Functional Small Molecule-phosphorothioate Nucleic Acid Conjugates:The Syntheses And Anti-tumor Applications

Conjugation strategy,such as small-molecular drug conjugation and oligonucleotide modification,is essencial in nanomedicine for cancer therapy.Functional drugs are usually conjugated to various carrier materials including polymers,antibodies,peptides,lipids,etc,for the improvement of pharmacokinetic properties,stability,and tumor targeting.However,challenges remain in this field,such as the uncontrollable molecular weight of polymeric materials,potential toxicity,immunogenicity,complexity of manufacture,imprecision of grafting sites and number,and low drug loading,etc.As a naturally existed biomacromolecule and therapeutics,nucleic acids can also serve as an ideal drug carrier material,owing to its precise and controllable molecular structure and sequence,biocompatibility,and biodegradability.Therefore,it is of great significance to investigate the conjugation of functional molecules on nucleic acids.Conventional conjugation strategies are usually based on solid-phase synthesis,which has many issues of complicated synthesis step,low drug loading,and change of the sequence-dependent functions,etc.Herein,inspired by the phosphorothioate modification of nucleic acids,we develop a general method for the syntheses of small molecule-nucleic acid conjugates through grafting functional molecules on phosphorothioate group.This method is simple and efficient without influencing the base pairing and molecule recognization property.Furthemore,owing to the precisely controllable introduction of phosphorothioate group on DNA during its synthesis,the precise conjugation of a series of functional molecules to nucleic acids with defined number and sites can be achieved.The obtained functional small molecule-nucleic acid conjugates are either directily employed for gene delivery or engineered into nanoparticles for drug delivery and antitumor treatment.The detail research contents are shown as follows:1.Maleimide-phosphorothioate nucleic acid conjugates to enhance cellular uptake of antisense oligonucleiotidesAntisense oligonucleiotides（ASOs）have been widely used due to their simple syntheses and efficient effect.However,their poor tumor targeting and cell penetration seriously restrict the druggability and clinical application.Traditional strategies to enhance cell penetration of ASOs by terminal modification with targeting molecule,such as cell penetrating peptides,and lipids,are complicated and unsafe.Therefore,we developed a new method to improve cell internalization and gene silencing effect of ASOs by grafting multitudes of maleimide onto the backbone of phosphorothioate ASO.The as-prepared maleimide-grafted ASO（ⁿMal-g-ASO）maintained the molecular recognization property and sequence-dependent functions of ASO.Mal-g-ASO also showed good biocompatibility.Through click conjugation with cell membrane thiols that triggers endocytosis-independent cellular internalization,Mal-g-ASO exhibited enhanced cellular uptake efficiency,resulting in a remarkable improvement of ASO-based gene silencing.2.Construction of photosensitizer-phosphorothioate nucleic acid conjugates based nanogels for combinational photodynamic-immunotherapyNucleic acids can not only use as therapeutics,but also be an ideal drug carrier.We further employ phosphorothioate nucleic acids as drug carrier for photosensitizer grafting to achieve combinational photodynamic-immunotherapy.Photodynamic therapy（PDT）and immune checkpoint blockade（ICB）combination is a crucial method in conquering tumor immunosuppressive environment for antitumor therapy.Nonetheless,challenges remain in co-delivery of photosensitizer and ICB due to the great difference of their physicochemical properties and functional locus.Hence,it is important to construct nanomedicines for PDT and ICB co-delivery.Herein,phosphorothioate desoxyribonucleic acid（DNA）is chosen as carrier for drug co-loading by grafting with photosensitizer and hybridizing with anti-PD-L1（programmed death-1 ligand）si RNA.Four DNA strands with dispersal phosphorothioate modification are grafted with photosensitizer（PPA）respectively to prepare water-soluble photosensitizer-nucleic acid conjugates（PPA-DNA）.The synthesized PPA-DNA conjugates can assemble into DNA tetrahedron with four tails at each vertex in 1×TAE/Mg²⁺buffer to generate PPA-grafted DNA tetrahedron（tailed-PPA-TET）.Furthermore,anti-PD-L1 si RNA with complementary tails is used as cross-linking agent to prepare DNA nanogel.In this co-delivery system,photodynamic effect of PPA will induce the immunogenicity cell death（ICD）of tumor cells and enhance immunogenicity.In the meantime,si RNA can downregulate the PD-L1 expression on tumor cells,which will enhance the tumor killing by cytotoxic T cells.Both agents work together to achieve better immune enhancement and antitumor therapy,which provides a new idea for co-delivery of various drugs.3.Construction of paclitaxel-phosphorothioate nucleic acid conjugates based multifunctional and tumor targeting spherical nucleic acidsThe above studies indicate that small molecular drugs can be grafted onto phosphorothioate nucleic acid backbone to synthesize drug-nucleic acid conjugates.Drug conjugation strategy is also an important method in construction of chemo-drug delivery system.However,challenges remain in chemotherapeutic drug delivery system with nucleic acid,including poor stability and low drug loading.In addition,the passive targeting property of nanomedicines to tumor tissues is still in dispute.Therefore,the exploration of new and facile strategies to construct targeting and multifunctional DDS is an unmet need for targeted tumor therapy.In order to stress the universality of phosphorothioate nucleic acid as drug carriers and to improve drug loading and tumor targeting,herein,diblock DNA strands(^PODNA-b-^PSDNA)containing both normal phosphodiester segment(^PODNA)and sequential phosphorothiolate segment(^PSDNA)are directly grafted with a multitude of paclitaxel（PTX）to abtain amphiphilic chemodrug-nucleic acid conjugates(^PODNA-blocked-(^PSDNA-grafted-PTX)).Then,the resulting amphiphilic conjugates assemble into PTX-loaded spherical nucleic acid（SNA）-like micellar nanoparticles（PTX-SNAs）with ^PODNA as micellular shell and hydrophobic PTX as core.The ^PODNA segment maintains its molecular recognition property and biological functions,which allows the as-prepared PTX-SNAs to be further functionalized with tumor-targeting aptamers,fluorescent probe strands,or antisense sequences.The results demonstrated that all PS groups could be consumed,resulting in higher drug loading and controllable of drug grafting.These multifunctional PTX-SNAs demonstrate active tumor-targeting delivery,efficient inhibition of tumor growth,and the reversal of drug resistance both in vitro and in vivo for comprehensive antitumor therapy.