolymer independently designed and synthesized by our team as the material to prepare PLEL injectable thermosensitive hydrogel,choose the hydrophilic chemotherapeutic drug mostly applied in clinical treatment of primary hepatocellular carcinoma——norcantharidin(NCTD)as a model drug,and loaded NCTD into PLEL hydrogel by physical mixing to prepare the drug-loaded hydrogel(NCTD/PLEL hydrogel).In the in vitro study,we studied the phase transition behavior of the drug-loaded hydrogel by the test tube inversion method and a rotational rheometer;The drug release behavior of the drug-loaded hydrogel was studied by high performance liquid chromatography(HPLC);The anti-tumor effect of the free drug solution was determined by the MTT assay,the anti-tumor effect of the drug-loaded hydrogel extracts were investigated by the MTT assay and flow cytometry-based apoptosis detection assay.In the in vivo study,we constructed a male BALB/c A-nu mouse human BEL-7402 hepatocellular carcinoma cells subcutaneous heterotopic xenograft tumor model,intratumoral injection administration was adopted,a fluorescent dye was used to simulate NCTD,the distribution uniformity and changes over time of the fluorescent dye in the tumor are studied through the cryo-sectioned tumor tissue slices and a small animal in vivo imaging system respectively;The anti-tumor effect of different administration groups was comprehensively evaluated by tumor growth inhibition and Ki-67,TUNEL immunohistochemical tests,the biological safety of different administration groups was evaluated by H&E stain.In the second part of this study,we used emulsion polymerization to prepare poly(N-isopropylacrylamide-co-methacrylic acid-co-polyethylene glycol methacrylate)(P(NIPAm-co-MAA-co-PEGMA))nanogels(NGs),choose the hydrophobic chemotherapeutic drug with broad-spectrum anti-tumor activity——10-hydroxycamptothecin(HCPT)as a model drug,co-loaded HCPT and magnetic iron oxide nanoparticles(MNPs)into NGs by emulsification to prepare the drug-loaded magnetic nanogel system(HCPT/MNP-NGs).In the in vitro study,we employed a transmission electron microscope(TEM)to observe the microscopic morphology of the system;A laser particle size analyzer was adopted to measure the particle size distribution and investigate the particle size changes with increasing or decreasing temperature of the system;The photothermal conversion assay of the system was performed by utilizing an UV-Vis spectrophotometer,an infrared digital thermometer and thermal imager;Comprehensively considering the actual drug loading and encapsulation efficiency measured by HPLC,the particle size distribution and stability,we screened the optimal ratio of HCPT and NGs;The drug release behavior of the system was studied by HPLC;A fluorescent dye was used to simulate HCPT,the MCF-7 uptake of the system was investigated by the slides of cells and a flow cytometry;The biological safety and anti-tumor effect of the system were determined by the MTT assay;Transwell assay was used to investigate the system stimulating the RAW264.7 to release ROS.In the in vivo study,we constructed a female BALB/c A-nu mouse human MCF-7 breast adenocarcinoma cells subcutaneous heterotopic xenograft tumor model,tail vein injection administration was adopted,the magnetic targeting action and imaging effect of the system were investigated by a MRI imager,the biological distribution of the system was inspected by inductively coupled plasma atomic emission spectroscopy(ICP-AES);The photothermal conversion performance of the system was studied by an infrared thermal imager;The anti-tumor effect of different administration groups was comprehensively evaluated by tumor growth inhibition and TUNEL immunohistochemical test,the biological safety of different administration groups was evaluated by body weight.We constructed a female BALB/c mouse highly metastatic mouse 4T1 breast adenocarcinoma cells subcutaneous heterotopic xenograft tumor model,tail vein injection administration was adopted,the anti-tumor effect of different administration groups was evaluated by tumor growth inhibition,the biological safety of different administration groups was evaluated by H&E stain,the metastasis suppression effect of different administration groups was evaluated by counting the number of metastatic tumor nodules visible in the lungs.Results:In the first part of this study,we prepared the NCTD-loaded PLEL injectable thermosensitive hydrogel(NCTD/PLEL hydrogel)and proceeded the in vivo and in vitro studies of it,the results are as follows:1.The addition of NCTD has no significant effect on the temperature-dependent rheological properties of PLEL hydrogel;The sol-gel phase transition temperature of the drug-loaded hydrogel with a PLEL content of 13 wt% is about 34 ℃,the gelation time under 37 ℃ is about 27 s,the system keeps its sol state at room temperature and shows a gel state at body temperature.2.The drug release behavior of the drug-loaded hydrogel fits the first-order kinetic equation,the drug release mechanism is dominated by drug diffusion,and the drug release is significantly slower than that of the free drug solution.3.None of the blank hydrogel extracts shows cytotoxicity,while the drug-loaded hydrogel extracts show obviously concentration-dependent and time-dependent cytotoxicity.4.Its precursor can evenly distribute in the tumor tissue before converting into a gel after being intratumorally injected.After transforming into a gel,it can significantly prolong the maintenance time of the concentrated state of the drug at the tumor site compared to the free drug solution.5.The different doses of drug-loaded hydrogel intratumoral injection groups show no obvious organ toxicity and the curative efficacy increase with the dose;At the same dose,the drug-loaded hydrogel group is better than the free drug groups,the free drug groups have obvious toxicity to lungs,liver and kidneys.In the second part of this study,we prepared the 10-hydroxycamptothecin and magnetic iron oxide nanoparticles co-loaded nanogel system(HCPT/MNP-NGs)and proceeded the in vivo and in vitro studies of it,the results are as follows:1.The system is in the form of spherical particles,with thermal shrinkage characteristics,and shrinks under physiological conditions with a particle size of about 200 nm.2.The absorption intensity of MNP-NGs in the near-infrared region is significantly higher than that of NGs;MNP-NGs has a remarkable photothermal response effect compared with normal saline.3.When preparing HCPT/MNP-NGs,the optimal ratio of HCPT and NGs is1∶20.4.Compared with the free drug,the drug release of HCPT/MNP-NGs is significantly slowed down,the release rate is slower and the release degree is smaller at physiological temperature compared to room temperature.5.After the system is uptaken by MCF-7,most of them locate in the cytoplasm,and the uptake of cells in the group with an external magnetic field significantly increased compared with the non-magnetic field group.6.MNP-NGs show low toxicity to NIH3T3 and MCF-7;The toxicity to MCF-7,MNP-NGs+Laser group is significantly higher than MNP-NGs group,IC50 value of HCPT/MNP-NGs group is slightly higher than free HCPT group,applying an external magnetic field can enhance the toxicity of HCPT/MNP-NGs.7.The fluorescence intensity of MCF-7 single culture group,MNP-NGs and MCF-7 co-culture group,MNP-NGs and RAW264.7 with MCF-7 co-culture group increase in sequence;The hydrogen peroxide and hydroxyl radical levels of MNP-NGs and MCF-7 co-culture group,RAW264.7 single culture group,MNP-NGs and RAW264.7 co-culture group,MNP-NGs and RAW264.7 with MCF-7 co-culture group increase in sequence and are all higher than MCF-7 single culture group,among which,the hydrogen peroxide level of MNP-NGs and RAW264.7 with MCF-7 co-culture group is 8 times higher than MCF-7 single culture group,the hydroxyl radical level is 7 times higher than MCF-7 single culture group.8.The T2-weight signal intensity at the tumor site of the mice in the external magnetic field group is 1.66 times stronger than that of the non-magnetic field group,the Fe element distribution is also more than that of the non-magnetic field group.9.After 5 minutes of near infrared laser irradiation,the temperature of the tumor site can rise to 48 ℃,10 minutes later,the temperature will reach 53 ℃.10.The curative effect of the free drug group is the worst and the body weight loss of the mice is the most significant;The body weight of the mice in the other groups do not decrease obviously;HCPT/MNP-NGs+MF group has the best curative effect;There is no significant difference between the curative effect of MNP-NGs+MF group and HCPT/MNP-NGs group,but they are all significantly different from HCPT/MNP-NGs+MF group.11.The curative effect on primary tumor of HCPT/MNP-NGs+MF group and MNP-NGs+MF+Laser group is significantly different from normal saline group,the number of tumor metastatic nodules is relatively less;The primary tumor in HCPT/MNP-NGs+MF+Laser group has almost completely disappeared after 10 days,although there is a slight recurrence in the later stage,the curative effect is still significantly better than that of the other groups,the lung tissue morphology of this group is the most intact and the number of tumor metastatic nodules is the fewest.Conclusions:NCTD/PLEL injectable thermosensitive hydrogel can maintain the concentrated state of the drug at the tumor site for a longer period of time,and the injection administration is easy to operate,which is expected to improve the tumor curative effect and reduce the toxic side effects in local chemotherapy.HCPT/MNP-NGs magnetic nanogel system can effectively deliver the drug to the tumor site under the action of an external magnetic field and maintain the concentrated state of the drug at the tumor site for a longer period of time.It is expected to improve the therapeutic effect of primary tumors,inhibit the distant metastasis of tumor cells,and reduce toxic side effects in systemic chemotherapy via the combined action of photothermal therapy,ROS and chemotherapy. |
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