Design Of Nanoplatforms Based On Core-shell Tecto Dendrimers For Cancer Theranostics

In recent years,the development of nanomedicine has promoted the progress of cancer theranostics.Dendrimer-based nanoplatforms are widely used in the theranostics of cancer via loading functional contrast agents or drugs due to their unique physical and chemical properties.However,single-generation dendrimers appear certain limitations with the deepening of research.For example,low-generation dendrimers have limited drug loading capacity and low gene delivery efficiency although low-generation dendrimers are simple to be easily synthesized and have low cytotoxicity.High-generation dendrimers with high gene delivery efficiency and increased drug loading capacity are complex and expensive due to tedious synthesis and highly cytotoxic.Recently,the development of supramolecular chemistry technology has brought a turn for the medical applications of dendrimer nanoplatforms.For instance,core-shell dendrimers(CSTDs) with higher complexity and controllable size were fabricated through simple methods by using different generation dendrimers as reaction blocks,which are expected to have similar properties of high-generation of dendrimers,enabling efficient load of one or more contrast or/and therapeutic agents as well as more flexible mulifunctionality.Therefore,CSTD-based nanocarriers allow a more accurate diagnosis and more efficient treatment of tumors than single-generation dendrimer based counterparts.In this thesis,G5-CD/Ad-G3 CSTDs with the generation 3(G3) poly (amindoamine)(PAMAM) dendrimers as the shell and generation 5(G5)PAMAM dendrimer as the core were synthesized via supramolecular self-assembly between adamantane(Ad) and ?-cyclodextrin(?-CD).And a series of functional nanoplatforms based on G5-CD/Ad-G3 CSTDs were constructed for the diagnosis and treatment of different tumors(especially triple-negative breast cancer and glioma with high recurrence,high mortality and low cure rate).The specific researches are as follows:(1)The CSTDs loaded with genotype inhibitors and anticancer drugs were designed for the combined gene therapy and chemotherapy of triple-negative breast cancer cells.Triple-negative breast cancer cells are aggressive and have a high risk of recurrence due to the lack of receptors necessary for effective treatment.On the one hand,CSTDs can achieve enhanced gene delivery and high-efficiency drug loading.On the other hand,the simultaneous integration of gene and drugs through CSTDs is expected to achieve synergistic and complementary combined therapy.Combination of these two aspects makes it possible to effectively treat triple-negative breast cancer.In chapter 2 of this thesis,the CSTDs nanoplatform was designed to co-deliver genotype inhibitors(micro RNA 21 inhibitors,miR 21i) and the anticancer drugs(doxorubicin(DOX)) for enhanced triple-negative breast cancer(MDA-MB-231 cells) treatment applications.First,CSTDs were prepared by supramolecular recognition of?-CD modified G5(G5-CD) and Ad conjugated G3(G3-Ad).Among them,each G5 dendrimer is surrounded by 4.2 G3 dendrimers.Subsequently,the synthesized CSTDs were used as gene carriers to deliver miR 21i.The results show that CSTDs can effectively transfect miR 21i into cancer cells in appropriate N/P ratios.Moreover,the transfection of miR 21i can inhibit the migration of cancer cells,regulate the expression of miR 21gene and its related target genes or proteins(including PTEN,PDCD4,p53 and Caspase-3).In addition,the CSTDs can further co-deliver miR 21i and DOX for improving in vitro therapeutic efficacy of triple-negative breast cancer cells.Based on these,the developed CSTDs are expected to be used as a universal platform to effectively load different therapeutic adjuvants and enhance the therapeutic efficacy of cancer through the combined therapy.(2)CSTDs chelated with gadolinium ions(Gd(?)) were designed as a nanoplatform for magnetic resonance (MR) imaging of breast cancer based on the amplified EPR effect.It is essential that nanoplatforms with enhanced passive tumor targeting were developed for precise cancer nanomedicine based on the enhanced permeability and retention (EPR)effects.Literature data have shown that gadolinium(Gd) chelated on dendrimers with higher molecular weight displays higher longitudinal relaxation rate(r₁)and MR imaging sensitivity.Thus CSTDs chelated with Gd(?) were designed as a nanoplatform to explore the permeability in the tumor microenvironment and the in vivo and in vitro tumor MR imaging ability of CSTDs in Chapter 3.First,by optimizing the reaction temperature(30?)on the basis of the previous work,each G5dendrimer was finally surrounded with 11.4 G3 dendrimers.Then,the optimized G5-CD/Ad-G3CSTDs are conjugated with tetraazacyclododecanetetraacetic acid(DOTA)-Gd(?) chelating agent and further acetylated to neutralize the remaining amino groups on the periphery of the CSTDs.The results suggest that the formed CSTD.NHAc-DOTA(Gd) complexes possess relatively uniform size distribution,colloidal stability,and good cell compatibility and biocompatibility in the studied concentration range.Compared with the G5.NHAc-DOTA/(Gd) complexes,the CSTD.NHAc-DOTA(Gd) complexes has a higher molecular weight and volume,leading to a longer rotation correlation time.The longitudinal relaxation(r₁) of CSTD.NHAc-DOTA(Gd) complexes is 7.34mM^-1s^-1,which is 1.5 times higher than that of G5.NHAc-DOTA/(Gd)(4.92mM^-1s^-1).More importantly,the CSTD.NHAc-DOTA/(Gd)complexes also displayed better permeability through fluorescence imaging in the in vitro 3D cell spheroid experiment and enhanced tumor MR imaging in vivo.Based on these,the developed CSTD.NHAc-DOTA(Gd)complexes can be used for enhanced tumor MR imaging on account of amplified EPR effect and improved r₁ relaxivity.(3)The multifunctional and acetylated CSTDs complexed with copper were used as a nanoplatform for targeted glioma MR imaging and chemodynamic therapy via crossing blood-brain barrier.The development of multifunctional nanoplatform to overcome the blood-brain barrier(BBB) and improve the diagnostic sensitivity and the treatment effect of glioma is still a serious challenge.Recently,researchers have paid more attention to chemodynamic therapy(CDT),which refers to the way of killing tumor cells through the Fenton-like reaction of transition valence metal with hydrogen peroxide(H₂O₂) to generate hydroxyl radicals(·OH).The CSTDs loaded with contrast and therapeutic agents and further multi-functionalized to cross the BBB and target gliomas can be applied for theranostics of glioma.Here,a multifunctional acetylated CSTD(M-CSTD.NHAc)nanoplatform were developed and further complexed with copper ions(Cu(?))for MR imaging and CDT of orthotopic glioma in chapter 4.First,G3.NH₂-Ad was covalently modified by pyridine(Pyr),dermorphin(DER)and arginine-glycine-aspartic acid(RGD)peptide respectively to obtain Ad-G3.NH₂-Pyr,Ad-G3.NH₂-PEG-DER,and Ad-G3.NH₂-PEG-RGD.Then,M-CSTDs were formed through the supramolecular assembly process between G5.NH₂-CD and functionalized G3.NH₂-Ad.Subsequently,the prepared M-CSTDs were further acetylated.Finally,the M-CSTD.NHAc was complexed with Cu(?)to obtain the product M-CSTD.NHAc/Cu(?)complexes.In this study,cell experiments showed that the CSTD.NHAc/Cu(?)complexes implemented CDT in glioma cells through the following mechanisms:(1)the formed M-CSTD.NHAc/Cu(?)complexes can across the BBB via binding?-opioid receptor due to the existence of DER;(2)GBM cells with overexpressed?_v?₃ integrin are targeted by RGD peptide modified on the surface of M-CSTD.NHAc;(3)after cellular uptake,glutathione is depleted by oxidation reaction with Cu(?),and then a Fenton-like reaction occurs to convert hydrogen peroxide in cells into toxic hydroxyl radicals,leading to lipid peroxidation,etc.Eventually,the GBM cells are killed under CDT.Furthermore,the T₁-weighted MR imaging of tumors was also explored owing to relaxation performance of M-CSTD.NHAc/Cu(?)complexes (0.7331 m M^-1s^-1).In brief,the efficacy of both MR imaging and CDT using brain glioma were evaluated in vivo.This study highlights the formation and mechanism of M-CSTD.NHAc/Cu(?)complexes used for MR imaging and CDT of glioma,which provides a theoretical basis for expanding the application of CSTD-based nanoplatforms in cancer theranostics.In summary,a series of functional nanoplatforms based on CSTDs are constructed and explored for combined therapy,accurate diagnosis and theranostics of cancers.The findings from the thesis related to CSTD-based carriers provide some theoretical basis and new ideas to develop nanoplatforms for precise diagnosis and efficient treatment of malignant tumors with extremely low cure rates and high recurrence rates.