Influence of Colonies' Morphological Cues on Cellular Uptake Capacity of Amino Modified Nanoparticles

Influence of Colonies' Morphological Cues on Cellular Uptake Capacity of Amino Modified Nanoparticles | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 31 January 2024 V1 Latest version Share on Influence of Colonies' Morphological Cues on Cellular Uptake Capacity of Amino Modified Nanoparticles Authors : Siyuan Huang , Yingjun Yang 0000-0003-2746-1192 [email protected] , Xiaoqiang Hou , Jingyi Chen , Guanjian Nie , Bingshe Xu , and Shukai Ding Authors Info & Affiliations https://doi.org/10.22541/au.170669877.75554281/v1 194 views 122 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Micropatterning techniques were widely applied to manipulate morphology of colony and to reveal the critical role of spatial factors in tumorigenesis and gastrulation. However, the effect of morphological cues on bioactive substances treatment during these biological processes was still not clear. Therefore, the influence of colony's morphology on cellular uptake capacity of insoluble bioactive substance should be another essential factor in biomolecular induced cellular behaviors' variation. In this study, PDMS stencils were applied to control colonies' size, and relationship between cellular uptake capacity of nanoparticles and morphological cues of colonies was analyzed. Consequently, benefited from more nanoparticles within micropatterns, the larger colonies endocytosed more nanoparticles. Additionally, concentrated cells with high cell seeding density or located at the peripheral region of micropatterned colonies have higher cellular uptake capacity. The improved cellular uptake capacity at the peripheral region was mainly caused by the uneven distribution of nanoparticles. In addition, the high cell density promoted cellular uptake was benefited from the cortical actin accelerated clathrin-mediated endocytosis in circular-shaped cells. Influence of Colonies’ Morphological Cues on Cellular Uptake Capacity of Amino Modified Nanoparticles Siyuan Huang 1,2& , Yingjun Yang 1&* , Xiaoqiang Hou 1,2 , Jingyi Chen 1,2 , Guanjian Nie 1,2 , Bingshe Xu 1,3* , Shukai Ding 1 * 1 Materials Institute of Atomic and Molecular Science, Shaanxi University of Science and Technology, Xi’an, 710021, China. 2 School of Materials Science & Engineering, Shaanxi University of Science and Technology, Xi’an, 710021, China. 3 Key Laboratory of Interface Science and Engineering in Advanced Materials of Ministry of Education, Taiyuan University of Technology, Taiyuan, 030024, China & These authors contributed equally to this work. * Corresponding author: Yingjun Yang, E-mail: [email protected] , Tel: 86-29-86611217 Bingshe Xu, E-mail: [email protected] , Tel: 86-29-86611217 Shukai Ding, E-mail: [email protected] , Tel: 86-29-86611217 Keywords: Morphological cues; Micropattern; Cellular uptake; Nanoparticles; Colony; Abstract Micropatterning techniques were widely applied to manipulate morphology of colony and to reveal the critical role of spatial factors in tumorigenesis and gastrulation. However, the effect of morphological cues on bioactive substances treatment during these biological processes was still not clear. Therefore, the influence of colony’s morphology on cellular uptake capacity of insoluble bioactive substance should be another essential factor in biomolecular induced cellular behaviors’ variation. In this study, PDMS stencils were applied to control colonies’ size, and relationship between cellular uptake capacity of nanoparticles and morphological cues of colonies was analyzed. Consequently, benefited from more nanoparticles within micropatterns, the larger colonies endocytosed more nanoparticles. Additionally, concentrated cells with high cell seeding density or located at the peripheral region of micropatterned colonies have higher cellular uptake capacity. The improved cellular uptake capacity at the peripheral region was mainly caused by the uneven distribution of nanoparticles. In addition, the high cell density promoted cellular uptake was benefited from the cortical actin accelerated clathrin-mediated endocytosis in circular-shaped cells. Introduction In vivo microenvironment, cells are surrounded by soluble biomolecules, adjacent cells and the extracellular matrix (ECM) [1]. Various biochemical and biophysical factors are provided by these components and simultaneously acting on cells [2, 3]. Nowadays, with well development of materials engineering, tremendous techniques were developed to mimic in vivo biophysical stimulations [4]. In particular, micropatterning techniques were utilized to investigate the role of cell or colony’s morphology in regulation of cellular behaviours [5]. It has been demonstrated that the morphologies of single cell including size[6], aspect ratio [7, 8], geometry [9] and chirality [10] were efficient in regulation of stem cell differentiation, DNA synthesis activity and cellular uptake capacity. Furthermore, compared with single cell, cell colony is more complex multicellular structures and valuable for organoid focused researches. In this case, morphology of colony also attracted extensive interest [11]. Recently, micropatterning techniques including microcontact printing[12], photolithography [13] and stencil-assisted patterning [14] were applied to manipulate colony’s morphology. In these researches, cells always showed various cell behaviours with different size and exhibited different phenotypes at central or peripheral region of micropatterned colony after induction of biochemical factors [15, 16]. In this case, the critical role of spatial factors in tumorigenesis [17, 18] and gastrulation [19, 20] has been indicated. These results were predicated upon the precondition of the homogeneous treatment of bioactive substances within micropatterned colony. However, the effect of spatial factors on bioactive substance treatment have not been considered at these researches. Although soluble biochemical molecules can evenly act on cells within micropatterned colony [21], there are tremendous insoluble amino acids and nanoparticles were also applied in various researches. Furthermore, the fully functioning of these insoluble substances is tightly related with successful transmembrane delivery [22]. Therefore, the influence of morphological cues on cellular uptake capacity should be another supplementary information for comprehensive understanding of spatial factors regulated cellular behaviours’ variation. In this study, the effects of colonies’ morphology on cellular uptake capacity were investigated by using PDMS stencils. The PDMS stencils containing circular micro-holes with 0.8 mm and 1.2 mm in diameter were prepared to accurately control colonies’ size. The melanoma cells with different seeding densities were cultured on the stencils to form micropatterned colonies with different sizes and cell densities. Then, the amino modified fluorescence polystyrene nanoparticles were incubated with micropatterned colonies to investigate the influence of colonies’ morphology on cellular uptake capacity. Firstly, the distribution of cells and endocytosed nanoparticles in micropatterned colonies was observed and analyzed. To explore the reason for morphological cues affecting on cellular uptake capacity of amino modified nanoparticles, the distribution of added nanoparticles within micropatterns and cytoskeleton structure of cells were also characterized. 2. Materials and Methods 2.1. Preparation of PDMS stencils The stencils were prepared by a simple punching process. In detail, the PDMS film with 100 μm in thickness was commercially purchased from Hangzhou Bald Advanced Materials Technology Co., Ltd. The PDMS films were firstly cut into a circular shape with 14 mm in diameter. Then, the holes with 0.8 mm or 1.2 mm in diameter were manufactured on the circular PDMS film by using a specific puncher. Before cell seeding, the prepared stencils were firstly be sterilized by immersed in 70% ethanol for 20 min and rinsed with PBS solution twice. Finally, the stencils were placed inner a 24-well plate for cell culture. 2.2. Cell culture Melanoma cells (B16) were purchased from Procell Lifer Science & Technology Co., Ltd. and subculture in DMEM medium (Mishu (Xi’an) Biotechnology Co., Ltd.) supplied with 10% FBS (Biological Industries Israel Beit Haemek Ltd.) and 1% penicillin-streptomycin (Mishu (Xi’an) Biotechnology Co., Ltd.). 1 mL cell suspension with 4×10 4 , 6×10 4 and 8×10 4 cells/mL in cell density was seeded on each stencil. After being cultured in a humidified CO 2 incubator for 6 h, the medium with suspending cells was refreshed. Then, the samples were further incubated for 18 h for the following experiments. 2.3. Nuclei staining and cell density analysis After cell seeding for 24 h, the samples were rinsed by prewarmed PBS and fixed by 4% cold paraformaldehyde (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 10 min. Then, the samples were permeabilized by 1% Triton X-100 (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 2 min and stained by 1‰ DAPI (Shandong Sparkjade Scientific Instruments Co., Ltd.) in PBS for 10 min. The fluorescence of DAPI was observed and recorded by a fluorescence microscope (MF52-N, Guangzhou Micro-shot Technology Co., Ltd.). The fluorescence images were applied to analyze cell distribution within micropatterned colonies. To count cells in different regions, the colony was firstly separated into the peripheral region and central region. To get the same area of the peripheral region and central region, the width of the peripheral region was set to 0.12 mm for micropatterned colonies with 0.8 mm in diameter and 0.18 mm for colonies with 1.2 mm in diameter. The cell numbers at peripheral or central regions were counted from DAPI staining fluorescence images by analyzing the particles process of ImageJ software. More than 50 fluorescent images were applied to get heatmaps and 5 representative fluorescence images were analyzed to get quantitative data. 2.4. Cellular uptake capacity analysis After the cell seeding process for 24 h, the medium was replaced by a fresh medium with 1% amino-modified fluorescent PS nanoparticles (100 nm, Xi’an ruixi biological Technology Co., Ltd.) and further incubated for another 24 h in a humidified CO 2 incubator. Then, the samples were harvested and incubated with 0.4% Trypan Blue (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 5 min to quench the fluorescence of extracellular nanoparticles. Finally, the samples were fixed by 4% paraformaldehyde and stained by 1‰ DAPI. The fluorescence of nanoparticles and DAPI was observed and recorded by fluorescence microscopy. The percentage and fluorescence intensity of nanoparticle positive cells were calculated to evaluate the cellular uptake capacity. To define nanoparticle positive cells, the integrated gray value (IGV) and area (A) of each cell were calculated by ImageJ. To get corrected IGV, the region without cell attachment was selected as background to calculate IGV background and A background . Then, the corrected IGV was calculated by (IGV/A-IGV background /A background )×A. The nanoparticle positive cells were defined as the cells with corrected IGV was 2 times higher than the IGV background . The percentage of nanoparticle positive cells were calculated by the number of nanoparticle positive cells divided by the total cell number. The corrected IGV of nanoparticle-positive cells were also recorded to evaluate the cellular uptake capacity. More than 30 fluorescent images of each group were analyzed. 2.5. Actin and nuclei staining After cells were cultured within stencils for 24 h, the samples were fixed by 4% paraformaldehyde for 10 min and permeabilized by 1% Triton X-100 for 2 min at room temperature. Then, the samples were blocked with 2% bovine serum albumin (BSA, Shanghai Aladdin Biochemical Technology Co., Ltd.) in PBS for 30 min at room temperature. Then, actin was stained by incubating the samples with Alexa Fluor-594 phalloidin (Beijing Solarbio Science & Technology Co., Ltd.) at a dilution ratio of 1:40 in PBS for 20 min at room temperature. Nuclei were stained with 1‰ DAPI at room temperature in the dark for 10 min. After being washed with PBS three times, the fluorescence images of each sample were observed and recorded by a fluorescence microscope. 50 fluorescent images were applied to obtain heatmaps. 2.6. Analysis of nanoparticles’ distribution To analysis the distribution of nanoparticles, 1 ml cell culture medium with 1% fluorescent nanoparticles was incubated with stencils in 24-well plates for 6 h. Then the fluorescence of nanoparticles was observed and recorded by fluorescent microscope. The percentage and fluorescence intensity of nanoparticle positive cells were also calculated as previously introduced. 5 fluorescent images were analyzed for quantitative results. 2.7. Blebbistatin treatment Blebbistatin (Shanghai Aladdin Biochemical Technology Co., Ltd.) was applied to disturb cytoskeleton organization. Detailly, after cell cultured on stencils for 16 h, the medium was refreshed by cell cultured medium with 1 ng/mL blebbistatin. After further cultured for 8 h, the medium was replaced by a fresh medium with 1% amino-modified fluorescent nanoparticles and 1 ng/mL blebbistatin. After further cultured in a humidified CO 2 incubator for another 24 h, samples were harvested and treated by 0.4% Trypan Blue for 5 min. Before fluorescence observation, the samples were stained by DAPI. The percentage and fluorescence intensity of nanoparticle positive cells were also analyzed as previously described. More than 30 fluorescent images were analyzed. 2.8. Dynasore treatment To inhibit dynamin activity, dynasore (Shanghai Aladdin Biochemical Technology Co., Ltd.) was incubated with melanoma colonies. In detail, after cell seeded on stencils for 16 h, unattached cells with the medium were refreshed by cell culture medium with 40 μM dynasore (Shanghai Aladdin Biochemical Technology Co., Ltd.) and further cultured for 8 h before addition of fluorescent nanoparticles. Nanoparticles were added by refreshing the medium with a cell culture medium containing 1% nanoparticles and 40 μM dynasore. Then the samples were further incubated for 24 h. After extracellular fluorescence was quenched by Trypan Blue, samples were fixed and stained by DAPI. The percentage and fluorescence intensity of nanoparticle positive cells were analyzed to determine cellular uptake capacity. More than 30 fluorescent images were analyzed. 2.9. Statistical analysis The significant difference among samples was performed using a one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons. The data are presented as means ± standard deviations (SDs). It is considered to be a statistically significant difference when p < 0.05. 3. Results 3.1. Preparation and characterization of stencils As shown in Figure 1.A, the PDMS stencils were prepared by punching process and applied for cell culture. The micro-holes with 0.8 mm or 1.2 mm in diameter were fabricated on a circular PDMS film with 14 mm in diameter by using a specific puncher (Figure 1. B and C). The diameter of the holes was 849.5 ± 33.9 μm and 1273.7± 62.6 μm. After sterilization, the PDMS stencils tightly adhered to TCPS (Tissue Culture Polystyrene) surfaces in 24-well plates for the following cell experiments. Since PDMS film’s hydrophobic properties do not encourage cell adhesion [23], cells were trends to adhere on TCPS surface that bared in micro-holes of PDMS stencils [24]. After the cell cultured with PDMS stencils, colonies were formed within micro-holes of the stencil and the morphology of colonies was controlled by PDMS stencils (Figure 1.D). After cell adhesion, the unadhered cells can be easily removed by the medium refreshing process. Additionally, the stencil associated micropatterning method had no significant influence on cell viability (Figure S1). 3.2. Cell distribution After the formation of micropatterned colonies, the diameter of micropatterned melanoma colonies was 835.9 ± 14.1 μm and 1262.2 ± 18.4 μm. Then, the nuclei were stained to characterize the cell distribution. As shown in Figure 2.A, the density of melanoma cells was controlled by seeding density. With few cells seeding, there was no significant difference of cell density between different size of colonies (Figure 2.B). With cell seeding density increased, the cell density of colonies with 0.8 mm in diameter was higher than the colonies with 1.2 mm in diameter (Figure 2.B). Furthermore, cells were predominantly adhered at the peripheral region of micropatterned colonies. To observe more clearly, more than 50 fluorescent images were applied to create heatmaps (Figure 2.B). The heatmaps showed the same phenomenon that melanoma cells concentrated at the peripheral region of micropatterned colonies, especially at low cell density. To quantitative analysis the cell density at a different region of colonies, the central and peripheral region with the same area was set as Figure 2.C shows. In detail, a radius of the central region and width of the peripheral region was set as 0.28 mm/0.12 mm for 0.8 mm colonies and 0.42 mm/0.18 mm for 1.2 mm colonies. As quantitative results showed in Figure 2.D, cell density at the peripheral region was significantly higher than in the central region. 3.3. Cellular uptake capacity Cellular uptake of amino group modified fluorescent PS nanoparticles was shown in Figure 3.A and B. The cellular uptake capacity was indicated by the percentage and fluorescence intensity of fluorescence positive cells. The colonies’ size did not have significant effect on percentage of fluorescence positive cells (Figure 3.C). On the other hand, the fluorescence intensity of micropatterned colonies with 1.2 mm in diameter was higher than the smaller colonies ( d = 0.8 mm)(Figure 3.D). Furthermore, within the colonies having same size, as cell seeding density increased, the percentage of fluorescence positive cells was decreased (Figure 4. A and E) but fluorescence intensity of each positive cell was increased (Figure 4. B and F). On the other hand, for the results of spatial factor regulated cellular uptake capacity, the percentage and fluorescence intensity of positive cells (Figure 4. C and G and Figure 4. D and H) at the peripheral region was significantly higher than the cells located in the central region. To explore the reason of these phenomena, the distribution of nanoparticles and structure of cells were analyzed. 3.4. Distribution of fluorescent nanoparticles To analysis the distribution of fluorescent nanoparticles within PDMS stencils, PDMS stencils were incubated with nanoparticles without cell culture. As shown in Figure 5, the total amount of fluorescent nanoparticles located within micropatterns with 1.2 mm in diameter was significantly larger than the micropatterns with 0.8 mm in diameter (Figure 5.B). Additionally, the fluorescent nanoparticles were also concentrated peripheral regions of circular PDMS micropatterns (Figure 5.A and C). 3.5. Structure of cytoskeleton Since previous researches have demonstrated the cellular uptake capacity is tightly related to the structure of the cytoskeleton [8, 10, 25], the actin structure of cells was characterized. The structure of the cytoskeleton was analyzed by actin-stained fluorescence images (Figure S3). The heatmaps of actin (Figure 6.A) revealed that actin was concentrated in the peripheral region of micropatterned colonies. This result had a good agreement with cell uneven distribution. As showed in magnified fluorescent images (Figure 5.B), more spindle-shaped cells (white arrow) were observed at the central region of the micropatterned colony with low cell density. In the contrast, more circular-shaped cells (green arrow) were found in peripheral regions with high cell density. In addition, actin was concentrated at a cortical region in circular-shaped cells and homogeneously distributed in spindle-shaped cells. 3.6. Influence of cytoskeleton on cellular uptake capacity To explore the function of cytoskeleton in cellular uptake process, the cytoskeleton was disrupted. As showed in Figure 7.A, actin was disturbed by blebbistatin before and during cellular uptake experiments. After blebbistatin treatment, there was no significant influence on the actin organization of spindle-shaped cells. In the contrast, actin at cortical region of cells with high density was disappeared and randomly distributed within the cytoplasm. In addition, the percentage (Figure 7.C and Figure S4.A) and fluorescence intensity (Figure 7.D and Figure S4.B) of fluorescence positive cells were decreased after blebbistatin treatment. 3.7. Influence of dynamin on cellular uptake capacity In addition, at the final step of CME, cortical actin is collaborated with dynamin to separate clathrin-coated pits from the plasma membrane [26, 27]. It means cortical actin is not only functionalized at the process of endocytic membrane’ invagination but also at the endocytic vesicles’ separation. Therefore, the function of cortical actin and dynamin in the regulation of cellular uptake capacity was also investigated by inhibition of dynamin activity. In this case, dynasore was applied to inhibit dynamin activity [28]. As shown in Figure 8.A, dynasore was incubated with melanoma colonies before and during cellular uptake experiments. Quantitative data revealed that the percentage (Figure 8.C and Figure S5.A) and fluorescence intensity (Figure 8.D and Figure S5.B) of fluorescence positive cells were significantly decreased after dynasore treatment. In addition, the fluorescence intensity has no significant difference with different cell seeding densities after dynasore treatment (Figure 8.D). 4. Discussion In this study, micropatterned PDMS stencils were applied to control the morphology of melanoma colonies. After micropatterned colonies formed, the size and morphology of colonies were easily controlled by PDMS stencils and the cell density was regulated by cell seeding process. For each colony, cells were predominately located at peripheral region. This phenomenon was similar to the cells cultured in microwell plates. This uneven distribution of cells can be explained by the effect of the meniscus. [29] Then, the results of cellular uptake capacity, cell density and nanoparticles distribution were comprehensively analyzed. Firstly, with similar low cell density (Figure 2.B), cellular uptake capacity of colonies with 1.2 mm in diameter (Figure 3.C) was higher than that in 0.8 mm micropatterned colonies. This phenomenon could be explained by more dropped nanoparticles in larger colonies’ area (Figure 5.B). As cell density increased, the fluorescence intensity was also increased with increasing of colonies’ size. This result was related by the average of nanoparticles per cell within colonies (Figure S2. B). Therefore, the larger colonies size can be indicated as a positive factor for enhancement of cellular uptake. In addition, the role of spatial factor in regulation of cellular uptake capacity was also analyzed. With cell density increased, the decreased percentage of fluorescence positive cells (Figure 4.A and E) may mainly be due to the smaller cell spreading area induced lower contacting probability between plasma membrane and nanoparticles at higher cell density (Figure S2.B) [30]. Furthermore, it is worth noting that the fluorescence intensity of positive cells was positively related to cell density. It means even the percentage of fluorescence positive cell decreased, the cellular uptake capacity of individual cell was enhanced by higher cell density. Simultaneously, with increasing of cell density, the decreased difference of fluorescence intensity between 0.8 mm and 1.2 mm colonies was also reveal the effect of cell density on cellular uptake capacity (Figure 3.D). Additionally, combined with the result of cell uneven distribution in micropatterned colonies (Figure 2), peripheral located cells have highest percentage of fluorescence positive cells and fluorescence intensity also indicated the higher cellular uptake capacity was related to higher density. In this case, the distribution of nanoparticles within micropatterned stencils and structure of cytoskeleton was characterized. With the effect of a meniscus, nanoparticles were also concentrated at a peripheral region of holes in PDMS stencils without cell culture. The concentrated nanoparticles at the peripheral region will lead to a higher contacting probability between plasma membrane and nanoparticles [31]. It can be the primary reason for the higher cellular uptake capacity of cells adhered to the peripheral region. However, it cannot be evidence to support the cellular uptake capacity can be enhanced by higher cell density. Therefore, the structure and function of the cytoskeleton in micropatterned colonies were also characterized. For the attractive result of actin structure that the cells with higher fluorescence intensity also showed more circular-shaped cells with typical cortical actin. As previously reported, the cortical actin was critical for invagination of the endocytic membrane in clathrin-mediated endocytosis (CME) [32, 33]. In addition, some research already revealed that CME is a primary approach for cellular uptake of amino modified nanoparticles [34]. Thus, cortical actin was considered as a critical factor in the regulation of the cellular capacity of amino group modified nanoparticles. Therefore, it indicated that the higher cell density enhances cellular uptake capacity through more cortical actin accelerated CME in circular-shaped cells. To demonstrate the functions of cortical actin in the regulation of cellular uptake capacity in micropatterned colonies, the actin organization was distributed. Blebbistatin is a specific myosin II inhibitor that can reduce cortical actin mediated cortex tension [35, 36]. For the results, with the disappearance of cortical actin, cellular uptake capacity was decreased by blebbistatin treatment. It can be another evidence to support the higher cellular uptake capacity of cells with a higher density is benefited from the cortical actin enhanced CME in circular-shaped cells. Additionally, since the cortical actin also takes effect on dynamin during endocytic membrane’ separation [37], the dynamin also been inhibited by dynasore treatment. The decreased cellular uptake capacity after dynasore treatment also indicated the cortical actin is necessary for the final step of CME. Furthermore, it can be predicted that the cell density-independent fluorescence intensity after dynasore treatment indicated the intermediate function of dynamin in cell density regulated cellular uptake capacity. In summary, the more endocytosed nanoparticles in colonies with larger size was related with more nanoparticles. Then, the higher cellular uptake capacity at the peripheral region of micropatterned colonies was mainly caused by uneven distribution of nanoparticles. On the other hand, the higher cell density enhanced cellular uptake capacity was reasoned by the cortical actin enhanced CME in circular-shaped cells with higher cell density. 5. Conclusions In this research, PDMS stencils were prepared and applied to control morphologies of melanoma colonies and the different cellular uptake capacity of colonies with different size or cells at different location of micropatterned colony have been revealed. At first, the enhanced cellular uptake capacity of colonies with larger size was related with more nanoparticles was dropped. In addition, With the effect of a meniscus, cells and endocytosed nanoparticles were predominately located at a peripheral region of micropatterned colonies. Furthermore, cellular uptake capacity was positively related to cell density. It has been demonstrated as a result of cortical actin accelerated CME in circular-shaped cells. Conflicts of interest There are no conflicts to declare. Acknowledgment This research was supported by Foundation of Shaanxi University of Science and Technology (Grant No. 126021993 and 126021823) and Natural Science Foundation of Shaanxi Province in China (Grant No. 2021JQ-545 and 2021JQ-536). Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References [1] Yang, Y., Wang, X., Huang, T. C., Hu, X. , et al. , Regulation of mesenchymal stem cell functions by micro-nano hybrid patterned surfaces. Journal of Materials Chemistry B 2018, 6 , 5424-5434. [2] Lane, S. W., Williams, D. A., Watt, F. M., Modulating the stem cell niche for tissue regeneration. Nature Biotechnology 2014, 32 , 795-803. [3] Emon, B., Bauer, J., Jain, Y., Jung, B., Saif, T., Biophysics of Tumor Microenvironment and Cancer Metastasis - A Mini Review. Computational and Structural Biotechnology Journal 2018, 16 , 279-287. [4] Huang, G., Li, F., Zhao, X., Ma, Y. , et al. , Functional and Biomimetic Materials for Engineering of the Three-Dimensional Cell Microenvironment. Chemical Reviews 2017, 117 , 12764-12850. [5] Falconnet, D., Csucs, G., Grandin, H. M., Textor, M., Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27 , 3044-3063. [6] Yang, Y., Wang, X., Wang, Y., Hu, X. , et al. , Influence of Cell Spreading Area on the Osteogenic Commitment and Phenotype Maintenance of Mesenchymal Stem Cells. Scientific Reports 2019, 9 , 6891. [7] Yang, Y., Wang, X., Hu, X., Kawazoe, N. , et al. , Influence of Cell Morphology on Mesenchymal Stem Cell Transfection. ACS Applied Materials & Interfaces 2019, 11 , 1932-1941. [8] Wang, Y., Yang, Y., Yoshitomi, T., Kawazoe, N. , et al. , Regulation of gene transfection by cell size, shape and elongation on micropatterned surfaces. Journal of Materials Chemistry B 2021, 9 , 4329-4339. [9] Bao, M., Xie, J., Piruska, A., Huck, W. T. S., 3D microniches reveal the importance of cell size and shape. Nature Communications 2017, 8 , 1962. [10] Wang, Y., Yang, Y., Wang, X., Yoshitomi, T. , et al. , Micropattern-controlled chirality of focal adhesions regulates the cytoskeletal arrangement and gene transfection of mesenchymal stem cells. Biomaterials 2021, 271 , 120751. [11] Hoang, P., Ma, Z., Biomaterial-guided stem cell organoid engineering for modeling development and diseases. Acta Biomaterialia 2021, 132 , 23-36. [12] Lee, L. H., Peerani, R., Ungrin, M., Joshi, C. , et al. , Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages. Stem Cell Research 2009, 2 , 155-162. [13] Au - Wisniewski, D., Au - Lowell, S., Au - Blin, G., Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging. JoVE 2019, e59634. [14] Sahni, G., Yuan, J., Toh, Y. C., Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates. J Vis Exp 2016. [15] Sahni, G., Chang, S. Y., Meng, J. T. C., Tan, J. Z. Y. , et al. , A Micropatterned Human-Specific Neuroepithelial Tissue for Modeling Gene and Drug-Induced Neurodevelopmental Defects. Advanced Science (Weinh) 2021, 8 , 2001100. [16] Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D., Brivanlou, A. H., A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nature Methods 2014, 11 , 847-854. [17] Lee, J., Abdeen, A. A., Wycislo, K. L., Fan, T. M., Kilian, K. A., Interfacial geometry dictates cancer cell tumorigenicity. Nature Materials 2016, 15 , 856-862. [18] Lee, J., Molley, T. G., Seward, C. H., Abdeen, A. A. , et al. , Geometric regulation of histone state directs melanoma reprogramming. Communications Biology 2020, 3 , 341. [19] Deglincerti, A., Etoc, F., Guerra, M. C., Martyn, I. , et al. , Self-organization of human embryonic stem cells on micropatterns. Nature protocols 2016, 11 , 2223-2232. [20] Morgani, S. M., Metzger, J. J., Nichols, J., Siggia, E. D., Hadjantonakis, A.-K., Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning. eLife 2018, 7 , e32839. [21] Savjani, K. T., Gajjar, A. K., Savjani, J. K., Drug solubility: importance and enhancement techniques. ISRN pharmaceutics 2012, 2012 , 195727-195727. [22] Iversen, T.-G., Skotland, T., Sandvig, K., Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011, 6 , 176-185. [23] Subramaniam, A., Sethuraman, S., Chapter 18 - Biomedical Applications of Nondegradable Polymers, in: Kumbar, S. G., Laurencin, C. T., Deng, M. (Eds.), Natural and Synthetic Biomedical Polymers , Elsevier, Oxford 2014, pp. 301-308. [24] Choi, J. H., Lee, H., Jin, H. K., Bae, J.-s., Kim, G. M., Micropatterning of neural stem cells and Purkinje neurons using a polydimethylsiloxane (PDMS) stencil. Lab on a Chip 2012, 12 , 5045-5050. [25] Wang, Y., Yang, Y., Wang, X., Kawazoe, N. , et al. , The varied influences of cell adhesion and spreading on gene transfection of mesenchymal stem cells on a micropatterned substrate. Acta Biomaterialia 2021, 125 , 100-111. [26] Merrifield, C. J., Feldman, M. E., Wan, L., Almers, W., Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nature Cell Biology 2002, 4 , 691-698. [27] Grassart, A., Cheng, A. T., Hong, S. H., Zhang, F. , et al. , Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis. Journal of Cell Biology 2014, 205 , 721-735. [28] Macia, E., Ehrlich, M., Massol, R., Boucrot, E. , et al. , Dynasore, a Cell-Permeable Inhibitor of Dynamin. Developmental Cell 2006, 10 , 839-850. [29] Freshney, R. I., Problem Solving, Culture of Animal Cells 2010, pp. 569-591. [30] Wang, X., Hu, X., Li, J., Russe, A. C. , et al. , Influence of cell size on cellular uptake of gold nanoparticles. Biomaterials Science 2016, 4 , 970-978. [31] Khetan, J., Shahinuzzaman, M., Barua, S., Barua, D., Quantitative Analysis of the Correlation between Cell Size and Cellular Uptake of Particles. Biophysical Journal 2019, 116 , 347-359. [32] Galletta, B. J., Mooren, O. L., Cooper, J. A., Actin dynamics and endocytosis in yeast and mammals. Current Opinion in Biotechnology 2010, 21 , 604-610. [33] Chaudhuri, A., Battaglia, G., Golestanian, R., The effect of interactions on the cellular uptake of nanoparticles. Physical Biology 2011, 8 , 046002. [34] Jiang, X., Dausend, J., Hafner, M., Musyanovych, A. , et al. , Specific Effects of Surface Amines on Polystyrene Nanoparticles in their Interactions with Mesenchymal Stem Cells. Biomacromolecules 2010, 11 , 748-753. [35] Tinevez, J. Y., Schulze, U., Salbreux, G., Roensch, J. , et al. , Role of cortical tension in bleb growth. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 , 18581-18586. [36] Lu, L., Oswald, S. J., Ngu, H., Yin, F. C., Mechanical properties of actin stress fibers in living cells. Biophysical Journal 2008, 95 , 6060-6071. [37] Loebrich, S., The role of F-actin in modulating Clathrin-mediated endocytosis: Lessons from neurons in health and neuropsychiatric disorder. Communicative & Integrative Biology 2014, 7 , e28740. Information & Authors Information Version history V1 Version 1 31 January 2024 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords biomaterials biophysics cell biology cellular uptake drug discovery mammalian cells micropattern morphological cues organoids science communication Authors Affiliations Siyuan Huang Shaanxi University of Science and Technology View all articles by this author Yingjun Yang 0000-0003-2746-1192 [email protected] Shaanxi University of Science and Technology View all articles by this author Xiaoqiang Hou Shaanxi University of Science and Technology View all articles by this author Jingyi Chen Shaanxi University of Science and Technology View all articles by this author Guanjian Nie Shaanxi University of Science and Technology View all articles by this author Bingshe Xu Shaanxi University of Science and Technology View all articles by this author Shukai Ding Shaanxi University of Science and Technology View all articles by this author Metrics & Citations Metrics Article Usage 194 views 122 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Siyuan Huang, Yingjun Yang, Xiaoqiang Hou, et al. 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