Electrotransfected Microneedle Patch Delivers CRISPR/Cas9 Targeted PLK1 For OSCC Treatment

Objective:This study intends to adopt a combination of CRISPR/Cas9 gene therapy and the microneedle（MN）patch:The backing of the MN was prepared with conductive materials.The needle of the MN was prepared with soluble polymers and plasmids.Then the in vivo gene electrotransfection experiment was carried out on the subcutaneous tumor animal model to achieve the targeted knockout of the PLK1 gene.The tumor-suppressive effect was evaluated by macro-observation,histochemistry,immunohistochemistry,molecular biology,and other analysis methods.This study provides some ideas for the clinical treatment of subcutaneous tumors.Methods:（1）Construction and extraction of CRISPR/Cas9 expression plasmids:Three kind of PLK1 knockout CRISPR/Cas9 plasmids were constructed,and the plasmids were extracted and amplified from Stbl3 receptor cells containing plasmids by alkaline lysis.（2）Preparation and characterization of the bilayered MN:First,dopamine-modified polypyrrole（DA-PPy）with good conductivity and good dispersibility in water was synthesized by chemical oxidation method and characterized by scanning electron microscope（SEM）and FT-IR.And then,the bilayered MN patch loaded with CRISPR/Cas9 plasmids and backing with good conductivity was prepared by a vacuum casting method.The surface morphology of the MN was characterized by microscope and SEM.The mechanical properties of the MN were tested by a universal testing machine,and the tip dissolution rate was measured by inserting the bilayered MN into the back skin isolated from pig.At the same time,the DNA content of MN tip,the conductivity of the MN base,and cytotoxicity of the MN were measured.（3）Construction of the oral squamous cell carcinoma（OSCC）animal model and gene transfection efficacy evaluation of the bilayered conductive MN:OSCC animal models were constructed by tumor transplantation and were divided into 5 groups,including Control group;Dox group;Syringe injection of plasmids and electric stimulation group（i.j.+E）;Microneedle injection of plasmids group（p-MN）;Microneedle injection of plasmids and electrical stimulation group（p-MN+E）.They were treated separately when the subcutaneous tumor diameter reached to 3-5mm or the tumor volume reached to 120 mm~3.The Control group was not treated.The Dox group was given injection of medication once a week,and the other groups were treated once every 2 days.The nude mice were sacrificed after 48 h of all treatment or when the tumor length reached to 2 cm.The main organs of nude mice were taken for in vivo biocompatibility test.Part of the tumor tissue was taken for H＆E staining.The changes of PLK1 and Ki67 relative contents were observed by immunohistochemistry.The knockout of PLK1 was detected by q PCR,and the animal weight and tumor volume were recorded every 3 days for a complete efficacy evaluation.Results:（1）Construction and extraction of CRISPR/Cas9 expression plasmids:PLK1knockout CRISPR/Cas9 plasmid vector was successfully amplified,and its purity and concentration were tested.High purity plasmids with a concentration greater than100 ng/μL were selected for subsequent experiments.（2）Preparation and characterization of the bilayered MN:First,DA-PPy was characterized by FT-IR.The results showed that the infrared characteristic spectrum of DA-PPy contained characteristic peaks of pyrrole（Py）and dopamine（DA）,which confirmed the successful synthesis of DA-PPy.SEM showed that DA-PPy appears as a fibrous rod,which was different from particle agglomerating polypyrrole（PPy）.The prepared MN patch were 121（11×11）cone microneedles array uniformly arranged on the flat base plate.SEM showed that the length of the MNs was about503.33±13.33μm,and the distance between each row of MN tip was about458.34±1.67μm.A multimeter was used to conduct conductivity tests and the current was roughly 0.7μA,which could be electrotransfected.The plasmids content of each MN patch was 24.656±9.9μg.The mechanical strength of the MN patch was strong enough to penetrate the corneum and release the drug into the skin.The tip of the MN could be dissolved within 5 min after insertion,and the final dissolved height accounted for 72.82%of the total height of the MN.The prepared MN patch had good biocompatibility as well.（3）OSCC animal model construction and efficacy evaluation of bilayered conductive MN gene transfection:Tumor volume can directly evaluate the efficacy of tumor therapy.In the Control group,tumor volume increased by 17.87 times compared to the initial volume,while in the p-MN+E group,the tumor volume decreased to 44.13%of the initial volume.This was confirmed by H＆E staining,TUNEL staining,Ki67 and PLK1 immunohistochemistry and q PCR results.Conclusions:The advantages of combining CRISPR/Cas9 with bilayered conductive MN patch prepared by vacuum casting method lied in that the MNs can painlessly deliver CRISPR/Cas9 plasmids to the subcutaneous tumor site,and the conductive substrate of the MN patch was conducive to the electrotransfection of tumor cells.It can increase the knockout rate of PLK1 gene to inhibit the proliferation of tumor cells.This study showed a broad application prospect in the treatment of subcutaneous tumors.