Multiparameter Quantification Of Lipid Nanomedicines By High-Sensitivity Flow Cytometry

The growing interest in the biomedical application of nanomedicine is largely attributable to its unique and appealing features in targeted delivery,controlled release and improved bioavailability.Currently,nanomedicines can be broadly divided into the following categories:lipid nanoparticles,polymeric nanoparticles,inorganic nanoparticles and virus nanoparticles.Among them,lipid based nanomedicine such as liposomes,solid lipid nanoparticles,lipid micelles and extracellular vesicles,which are characterized by high biological compatibility and substantial drug-loading capability,are considered the most successful drug delivery systems known to date.To achieve sufficient quality control and highly-efficient drug delivery,properties such as particle size and size distribution,particle concentration,drug content,fraction of drug encapsulation and surface ligand density must be precisely characterized.Because of the complexity and the large intrinsic heterogeneity of lipid nanomedicines in particle size and composition,rapid and rigorous characterization at the single-particle level is of fundamental importance to reveal the individual difference that would be otherwise averaged out in ensemble measurements.However,it is very challenging to achieve multiparameter and quantitative analysis of lipid nanomedicines at the single particle level on account of their small sizes and poor structure stability.Whereas cryo-TEM can provide valuable size and morphology information with maximum structure preservation of lipid nanomedicines,its routine application is prevented due to the poor accessibility,low throughout and the time-consuming sample preparation.Other commercialized single-particle techniques also fall short in fulfilling the requirement for nanomedicine characterization owing to the limited sensitivity and resolution or the lack of multiparameter analysis ability.Thus,it is of vital importance to develop a rapid,sensitive and high-throughput quantitative characterization method at the single particle level for the analysis and development of lipid nanomedicines.Flow cytometry is a single particle detection technique for rapid,multiparameter and quantitative analysis and sorting of individual cells or cell-size particles in suspension.Due to the limitation of sensitivity,conventional flow cytometer can only be applied to the analysis of particles with large size or strong fluorescence.However,it is extremely difficult for conventional flow cytometer to achieve multiparameter analysis of drug-loading lipid nanoparticles smaller than 200 nm.Integrating light scattering with strategies for single-molecule fluorescence detection in a sheathed flow,our laboratory have developed high sensitivity flow cytometry(HSFCM)that enables light-scattering detection of single nanoparticles with light-producing power below the level of single fluorescent molecules for the first time.The light-scattering based size detection limits for single nanoparticles are 24 nm and 7 nm in diameter for silica and gold nanoparticles,respectively.And the fluorescence detection limit is three fluorescent molecules of alexa fluor 555.By offering high-resolution analysis of single nanoparticles in liquid suspensions at a throughput of up to 10 000 particles per minute,HSFCM can reveal the size distribution in minutes which would take hours for cryo-TEM to accomplish.Moreover,through fluorescence detection,the multiple biochemical properties of single particles and their correlation can be quantitatively characterized.Taking advantage of the superior resolution,sensitivity,detection speed and the capability of multi-parameter analysis of HSFCM,we develop a rapid characterization method for lipid nanomedicines in this dissertation,which allows the absolute quantification of particle size,drug content,fraction of drug encapsulation,particle concentration and the surface ligand density of liposomal nanomedicines at the single-particle level.This novel method aims to meet the characterization challenge of lipid nanomedicine and to promote its development.This dissertation consists of the following sections:In chapter one,the research background of nanomedicine and the single particle detection techniques for nanomedicine analysis are briefly introduced.Chapter two introduces the side scattering based size measurement of lipid nanomedicines by HSFCM.Using silica nanoparticles with various size as the standards,we successfully characterized the size distribution of doxorubicin-loaded liposomes.Furthermore,we discuss the influence of the particle refractive index on the light-scattering based detection of liposomal nanoparticles.We propose that a calibration of scattering intensity should be applied via Rayleigh scattering theory when sizing nanoparticles with refractive index different from the standards.At last,to achieve a perfect match of particle refractive index,we developed a strategy by using well prepared and size characterized doxorubicin-loaded liposomes as standards for the measurement of commercial nanomedicine products.Chapter three presents the side scattering and fluorescence dual-parameter detection of lipid nanomedicines.By means of that,drug encapsulation fraction,serum stability and particle concentration of doxorubicin encapsulated liposomes could be characterized.Furthermore,by using fluorescent silica nanoparticles with various molecules of equivalent soluble fluorochrome(MESF)or the well-prepared doxorubicin-loaded liposomes as standards respectively,we revealed the drug content distribution of doxorubicin-loaded liposomes.Applications of this analysis method for the multiparameter characterization of commercial.doxorubicin-loaded liposomes(Doxoves and gDoxil)and siRNA encapsulated lipid nanoparticles were also demonstrated.Chapter four describes the preparation and multiparameter characterization of targeted liposomes.Firstly,using folate coupled liposomes as a model,we develop a quantitative characterization method for the surface ligand density and studied its biological impact on the cell binding ability of liposomes.Besides,the influence of preparation methods and PEG length on the ligand density distribution and heterogeneity is also elucidated.Finally,the method is applied to the transferrin or antibody coupled liposomes,and a novel evaluation method for the proportion of available ligand number in total protein content is established.In chapter five,the work of this thesis is summarized and the future prospect in single lipid nanoparticle analysis is discussed.