Establishment Of The Method For Quantifying Intracellular Iron And Its Application In Studying The Fate Of Nanoparticles In Cells

Iron-based nanoparticles（IbNPs）have been widely applied in biomedical research due to their excellent biocompatibility and unique magnetic,optical,mechanical,and electrical properties.Engineered functional IbNPs are frequently introduced into cells to enable cell tracking,directed cell migration,and cell function regulation.In practical applications,it is a major challenge to achieve efficient and controlled delivery of IbNPs to specific organelles.In depth understanding of IbNPscells interaction can optimize the design of IbNPs,improve the cell labeling efficiency,and regulate cell function more precisely.The study of intracellular fate of NPs is generally consisted of two aspects: the accurate quantification of intracellular IbNPs content and the dynamic tracking of IbNPs（IbNPs localization and transportation）.A variety of techniques have been developed for quantifying the content of intracellular IbNPs,such as optical properties-based,magnetic properties-based and magnetic ionization characteristics-based method.However,most of these approaches rely on specialized instruments and can not provide iron mass distribution inside cells.Microscopy-based approach is a widely applied strategy in visualizing intracellular IbNPs dynamics at single-cell level,including fluorescence-based,scattering-based and absorption-based microscopy,while none of these imaging methods could perform the absolute quantification of IbNPs in cells.Based on these backgrounds,we found that there is a lack of tools that could satisfy both requirements（accurate quantification and dynamic processes of intracellular IbNPs）at single-cell level.In this paper,a method that combines optical microscopy and Beer–Lambert’s law is proposed for quantifying intracellular iron content based on optical images,which is named as “pixel-based colorimetry” method（PbC method）.The PbC method was applied in quantifying intracellular superparamagnetic iron oxide nanoparticles（SPIONs）in human umbilical cord mesenchymal stem cells（h UC-MSCs）and iron deposition in erythroblasts.At last,combining the PbC method with live cell imaging system,the fate of Prussian Blue nanoparticles（PBNPs）in cells was dynamically tracked and quantitatively analyzed.We clarified the contributions of each factor（asymmetric cell inheritance,lysosomal digestion,and exocytosis）to the reduction of intracellular PBNPs.（1）The establishment of the principles of the quantification method（PbC method）The principle of Perls staining is to transform the intracellular Fe³⁺ into Prussian blue（PB）.Therefore,at first,we established the relation between optical characteristics of PB solution image（the ratio of adjacent channels in a pixel）and PB mass per pixel by combining Beer-Lambert’s law and principles of microscopic imaging.Then,we verified the reliability of the principles and obtained the standard curve.The accuracy of the method can reach femtogram per pixel(10^-2 fg PB per pixel)and the detection range spans over 4 decades(10^-2 fg-10² fg PB per pixel).The study into the influence of imaging parameters on the measurements showed that the magnification has no real impact on the standard curve.However,the intensity of exposure has a slight effect on the measurement,which can be circumvented by ensuring the R/G/B signals(Q_i0)in establishing standard curves are identical with which in cell sample measurement.（2）Quantification of iron in SPIONs-labeled stem cells and iron-deposited erythroblasts based on PbC methodIn this section,we investigated the feasibility of PbC method in quantifying intracellular stainable iron,SPIONs-labeled h UC-MSCs were first selected as the model system,we quantified the iron content after cell labeling and obtained the iron mass distribution in cells.Furthermore,the single cell-level analysis enables the investigation of heterogeneity in the uptake of SPIONs by cells.At last,we proved that the method allows for studying the internalization and degradation of SPIONs in stem cells.In addition,the evaluation of iron in erythroblasts is important for the diagnosis of anemia,however,there is lack of tools for quantifying the deposited-iron in erythroblasts,the existing evaluation method is time-consuming and inaccurate.Hence,we expanded the PbC method in quantifying the iron deposition within erythroblasts.The results show that compared with the traditional grading method,the PbC method is more accurate and efficient,and is expected to be an improved strategy with high precision for bone marrow iron quantification.（3）The study into the fate of intracellular PBNPs based on PbC methodIn this section,combining the PBNPs,the live cell imaging system and the proposed PbC method,we continuously monitored the intracellular PBNPs dynamics up to three cell divisions for overall 60 h.We precisely captured the fine changes of intracellular PBNPs content during interphase,and the asymmetric inheritance of PBNPs in two daughter cells during mitosis.As supported by the investigation of PBNPs lysosomal digestion and exocytosis,we concluded the percentage of each factor that contribute to the reduction of intracellular PBNPs content within one cell cycle:asymmetric cell inheritance（92.78 %±1.78 %;59.19 % ±10.09 % vs 33.59% ±10.09 % in two cells）,lysosomal digestion（6.93%±0.06%）and exocytosis（0.29%± 0.06 %）.