| ObjectiveThe 5-year survival rate of pancreatic ductal adenocarcinoma is less than 5% due to the lack of effective treatment. Even if treated with the current first line chemotherapeutic drug, Gemcitabine(Gem), the median overall survival of patients only ranged from 5.0 to 7.2 months. Therefore, new therapy approaches are urgently needed for the vast majority of pancreatic cancer patients.Because of inefficient tumor vascular supply, there are large hypoxic areas in pancreatic cancer tissues. Tumor cells can survive under hypoxic conditions by activating many signaling pathways, in which HIF1(hypoxiainducible factor 1, consist of oxygen-regulated HIF1αand constitutively expressed HIF1β) was the most important transcription factor. In recent years, targeting HIF1α has become a novel and efficient strategy for cancer therapy.Many HIF-1α inhibitors have been identified, in which the vast majority were chemical inhibitors. However, the relative lack of specificity led to many side effects in clinical trials of HIF-1 chemical inhibitors. RNA interference(RNAi) mediated by small interfering RNA(si RNA) is an effective method to selectively inhibit expression of a target gene. However, naked si RNA has a half lifetime of less than an hour in the bloodstream and hardly penetrate across cell membranes. These issues suggest that the development of safe and effective delivery systems is essential for therapeuticsusing si RNA in vivo.Two major classes of biomaterials that have been employed for si RNA delivery are liposomes and polymers. Although many different types of biodegradable polycations and cationic liposomes have been explored, the high chargedensities-induced toxicity is stilla challenge. Recently, lipid-polymer hybrid nanoparticles have emerged as effective vehicles for si RNA delivery. Lipid-polymer hybrid nanoparticles, with a single layer or bilayer lipid shell around a polymeric core, may combine the advantages of polymers and liposomes. However, the studies using lipid-polymer hybrid nanoparticles for co-delivery of drug and si RNA were still few.Herein, we deaigned anovel biocompatible lipid-polymer hybrid nanoparticle to co-deliver si RNA agaist HIF1α(si-HIF1α) and Gemfor pancreatic cancer treatment. All of component materials have been approved by the US Food and Drug Administration(FDA) for clinical uses or as food additives.Except the synthesis of this nanoparticle, we will focus on the effect of outer lipid bilayer coating on the stability, drug release, efficacity of si RNA deliveryof our nanoparticle and the anti-tumor effect in subcutaneous and orthotopic tumor models.Methods1.The role ofHIF1α in the proliferation and Gem chemosensitivity of pancreatic cancer cells1)The si-HIF1α was transfected into Panc-1 cells using Lipofactamine 2000, and the expression of HIF1α was determined using westernblot.2) After knockdowning HIF1α, the proliferation of Panc-1 cells was determine by CCK-8 assay.3) The Panc-1 cells transfected with si-HIF1α and negative control si RNA(si-ctrl) were treated by Gem, and the cell survival was examined using CCK-8 assay, to reveal the effect of HIF1α on Gem chemosensitivity.2. The design, synthesis and characeteration of nanoparticles.1) The design of novel biocompatible lipid-polymer hybrid nanoparticle for co-delivery of Gem and si-HIF1α: The designed nanoparticles are composed with a cationic polymeric core and anouter PEGylated lipid bilayer shell. The inner is EPL-modified m PEG-PLGA nanoparticles(ENPs) with high positive charge, which can effectively encapsulate Gem into its hydrophilic core and adsorb negatively charged si-HIF1α on the surface. After si RNA adsorbtion, the outer PEGylated lipid bilayer shell is coated on the surface of EPL(LENPs).The PEGylated lipidbilayer shell can decrease leakage of Gem and si RNA and improve stability of LENPs in the bloodstream.2)LENPs were fabricated reproducibly by combining a doubleemulsion method for polymer nanocore and ultrasound assistedself-assembly of a lipid film for lipid shell.3)The nanoparticle size(diameter, nm), size distribution and surface charge(zetapotential, m V) were determined using DLS measurement.4)The structure of nanoparticles was observed using TEM.5) The Gem encapsulation efficiency of ENPs was determined using HPLC, and nucleic acidadsorption efficiency of ENPs was detected via the change of surface charge and an electromobility shift assay.6) The stability of nanoparticles was determined via the change of size distribution after incubation in FBS for different time.7) The released Gem and nucleic acidfrom nanoparticles were determined using HPLC and electrophoresis, respectively.3. Characateration of the effect of siRNA delivery of nanoparticles in vitro and in vivo.1)The cellular uptake and intracellular location of LENP-deleveried si RNA were detected by observation of fluorescence labeling si RNA using confocal microscope.2) The knockdown effect of si RNA against Actin(si-Actin) was examined by observation of actin labeled with phalloidin-FITC usingconfocal microscope, and the knockdown effect of si-HIF1α was examinedby western blot and realtime RT-PCR.3)The circulating time and tumor target effect in vivo of fluorescence labeling si RNA delivered by nanoparticles were detected via measurement of fluorescence intensity of blood and organs using in vivospectrumimaging system.4. The anti-tumor effect of different nano-drug formulations in vitro and in vivo.1)The anti-tumor effect in vitro was determined via detecting the cell survival after different treatments using CCK-8 assay.2) The anti-tumor effect in vivowas examined in subcutaneous and orthotopic tumor models in mice.ResultsKnockdown of HIF1α significantly inhibited the proliferation and enhanced the Gem chemosensitivity of Panc-1 cells. The ENPs were successfully synthesized via a double emusion mothed, and had a high encapsulation efficiency of Gem(highest is 42% at the mass ration of m PEG-PLGA:Gem with 80:1) and adsorption efficiency of nucleic acid. After surface coating with lipid bilayer, compared to ENP-Gem-DNA, the size and structureof LENP-Gem-DNA had no obvious change and the surface charge changed to-34 m V. Due to the surface coating with lipid bilayer, LENP-Gem-DNA exhibited better stability and lower DNA release in FBS than ENP-Gem-DNA, and LENP-Gem exhibited lower Gem release than ENP-Gem either in p H7.4 or in p H4.4.LENP-deliverd si RNA can be uptaken by cells and escape from the lysosomes, and exhibited higher knockdown efficacy than ENP-deliverd si RNA. LENPs showed betterstability and longer circulating time in the bloodstream than ENPs.These characteristics endow LENPs improved drug targetingproperties via the enhanced tumor vasculature access effects intumor tissues. Consequently,LENP-Gem-si-HIF1αexhibited significantly better synergistic anti-tumor effects than ENP-Gem-si-HIF1α inthe subcutaneoustumor model. More importantly,LENP-Gem-si-HIF1αalso showed excellent capability to inhibittumor metastasis in the orthotopic tumor model.ConclusionIn summary, a novel biocompatible lipid-polymerhybrid nanoparticleencapsulating si-HIF1αand Gem were fabricated for pancreatic cancer treatment andexhibitedexcellent anti-tumor effects in subcutaneous and orthotopic tumor models. This novel lipid-polymerhybrid nanoparticlepossesses excellent stability in bloodstream and property in co-delivery of si RNA and drugin vivo. The current work lays thefoundation for a combination therapy strategy with si RNA andchemotherapy drugs using lipid-polymer hybrid nanocarriers. |
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