Host-Guest Nanomaterials Based On Cyclodextrins For Imaging And Combined Immunotherapy

As a branch of supramolecular chemistry,host-guest chemistry is based on the specific recognition between host molecule and guest molecule by non-covalent bonding such as hydrophobic interaction,hydrogen bond orπ-πstacking to self-assemble the supramolecules.With the rise of supramolecular chemistry in recent years,the concepts and principles of supramolecular have been introduced into the self-assembly of polymers and nanomaterials.Due to the characteristics of dynamic reversibility,diversified modification and multi-function,the host-guest nanomaterials have been widely used in biological imaging and sensing,gene therapy and drug delivery.Traditional methods of cancer therapy include surgery,chemotherapy,photodynamic therapy and photothermal therapy.In recent years,immunotherapy,which depends on the whole immune system,has attracted the attention of researchers.Immunotherapy is main to enhance the immunogenicity of tumor cells or prevent the tumor immunosuppression by using immunocheckpoint blockers,vaccines or small molecule inhibitors.However,nanodelivery system can improve the solubility,stability and targeting of immune agents,enhance the antitumor immune response,and reduce the side effects on normal tissues.Based on the research background,we use a suitable small molecular inhibitor as the guest to construct the host-guest nanomaterial,which combining PDT and immunotherapy to increase the tumor cell immunogenicity and overcome the tumor immunosuppression.The main work of this dissertation is to synthesize cyclodextrin derivatives with CD44 targeting and the conjugate of small molecule inhibitor（NLG919）with photosensitizer（PPa）,and use the host-guest interaction betweenβ-cyclodextrin（β-CD）and NLG919 by supramolecular self-assembly for fluorescence imaging and combination immunotherapy.The contents of this dissertation are as follows:Chapter 1.IntroductionIn this chapter,we firstly introduced the research status and prospect of cyclodextrin,calixarene and cucurbituril in host-guest chemistry.Then we described the characteristics of tumor microenvironment such as the enzyme,reducing environment and immunosuppressive microenvironment.Next,the application and prospect of nanomaterials in immunotherapy were introduced.And finally,we expressed the aim and meaning of the work.Chapter 2.Synthesis and Characterization of HCNSPIn this chapter,we prepared supramolecular nanoparticles HCNSP with CD44 targeting and GSH responsibility by ultrasonic method.We synthesized HA-CD with NH₂-β-CD and hyaluronic acid（HA）by amidation reaction,and synthesized NLG919-S-S-PPa by coupling PPa and NLG919 with disulfide bond.Then,we constructed HCNSP by self-assembly of host-guest interaction in aqueous solution.NLG919 was used as host molecule for supramolecular assembly for the first time.According to molecular docking and simulation,NLG919 was easier to enter the cavity ofβ-CD than PPa.The inclusion ratio of NLG919 toβ-CD was 1:1 and the binding constant was 4.2 x 10² M^-1,which indicated that the system is stable.The DLS and TEM showed that the morphology of HCNSP nanoparticles were uniform,the shape was round and the particle size was about 50 nm.The stability of HCNSP was high in PBS or 10%FBS solution.In the presence of surfactant（sodium dodecyl sulfate,SDS）,HCNSP had high sensitity with GSH,which fluorescence mostly recovered in the presence of GSH.Compared with the control group,HCNSP also had strong photodynamic activity.In conclusion,we had successfully prepared stable supermolecular nanoparticle HCNSP,which provide strong support for subsequent cell and vivo experiments.Chapter 3.Antitumor Mechanism of HCNSP in VitroThis chapter mainly studied the cellular uptake,photodynamic activity,cytotoxicity and antitumor mechanism of HCNSP at the cellular level.HA could specifically bind to CD44receptor,which highly expressed on the surface of CT26 tumor cells,and the CD44 receptor promoted the cellular uptake of HCNSP.The uptake capacity of HCNSP in CT26 cells was1.7 times higher than that HA pretreated.Under 671 nm laser irradiation,the ROS produced by PPa could not only cause the apoptosis or necrosis of cell,but also induce immunogenic cell death（ICD）of tumor.Through the detection of ICD markers such as calreticulin（CRT）and high mobility group box 1（HMGB1）,it was confirmed that ROS could induce ICD in tumor cells and transport CRT to cell membrane surface and release nucleus HMGB1.Therefore,HCNSP could enhance the immunogenicity of tumor cells and the maturation degree of dendritic cell（DC）.Then,we realized that the secretion of IFN-γcan increase the expression of indoleamine 2,3-Dioxygenase（IDO-1）.In order to alleviate the immunosuppressive tumor microenvironment caused by IDO-1,we found that NLG919reduced by GSH significantly inhibited the activity of IDO-1.Chapter 4.Fluorescence Imaging and Combined Immunotherapy of HCNSP in VivoIn this chapter,we mainly used HCNSP to study the fluorescence imaging,antitumor therapy and immune response analysis in vivo.Compared with control group,HCNSP showed better tumor targeting,higher fluorescence intensity and more aggregation in tumor site even after 48 hours.In the antitumor treatment of subcutaneous tumor and abscopal tumor,HCNSP with laser both had significant antitumor effect,slower tumor growth rate,long survival time and well biological safety.During the analysis of tumor immune microenvironment,it was also proved that HCNSP have good ability of inducing DC maturation,activating T cells for cellular immune response and reducing negative feedback immunosuppressive cells.