Design And Evaluation Of Afatinib-loaded Immunoliposomes With Antibody-mediated For Targeting The Epidermal Growth Factor Receptor

Non-small-cell lung cancer(NSCLC)accounts for ~85% of all lung cancers,and is the leading cause of cancer-related deaths worldwide.Seventy-five percent of patients with NSCLC are diagnosed at an advanced stage,and most die within 1 year.With the discovery of epidermal growth factor receptor(EGFR)overexpression in NSCLC,various EGFR tyrosine kinase inhibitors(TKIs)and anti-EGFR monoclonal antibodys have been applied in clinical therapy.Unfortunately,adverse reactions caused by combination therapy have affected patient compliance,and have resulted in treatment discontinuation in severe cases.In the present study,afatinib(AFT),the EGFR-TKI drug,was encapsulated in “liposomes”(LPs)to achieve longer circulation in the blood and an enhanced permeability and retention effect in tumors.Concomitantly,CTX was designed to bind to drug-loaded LPs to form “immuno-LPs”(LP-CTX)for tumor-cell selectivity and therapeutic activity.Ammonium sulfate gradient method was used to prepare AFT-LPs,which were then co-incubated with thiolated cetuximab(CTX)to prepare LP-CTX.Both LP formulations were characterized and tested in vitro using two NSCLC cell lines to study the effect of CTX-EGFR association on active targeting.The pharmacokinetics of the LPs and immuno-LPs were evaluated in rats and the tumor retention and the antitumor efficacy of the two formulations was compared in NSCLC xenografted mice.1.Preparation of AFT-LPFilm-dispersion method,reverse evaporation methods,improved ethanol injection methods,and ammonium sulfate gradient methods were employed to prepare LPs.Sephadex gel microcolumn centrifugation was performed to purify the LPs.The encapsulation efficiency of the LP was measured by ultraviolet spectrophotometry,and the particle size was measured by a laser particle size analyzer.Compared with different preparation methods,liposomes prepared by ammonium sulfate gradient method have higher encapsulation efficiency and smaller particle size.Moreover,the formulation parameters were further optimized by single factor investigation and response surface analysis to determine the optimal preparation method of AFT-LP.The AFT-LP encapsulation efficiency obtained by the optimal formulation was 90.25% ± 8.42%,and the particle size was 112.5 ± 1.8 nm.2.Preparation of AFT-LP-CTXCTX was thiolated by reaction with excess Traut's reagent,purified and incubated with to prepare LP-CTX.The key parameters in the preparation process were evaluated with the connection efficiency and particle size.Finally,LP-CTX was prepared by selecting CTX: lipid(1:500 mol/mol)as the optimal condition,with 20 h for the incubation.The binding efficiency was about 70% and the particle size was about 117 nm.Sepharose CL-4B gel column was employed to separate and identify LP-CTX.The elution curve following elution of LP-CTX and free CTX demonstrated successful linking of CTX to LPs.3.Characterizations of LP and LP-CTXThe morphology of LP and LP-CTX was observed by transmission electron microscopy.The particle size and zeta potential were measured,as assessed using DLS.The encapsulation efficiency and drug loading were calculated.Using transmission electron microscopy,the morphology of LPs and LP-CTX was observed to be spheroidal “nanocapsules”.The mean particle size of LPs and LP-CTX was about 110 nm.The particle size of LP-CTX did not change significantly compared with that of LPs.In addition,the surface of LP-CTX demonstrated a slightly positive charge because of CTX incorporation,which could improve tumor accumulation and facilitate targeting to the lungs compared to negatively charged LPs.The encapsulation efficiency and drug loading of LPs and LP-CTX were about 90% and about 7%.AFT release from LPs and LP-CTX in PBS or PBS containing 50% FBS during 24 h in vitro was investigated.AFT release from the two formulations was similar,with ~40% release observed at 8 h and maintained to 24 h.The presence of FBS increased the release rate during the initial phase.This may have been because serum proteins affect LP stability,albeit minimally.4.In vitro studies of LP and LP-CTXThe toxicity of LP-CTX was compared with that of AFT solution and LPs in A549 and H1975 cells.The cellular uptake of A549 and H1975 cells exposed to Dil-LP and Dil-LP-CTX were obtained and quantified,and the effect of the EGFR on targeting was examined in additional,quantitative parallel experiments.The toxicity of LP-CTX was higher and the relative internalization rate was faster.Specific EGFR association was demonstrated through competition with free CTX,which reduced the cellular uptake to levels similar to those of the liposomes.5.LC-MS/MS method for the determination of AFTA sensitive quantitative LC-MS/MS method for AFT in rat plasma was developed and validated using AFT dimaleate as a standard.The Agilent Eclipse XDB-CN column(100 × 2.1 mm,3.5 ?m)was applied for chromatographic separation.The mobile phase was water and methanol(15:85,v/v)containing 0.1% formic acid with a flow rate of 0.5 mL/min.The linear range was 0.5–200 ng/mL.This method meets the regulatory requirements of international guidelines for selectivity,sensitivity,precision,accuracy,and stability.6 In vivo studies of LP and LP-CTXThe plasma samples from rats at different time intervals after treatment were collected to determine the drug concentration using the LC-MS/MS assay and pharmacokinetic parameters were calculated to investigate the delivery of AFT in vivo.A tumor-bearing mouse model was established to study the tissue distribution and tumor grow inhibition.Moreover,DiR-labeled LP and LP-CTX were developed to track the distribution of the two formulations in vivo by using small animal in vivo imaging.The plasma concentration of LPs and LP-CTX was nearly 400-fold higher than that of AFT solution for the first sampling time.And LPs and LP-CTX behave similarly in healthy rats.Compared with LPs,obviously higher AFT retention in tumors and therapeutic effects were detected with LP-CTX in tumor-bearing mice.However,in the small animal in vivo imaging experiment,the distribution of DiR in mice was not completely consistent with the results of tissue distribution measurement of AFT.The reason may be that DiR released from the liposome still has a fluorescent effect and is easily accumulated in the liver and spleen.Therefore,the imaging can not represent the distribution of AFT in vivo.We designed and evaluated AFT-loaded immuno-LPs with CTX for targeting the EGFR.LP-CTX showed a significant increase in cellular uptake in EGFR-positive and EGFR-mutant NSCLC cell lines.In pharmacokinetic studies,the extended elimination half-life indicated that the dose of LPs and LP-CTX should be reduced appropriately upon clinical application so that the risk of an adverse reaction may be reduced.In the NSCLC xenograft model,immuno-LPs exhibited strongly enhanced drug delivery capacity and growth inhibition effect for tumors.Therefore,EGFR-targeting immuno-LPs appear to be promising for NSCLC treatment.