The Study Of A Novel Compositive Hydrogel System Co-loading TPZ-loaded Micelles And PD-1 Antibody Combined With Radiotherapy In The Treatment Of Triple-negative Breast Cancer

Background and objective:Triple-negative breast cancer（TNBC）is a large type of tumors.TNBC are negative for estrogen receptor（ER）,progesterone receptor（PR）and human epidermal growth factor receptor 2（HER2）.TNBC accounts for about 10-20%of breast cancer.And the local recurrence rate and distant metastasis rate of TNBC are higher than any other breast cancer subtypes.TNBC is also the one with the worst prognosis and the most limited treatment strategy among all breast cancers,so it’s urgently to explore new treatment modes for TNBC.Hypoxia is very common in solid tumors.And hypoxia is one of the main reasons for the failure of radiotherapy or chemotherapy.Tirapazamine（TPZ）is one of the typical representatives of hypoxia activated prodrugs（HAPs）.TPZ is activated at the hypoxic tumor site,and it has certain advantages over traditional chemotherapy.However,problems such as large dosage of TPZ,poor water solubility,and complicated administration,etc.restrict the further application of TPZ.Radiotherapy is been widely used in tumor treatment,and it is also one of the important treatments for TNBC.Radiotherapy can aggravate the local hypoxic state of the tumor,thus can activate and increase the efficiency of TPZ,theoretically.Although in both basic experiments and clinical phaseⅠ/Ⅱtrials,TPZ combined with radiotherapy or chemotherapy had been proved to have a good application prospect,some key phaseⅢclinical trials showed that TPZ could not increase the efficacy on the basis of concurrent chemo-radiation or combined chemotherapy.Even so,a few phaseⅢclinical trial and subgroup analysis,still support the application of TPZ in patients with hypoxic tumors,suggesting that the optimization of TPZ administration ways may contribute to the improvement of its effect.Immunotherapy has changed the treatment of cancer,and become another important treatment method besides surgery,radiotherapy and chemotherapy.However,problems such as poor control of large tumors,low single-drug efficiency,and potentially fatal adverse events of immunotherapy can’t be ignored.At present,the research of immunotherapy in breast cancer mainly focuses on TNBC.In the neoadjuvant setting of TNBC,it has been reported that,antibodies against PD-1 or PD-L1（αPD-1/αPD-L1）can enhance the efficacy of chemotherapy,but the adverse events have also increased.Besides,the combination of immunotherapy and radiotherapy has also been reported to increase the efficacy,and may happen the abscopal effect.Although the combination of immunotherapy and chemotherapy or radiotherapy show good effects and prospects,the optimal combination of immunotherapy still needs further research.The drug delivery system（DDS）has gradually become a powerful tool for combination therapy of cancer.The micelle can encapsulate hydrophobic drugs（such as TPZ）,improve the dispersibility in water of these drugs,and increase their efficacy.But micelle have shortcomings,such as too short residence time,sudden drug release,etc.Hydrogel can load hydrophilic drugs（such asαPD-1）,and has advantages such as sustained drug release,etc.,and the combination of micelle and hydrogel may complement each other.On a same carrier,it’s still difficult to co-deliver hydrophobic micromolecular drugs and hydrophilic macromolecular ones simultaneously and efficiently at present.Based on all these,in the first part of this study,we used monomethyl polye-thylene glycol-polycaprolactone（MPEG-PCL）to encapsulate the hydrophobic drug TPZ firstly,thus TPZ-loaded micelles（TPZ-M）was made,in order to increase the dispersibility of TPZ in water.In the second part,we synthesized a new type of hydrogel,N,O-carboxymethyl chitosan-aldehydated hyaluronic acid（NOCC-AHA）,and simultaneously encapsulated TPZ-M and hydrophilicαPD-1 to make a co-loading novel compositive hydrogel system,namely NOCC-AHA/（TPZ-M+αPD-1）,thus ingeniously used the same carrier to efficiently deliver the hydrophilic drugαPD-1 and the hydrophobic drug TPZ.Theoretically,when the novel compositive hydrogel system was injected into the local tumor,it could increase the local drug concentration of the tumor,while reducing the doses of TPZ andαPD-1.At the same time,it could avoid the direct passage through the blood system,which could reduce adverse events.In the third part of the study,we constructed three subcutaneous triple-negative breast cancer,namely 4T1 xenograft mice models,studied the efficacy and safety of the novel compositive hydrogel system[NOCC-AHA/（TPZ-M+αPD-1）],and when it combined with radiotherapy in the models,and preli-minarily explored the influence of this combination of treatment.Materials and Methods:In the first chapter,we synthesized MPEG-PCL（the micelle material）by ring-opening polymerization,verified whether its structure is correct through nuclear magnetic resonance spectroscopy.And conducted in vitro cytotoxicity experiments to verify its safety through MTT method.Then encapsulated TPZ with MPEG-PCL,made TPZ-M by film hydration method.The prescription was screened to determine the best prescription of TPZ and MPEG-PCL through theoretical drug loading of 5%,7.5%,10%,12.5%,15%,etc.In order to characterize the micelles,we conducted experiments of particle size,zeta potential,morphology,encapsulation efficiency,drug loading,and TPZ release in vitro,etc.,respectively.Under hypoxic and normal oxygen conditions,we detected the cytotoxicity of TPZ-M and free TPZ,respec-tively,through MTT method.In the second chapter,synthesized aldehyde hyaluronic acid（AHA,the theore-tical degree of oxidation was 40%）through oxidating hyaluronic acid（HA）.We determined its actual degree of oxidation through the hydroxylamine hydrochloride method,and verified whether the structure of AHA is correct through nuclear magnetic resonance spectroscopy and fourier transform infrared spectroscopy.The chemical reaction between the amino group on N,O-carboxymethyl chitosan（NOCC）and the aldehyde group on AHA,was used to prepare the black hydrogel（NOCC-AHA）,schiff base was generated at the same time.We determined the final gelation concentration after exploring the component（AHA and NOCC）concentration,through different concentration ratios of 20,25,30 mg/m L.TPZ-M andαPD-1 were encapsulated into NOCC-AHA by one step,to construct the novel compositive hydrogel system,namely NOCC-AHA/（TPZ-M+αPD-1）.The blank material（NOCC-AHA/M）was prepared by encapsulating blank micelles（M）.Characterized the hydrogel from the aspects of gelation,rheology,scanning electron microscopy,and TPZ release in vitro,etc.,respectively.Conducted in vitro cytotoxicity experiments on NOCC-AHA and NOCC-AHA/M by MTT method,in order to verify the safety of the materials been used.In the third chapter,the unilateral,bilateral and rechallenged subcutaneous 4T1xenograft mice models of BALB/c mice were constructed respectively,which were all divided into 2 parts,namely the curative effect experiment and preliminary mechanism exploration experiment.Through injection of NOCC-AHA/（TPZ-M+αPD-1）,and injection of NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy,the tumors on mice were treated locally.In the curative effect experiment of the unilateral subcutaneous 4T1 xenograft mice model,the tumor size and the weight of mice,etc.were monitored.After the experiment,the mice were sacrificed,and the tumor,main internal organs and blood,etc.were collected,in order to explore the efficacy and safety of NOCC-AHA/（TPZ-M+αPD-1）and NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy,and its impact on tumor proliferation and apoptosis,etc.In the curative effect experiment of the bilateral and rechallenged subcutaneous 4T1 xenograft mice models,the tumor size and mice body weight,etc.were monitored.When the monitoring of tumor volume was completed,the mice were raised to observe the survival.Thus to explore the efficacy and safety of NOCC-AHA/（TPZ-M+αPD-1）,and NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy.In the preliminary mechanism experiment,the research design depends on different mice models.The treated tumor,the contralateral of treated tumor,inguinal lymph node on the treated side,spleen,blood and other specimens were collected,respectively.The preliminary possible mechanism of the novel com-positive hydrogel system,and the novel compositive hydrogel system combined with radiotherapy were explored,mainly from T cells in the tumor on the treatment side and the contralateral of treated side（including Treg cells）,DC cells in the inguinal lymph node on the treated side,T cells in the spleen(including effector memory T cells,namely T_EM),and IFN-γin mice serum,etc.Results:In the first chapter,the MPEG-PCL was successfully synthesized by ring-opening polymerization firstly,its molecular weight was 3 946 Dalton（PEG/PCL=2000/1 946）,and its structure was verified to be correct by nuclear magnetic reso-nance spectroscopy.Then,MPEG-PCL was used to encapsulate TPZ,and TPZ-M was successfully formed by the film hydration method.After the prescription screening,the ratio of TPZ:MPEG-PCL polymer（w/w）of 10:90 was the final formulation.Under this ratio,the particle size of TPZ-M was 24.46±0.56 nm,the polydispersity was 0.171±0.043,the zeta potential was-4.34±1.92 m V,the encap-sulation efficiency was 90.82±0.44%,and the drug loading was 9.42±0.05%.TPZ-M was a uniform spherical nanoparticles showed by transmission electron microscopy.TPZ-M could release TPZ slower than free TPZ,about 76.38%of TPZ were released in 96 h,which was showed by in vitro release experiments.The results of MTT experiments showed that,the cell viability of both 4T1、3T3、L-O2 and 293T cells could still be maintained above 92%after incubated with black micelles under the concentration of 1 000μg/m L of the micelle material.Under normoxic conditions,neither TPZ-M nor free TPZ had obvious toxicity to cells.Under hypoxic conditions of 1%oxygen concentration,the toxicity to cells of TPZ-M and free TPZ had increased significantly,and were time-dependent,for both could kill nearly 50%of4T1 cells at 24 h at a concentration of 16μg/m L,and about 70-80%of 4T1 cells could be killed at 48 h,and the cytotoxicity of TPZ-M was higher than free TPZ.In the second chapter,we designed a novel compositive hydrogel system co-loading TPZ-loaded micelles and PD-1 antibody.At first,AHA,the hydrogel component material,was successfully synthesized by oxidizing HA,its actual oxidation degree was 29.69%±2.71%（theoretical oxidation degree was 40%）,and its structure was verified to be correct by nuclear magnetic resonance spectroscopy and fourier transform infrared spectroscopy.Then,NOCC-AHA was successfully synthesized by the chemical reaction between the amino group on NOCC and the aldehyde group on the AHA,which produced schiff base at the same time.After the exploration of the gelation experiment,the final determination of the concentrations of the gelation component were NOCC,25 mg/m L,and AHA,30 mg/m L.The novel compositive hydrogel system,namely NOCC-AHA/（TPZ-M+αPD-1）was prepared by adding TPZ-M andαPD-1 at the same time.And the blank material（NOCC-AHA/M）was prepared by adding blank micelles,as a control group.At the experi-mental concentration,rheological experiments showed that the gelation time of NOCC-AHA/（TPZ-M+αPD-1）was about 71 s,and the strength were reasonable.The synthesized NOCC-AHA/（TPZ-M+αPD-1）,NOCC-AHA and NOCC-AHA/M all had a porous,sieve-like three-dimensional structure showed by scanning electron microscopy.NOCC-AHA/（TPZ-M+αPD-1）could release TPZ slower than TPZ-M and free TPZ,about 65.99%of TPZ were released in 96 h,which was showed by in vitro release experiments.The cell viability of 4T1、3T3、L-O2 and 293T cells could be maintained above 95%after incubation with hydrogel extracts of NOCC-AHA and NOCC-AHA/M for 24 and 48 h,which was showed by MTT.In the third chapter,at first,the unilateral、bilateral and rechallenged subcutane-ous 4T1 xenograft mice models in BALB/c mice were constructed respectively.The novel compositive hydrogel system co-loading TPZ-loaded micelles and PD-1antibody,and the novel compositive hydrogel system combined with radiotherapy was performed locally,and the effect on subcutaneous 4T1 xenograft mice models was studied.Regardless of whether it’s the treated tumor,the contralateral of treated tumor,or the rechallenged tumors,the results of efficiacy showed that,NOCC-AHA/（TPZ-M+αPD-1）was more effective in suppressing tumors than the NS group.While NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy had a better anti-tumor effect than NOCC-AHA/（TPZ-M+αPD-1）or radiotherapy alone.NOCC-AHA/（TPZ-M+αPD-1）or radiotherapy alone could prolong the survival of mice to a certain extent,while in NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy group,the survival of mice was the longest among all groups.For in vivo safety,there was no obvious abnormalities in the blood biochemical parameters of each group of mice.The kidney,spleen,lung,heart,liver and other major organs of the mice in each group were all normal.And in all the modles,no obvious change of body weight of the mice were found.In the preliminary mechanism exploration experiment,there were less prolifer-ation cells in NOCC-AHA/（TPZ-M+αPD-1）than that of the NS group,while in the NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy group,there were the lest proliferation cells,which was showed by Ki67 immunohistochemical staining.There were more apoptosis cells in NOCC-AHA/（TPZ-M+αPD-1）compared with NS group,while the most apoptosis cells were found in NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy group,which was showed by TUNEL experi-ment.The results of flow cytometric showed that,in the treated tumor,NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy could increase the ratio of CD4⁺T or CD8⁺T cells,and increased the ratio of early activated CD4⁺CD69⁺T or CD8⁺CD69⁺T cells.At the same time,the combination reduced the ratio of Treg cells（CD4⁺CD25⁺Foxp3⁺）in the treated tumor.In the contralateral side of treated tumor,NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy could increase the ratio of CD8⁺T cells,while no influence on CD4⁺T cells was found.In the inguinal lymph nodes on the tumor side,NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy could increase the mature ratio of CD80⁺CD11c⁺、CD86⁺CD11c⁺and CD80⁺CD86⁺CD11c⁺DC cells.In the spleen,NOCC-AHA/（TPZ-M+αPD-1）com-bined with radiotherapy could increase the ratio of CD4⁺T or CD8⁺T cells.At the same time,the combination could increase the ratio of effector memory T cells,namely T_EM（CD8⁺CD44⁺CD62L^-）in the spleen.In the serum of mice,the results of ELISA showed that,NOCC-AHA/（TPZ-M+αPD-1）combined with radiotherapy relatively increased the IFN-γcontent.Conclusion:MPEG-PCL showed no cytotoxicity to cells in vitro,TPZ encapsulated by MPEG-PCL could prepare nanoparticles,namely TPZ-loaded micelles（TPZ-M）,TPZ-M increased the dispersibility of TPZ in water,and could form nanoparticles with uniform size.TPZ-M could release TPZ more slowly.In hypoxia conditions,the cytotoxity to 4T1 cells of TPZ-M was stronger when compared with free TPZ.The novel compositive hydrogel system designed in this research could load and deliver hydrophobic TPZ and hydrophilicαPD-1 at the same time.The novel compositive hydrogel system was temperature sensitive,and it was a porous,mesh-like three-dimensional structure,could release TPZ more slowly,and all the materials been used showed no obvious toxicity to cells.The novel compositive hydrogel system had a certain anti-tumor effect in subcutaneous 4T1 xenograft mice models,and could prolong the survival of mice to some extent.While the novel compositive hydrogel system combined with radiotherapy could further improve the therapeutic effect in subcutaneous 4T1 xenograft mice models.Abscopal effect and immune memory effects had also been observed,the survival of mice were improved significantly without increasing toxicity and adverse events.The effects of the novel compositive hydrogel system combined with radiotherapy on the 4T1 tumor-bearing mice may include:the reduced proliferation and the increased apoptosis of tumor cells,the decreased Treg cells,the increased DC cells and T cells(including T_EMcells),and the increased secretion of IFN-γin the serum of mice.