Microfluidic Based Preparation Of Multifunctional SiRNA Lipid Nanoparticles For Cancer Therapy

Small interfering RNA?siRNA?based therapeutics have been demonstrated as an effective strategy for cancer therapy due to its sequence-specific gene silencing effect.However,its poor stability,short blood circulation,low transfection efficiency,as well as the inefficient endosome escape are still significant challenges for the use of siRNA in the clinical studies.Thus,the development of novel delivery systems has been emerged as promising tools for solving the issues above,thereby enhancing the silencing effect of siRNA-based therapies.In this study,a platform developed through the combination of engineering approach and material science was proposed for the enhancement of siRNA delivery.Furthermore,Cell division cycle protein 20?CDC20?was selected as target gene for breast cancer therapy through the use of bioinformatic analysis,which would improve the efficiency of cancer therapy,and reduce the side effect due to the non-specific binding to healthy cells.Microfluidic?MF?refers to precise manipulation of fluids within nano or micro-scale channels under laminar flow conditions.The well-designed microfluidic channel allows for the production of nanoparticles with a narrower size distribution in a more reproducible manner compared to the conventional methods,providing new opportunities for the evaluation of drug delivery systems.'Smart' stimuli-responsive materials are sensitive to biological stimuli,including pH,redox,enzyme,glucose,hypoxia,and ATP.The tunable physical or chemical responses of these materials to stimuli,such as swelling,shrinking,dissociation or degradation,can be utilized to achieve precise control of the cargo release to specific cells or tissues,thus improving the therapeutic efficiency while minimizing the side effects of drugs.Herein,hypoxia and pH-responsive multifunctional siRNA nanoparticles were prepared by the microfluidic device to investigate its potential anti-tumor efficiency in this study.The details are given as follows:1.Single-step synthesis of targeted and pH responsive lipid nanoparticles for siRNA delivery by using microfluidic devicesA single-step process was employed to synthesize the transferrin-conjugated lipid nanoparticles?Tf-LNPs?based on pH responsive lipid by using MF method,as opposed to the multi-steps bulk mixing?BM?method.The results indicated that this single-step MF process enabled a rapid and efficient synthesis of Tf-LNPs,named as Tf-LNPs-MF.Tf-LNPs-MF displayed smaller size profile and more uniform structures when compared to the LNPs produced by multi-steps BM method?Tf-LNPs-BM?.Furthermore,Tf-LNPs-MF efficiently delivered siRNA in the in vitro cellular uptake studies as well as in vivo tumor inhibition studies.In short,this single-step preparation assisted by microfluidic significantly simplified the Tf-LNPs production process while improving the efficiency of drug delivery,which could potentially serve as a valuable tool for the development of novel cancer therapeutic strategies.2.Synthesis of hypoxia-responsive polypeptide and bioinformatic analysis of therapeutic targetHypoxia-responsive 6-?2-nitroimidazole?hexamine?NIHA?was synthesized via the coupling reaction of 2-nitroimidazole?NI?and 6-?t-Boc-amino?hexyl bromide?BAHB?.After that,ring-opening polymerization of?-benzyl-L-glutamate N-carboxyanhydride?BLG NCA?was initiated by using mPEG-NH₂ as the macromolecular initiator,while the diblock copolymer mPEG-b-PLG was synthesized by the removal of benzyloxy protection.Finally,EDC·HCl and NHS were used as condensation agent and catalyst,respectively.mPEG-b-P?LG-g-NI?was synthesized by the combination of the synthesized NIHA with the side carboxyl group in mPEG-b-PLG.NMR spectra showed that all the signal peaks were assigned accurately.The component ratios of the signal peaks were consistent with the hydrogen ratios in the structural formula,which proved the feasibility of NIHA,mPEG-b-PLG,and mPEG-b-P?LG-g-NI?synthesis.In addition,the result of bioinformatic analysis demonstrated that the overexpression of CDC20 in tumor was positively correlated with hypoxia and contributed to poor prognosis in breast cancer therapy.3.Synthesis of hypoxia-responsive siRNA nanoparticle for cancer therapyUnder the hypoxic tumor environment,the hydrophobic NI group was converted to hydrophilic 2-aminoimidazole?AI?via a single-electron reduction catalyzed by a series of nitro-reductases,leading to the disassembly of nanoparticles,and subsequent rapid released cargo in tumor cells,which were used widely in molecular probe and pro-drug.A self-assembled hypoxia-responsive nanoparticle?HRNP?platform was developed by NI-modified polypeptide and cationic lipid-like compound for the delivery of siRNA to specifically target CDC20 positive human breast cancer cells?MCF-7?in vitro and in vivo.The in vitro results have shown that⁹0%of the luciferase expression was knocked down after being treated with HRNP/siLuc at a siRNA dose of 10.0 nM with no apparent cytotoxicity.Moreover,the HRNP/siCDC20 sufficiently accumulated and silenced the expression of CDC20 in tumor tissues and therefore suppressed the growth of xenograft breast cancer tumors at an inhibition rate of⁸5%,indicating the enormous potential of this hypoxia-targeting RNAi nanoparticle for breast cancer therapy.4.Synthesis of hypoxia and pH dual-responsive lipid nanoparticle by using microfluidic devices for siRNA deliveryBased on results listed above,we successfully synthesized the hypoxia and pH dual-responsive lipid nanoparticle?hpLNP?by using microfluidic devices in one step.The size of hpLNP/siRNA was uniform and its surface charge increased as a function of the PEG detachment and phospholipids protonization at pH 6.5,which enhanced the high cellular uptake of hpLNP/siRNA,as well as the rapid cargo release triggered by hypoxic conditions,facilitating effective gene silencing and anti-tumor effects.In summary,the hypoxia and pH dual-responsive lipid nanoparticle with a narrower size distribution was achieved by using novel microfluidic devices,enhancing a significant gene silencing effect.The strategy of combining of engineering approach and material science has the advantage of high controllability and repeatability,which could serve as a solid foundation for future siRNA-based therapies.