Pvt1-targeting Biomimetic Nanosystems Delivered By A Bio-scaffold For Postoperative Colorectal Cancer Therapy

Background: Colorectal cancer(CRC)recurrence and metastasis after surgical resection result in poor clinical outcomes,thus it is urgent to develop a safe and effective postoperative treatment strategy.Due to low side-effects,targeted clearance of tumor cells and formation of immune memory,cancer immunotherapy is highly anticipated to combat tumor recurrence and metastasis,but often suffers from low response rates.Long noncoding RNA plasmacytoma variant translocation 1(Pvt1)mediates CRC progression and immunosuppression of tumor microenvironment,expecting to be a potential target for immunotherapy.Nanoparticles encapsulating therapeutics can both improve biostability and intracellular uptake of the loaded drugs.Further,biomimetic nanoparticles containing arrays of autologous tumor-associated antigens(TAAs)can not only target the local tumor,but also contribute to antitumor immune reponse activation.Based on these,we develop a bio-scaffold encapsulating lnc RNA-targeting biomimetic nanosystems against CRC postoperative recurrence.Methods: Here,we fabricated a bio-scaffold encapsulating lnc RNA-targeting nanosystems and chemo-therapeutics.A plasmid encoding short hair-pinned RNA against Pvt1(termed as “shPvt1”),CRC cells’ membrane(termed as “CM”),(2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride(DOTAP,termed as “D”)and cholesterol were assembled and extruded via a polymeric membrane,forming a shPvt1-CM-D nanosystem.This nanosystem and Oxaliplatin(termed as “Oxa”)were both encapsulated into a hyaluronic acid and alginate-based hydrogel,thereby fabricating a shPvt1-CM-D/Oxa-loaded bio-scaffold(shPvt1-CM-D/Oxa@gel).Then,the morphology of the bio-scaffold was characterized by transmission electron microscopy and scanning electron microscopy,the drug release was detected by atomic absorption spectrometer and fluorescence tracing technique in vivo.Afterwards,the triple immune effects mediated by the bio-scaffold were verified.The release of damage associated molecular patterns(DAMPs)from CRC cells was detected by ELISA analysis,the expression of CD80/CD86 on the surface of bone marrow-derived dendritic cells(BMDCs)was tested by flow cytometry analysis,and ameliorating of immunosuppression in granulocytic myeloid-derived suppressor cells(G-MDSCs)was validated by co-culture experiment.Finally,postoperative in situ recurrence model,ectopic secondary invasion model,metachronous liver metastasis model and synchronous distal metastasis model of CRC were established in mice,and the bio-scaffold was implanted in the surgical bed to evaluate therapeutic efficacy against CRC recurrence and metastasis.Results: A shPvt1-carrying nanoparticle shPvt1-CM-D(diameter of about 270 nm)was synthesized,which was wrapped in OA/HA hydrogel scaffolds.The bio-scaffold had a good biodegradability,and sustainedly released Oxa and shPvt1-CM-D for 5 and 10 days respectively in vivo to induce triple immune responses.First,shPvt1-CM-D enhanced immunogenic cell death(ICD)induced by Oxa and further promoted the release of calreticulin,ATP and HMGB1 in tumor.Second,the enhanced ICD with TAAs in CM jointly vaccined DC and promoted the up-regulation of CD80/CD86 and secretion of TNF-α,IL-6.Third,shPvt1-CM-D inhibited the secretion of ROS and Arg in G-MDSCs and alleviated T cell retardation.In animal experiments,compared with modest inhibition on local tumor recurrence in Oxa@gel,CM-D@gel,and shPvt1-CM-D@gel(inhibition rate(IR): 33.3%,16.6% and 33.3%,respectively),shPvt1-CM-D/Oxa@gel elicited the strongest suppression,and 5 of 8 mice showing no detectable tumors(IR: 62.5%).Additionally,shPvt1-CM-D/Oxa@gel generated potent immune memory responses to significantly suppress metachronous metastasis,especially for liver metastasis.Futhermore,the bio-scaffold inhibited synchronously distant tumors by activating systemic immune responses.Conclusions: We developed a bio-scaffold loading lnc RNA Pvt1-targeting biomimetic nanosystems and chemotherapeutic agents Oxa.The bio-scaffold notably inhibited postoperative CRC recurrence and distant metastasis,opening a new avenue of utilizing lnc RNA targets for postoperative CRC immunotherapy.