Application Of Albumin Nanoparticles Modified With CTLA-4 Nucleic Acid Aptamers And Loaded With Antihistamines In Tumor Immunotherap

Purpose:Immune checkpoint blockade(ICB)is a promising strategy for cancer treatment and has generated remarkable clinical results against multiple malignancies.Exploration of new technical approaches to further boost the therapeutic efficacy of ICB is therefore of potential medical importance.In addition to antibodies,other newly developed ligands,including aptamers,may also serve as alternative ICB agents.A previously developed CTLA-4-antagonizing DNA aptamer can significantly inhibit tumor growth in animal models.It has been recognized that tumor immune microenvironment(TIME)is play an important role in anticancer immunity.Hence,it is reasonable to combine ICB therapeutics with TIME-modulating agents.Fexofenadine,a histamine receptor H1(H1R)blocker,appears to modulate TIME and facilitate antitumor immunity by inhibiting the polarization of macrophages to M2 phenotype.Based on the above information,in this study,a novel nanotherapeutics(Apt-NP-FEXO)was constructed by encapsulating FEXO into albumin nanoparticles that were functionalized with CTLA-4 aptamers.So far as we know,this is the first report on using nanotechnology with FEXO to enhance the therapeutic function of CTLA-4 blockade.Methods:Thiol-modified CTLA-4 aptamer was linked to the amino groups of BSA via sulfo-SMCC to form Apt-BSA.To evaluate whether the CTLA-4 aptamer was conjugated with BSA to form Apt-BSA,agarose gel electrophoresis,SDS-PAGE,and DLS were performed.NP,Apt-NP,NP-FEXO,and Apt-NP-FEXO were fabricated with a modified self-assembling method reported previously.The average sizes and zeta potentials of nanoparticles were determined by DLS.The morphology of nanoparticles was observed by TEM.To estimate the drug loading efficiency,encapsulation efficiency,and FEXO release pattern,HPLC was conducted.To evaluate whether the aptamer-modified NP could still recognize its target,the bindings of Apt-NP or NP to CTLA-4 positive cells were evaluated by flow cytometry,fluorescent microscopy and confocal microscopy.CFSE-MLR was applied to evaluate the proliferation of lymphocytes treated by CTLA-4 aptamer and aptamer-modified NPs.MTS assay was used to study whether the CTLA-4 aptamer or aptamer-modified NPs could boost lymphocyte-mediated cytotoxicity.Animal studies were performed to assess in vivo antitumor efficacy of the nanoparticles.Results:CTLA-4 aptamer was conjugated with BSA,forming Apt-BSA.The average sizes of the nanoparticles were as follows:NP(117.8 nm),Apt-NP(149.0 nm),NP-FEXO(139.3 nm),and Apt-NP-FEXO(159.0 nm).The apparent zeta potentials of the nanoparticles were as follows:NP(-12 mV),Apt-NP(-18.5 mV),NP-FEXO(-0.371 mV),and Apt-NP-FEXO(-5.36 mV).The FEXO encapsulation efficiencies were 31.06%for NP-FEXO and 32.75%for Apt-NP-FEXO.Both FEXO-encapsulating NPs exhibited typical sustained drug release profiles.CTLA-4 aptamer modified NP could bind with CTLA-4 expressing cells,and stimulate the proliferation of lymphocytes to some extent.Similar to free CTLA-4 aptamer,both Apt-NP and Apt-NP-FEXO remarkably enhanced PBMC-mediated cytotoxicity against the tumor cells.Animal studies revealed that although free CTLA-4 aptamers suppressed tumor growth to some extent,Apt-NP generated a more prominent antitumor effect.Notably,Apt-NP-FEXO further enhanced tumor suppression vs.Apt-NP,and did not generate extra systemic toxicity in vivo.Conclusion:In this study,we combined ICB therapeutics with TIME-modulating agents and designed a novel nanotherapeutics(Apt-NP-FEXO)for co-delivery of FEXO and CTLA-4 aptamers.Apt-NP-FEXO significantly improved the antitumor efficacy in vivo,and may have application potential in cancer immunotherapy.