A. Formation of polymeric micelles

Polymeric micelles that are spontaneously formed by self-assembly of amphiphilic macromolecules have demonstrated a variety of favorable properties including biocompatibility, longevity, high stability, the capacity to effectively solubilize a variety of poorly soluble drugs, triggered drug release, and the ability to accumulate at the site of interest due to the EPR effect.3131. S. Movassaghian, O. M. Merkel, and V. P. Torchilin, Nanomed. Nanobiotechnol. 7, 691 (2015). https://doi.org/10.1002/wnan.1332 Moreover, the shell of the micelles further allows the possibility of decorating the surface with targeting moieties to promote the development of smart multifunctional micelles.3232. A. Varela-Moreira, Y. Shi, M. H. A. M. Fens, T. Lammers, W. E. Hennink, and R. M. Schiffelers, Mater. Chem. Front. 1, 1485 (2017). https://doi.org/10.1039/C6QM00289G In this work, P(HPMA22-co-MAA10)-b-PMMA50 was synthesized and micelles were prepared by dialysis of the polymer against Milli-Q water. The DLS measurements revealed the hydrodynamic diameters of the blank micelles to 28.82 nm with a polydispersity value of 0.34 [Fig. 3(a)], while the zeta-potential of colloidal suspension was around −10.9.WST-1 works by the reduction of the stable tetrazolium salt WST-1 to produce water-soluble formazan by cellular dehydrogenases on the cell surface. The generation of the dark yellow formazan dye can be quantified at 420 – 480 nm (optimal at 440 nm) with a spectrophotometer and is directly correlated to the number of viable cells. The WST-1 assay was performed to confirm the cell viability, and the results showed that the cells displayed high viability after treatment with the HPMA micelles at the studied concentrations [Fig. 3(b)]. In this work, the micelles did not produce any cell toxicity after 72 h of incubation even when the concentration reached 1 mg/ml. We measured the toxicity of HPMA micelles to the fibroblasts, and we found that the micelles are nontoxic to Hs-27 and MRC-5 cells (Fig. S1 in the supplementary material).5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1.

B. Characterization of AzPhPVA

PVA was chosen as a nonbiofouling material because of its superior ability in protein adsorption resistance and cell adhesion in an aqueous culture medium.3333. P. Pavli, P. S. Petrou, A. M. Douvas, D. Dimotikali, S. E. Kakabakos, and P. Argitis, ACS Appl. Mater. Interfaces 6, 17463 (2014). https://doi.org/10.1021/am5053224 The photoreactive polymer was synthesized by coupling the hydroxyl groups of PVA with 4-azidobenzoic acid via a typical esterification reaction. The introduction of photoreactive azido groups in AzPhPVA was confirmed by 1H NMR analysis, as shown in Fig. 1(b). The appearance of peaks assigned to the azidophenyl protons in photoreactive PVA was at around 7.2 and 8.0 ppm, and that of methylene protons was at 1.4 ppm. The percentage of the hydroxyl groups in PVA coupled with the azidophenyl groups determined by peak integration was 2.3%.

C. PVA-micropatterned polystyrene surfaces

Photolithography is a process of transferring geometric features drawn on a mask to the surface of a wafer or substrate via ultraviolet illumination.3434. F. L. Yap and Y. Zhang, Biosens. Bioelectron. 22, 775 (2007). https://doi.org/10.1016/j.bios.2006.03.016 The mask is generally made of a quartz/glass plate coated with a thin layer of nontransparent chromium. Herein, the synthesized AzPhPVA was immobilized on cell-culture polystyrene plates to facilitate the creation of surfaces with spatial control of their nonfouling properties. Upon UV irradiation, the photolyzed azidophenyl groups in AzPhPVA generated a highly reactive intermediate, phenyl nitrene, which can react with neighboring atoms to form a covalent bond. Thus, AzPhPVA can be cross-linked to the cell-culture plate using UV light.The PVA-coated plates were covered with a photomask composed of microdots with diameters of 20, 30, 40, and 50 μm to control the spreading area of cells (Fig. 4). Micropatterns of different shapes (round, triangle, and square) were used to control cell shapes (Fig. 4), with the shapes having an equal area of 1256 μm2. The photomask used in this work consists of UV-nontransparent dark microdots and surrounding UV-transparent domains. The AzPhPVA in the UV-transparent areas was cross-linked and grafted onto the polystyrene surface. The photoreactive PVA covered by the chromium microdots that were protected from the UV light remained unreacted and could be washed away to support cell adhesion. Observation with an optical microscope demonstrated that the formation of a PVA micropattern on the polystyrene plate correlated with the sizes and shapes of the coated chrome photomask [Fig. 4(c)]. We measured the sizes of micropatterns and listed them in Table S1 in the supplementary material.5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1. The sizes are similar to the sizes of photomasks.

D. Cell adhesion on PVA-micropatterned PSt surface

MCF-7, A549, HS-27, and MRC-5 cells were seeded on the plate and distributed evenly across the whole plate. However, the cells only adhered to the cell-adhesive polystyrene regions and formed a micropatterned cell array because the PVA effectively prevented protein adsorption and cell adhesion.33,3533. P. Pavli, P. S. Petrou, A. M. Douvas, D. Dimotikali, S. E. Kakabakos, and P. Argitis, ACS Appl. Mater. Interfaces 6, 17463 (2014). https://doi.org/10.1021/am505322435. A. Mühlebach, B. Müller, C. Pharisa, M. Hofmann, B. Seiferling, and D. Guerry, J. Polym. Sci., Part A: Polym. Chem. 35, 3603 (1997). https://doi.org/10.1002/(SICI)1099-0518(19971130)35:16<3603::AID-POLA28>3.0.CO;2-I After 1 day of culture, cell adhesion and distribution were simultaneously regulated on the PVA-micropatterned polystyrene plate. The dot has an appropriate size for single-cell adhesion; therefore, most of the pits of micropatterns were occupied by a single cell. Only micropatterns with single cells were used for fluorescence analysis.

E. Influence of cell spreading on micelle uptake

Cell spreading areas can be systematically manipulated using micropatterns to study the effects of cell spreading on cell behaviors and functions.3636. I. Poudel, D. E. Menter, and J. Y. Lim, Biomed. Eng. Lett. 2, 38 (2012). https://doi.org/10.1007/s13534-012-0045-z The diameters of the chrome microdots on the photomasks were 20, 30, 40, and 50 μm with surface areas of 314, 707, 1256, and 1967 μm2, respectively. To compare the influence of spreading area on the cellular uptake of micelles, a semiquantitative analysis of the fluorescence images was performed to calculate the total fluorescence per cell and fluorescence intensity per unit area. In the MCF-7 cells, the fluorescence intensities per unit area were 23.52 ± 6.05, 29.84 ± 8.55, and 55.94 ± 10.92 for the 20-, 30-, and 40-μm micropatterns, respectively, while the total fluorescence intensities were 7296.88 ± 5492.17, 13665.71 ± 9068.01, and 55 332.66 ± 19 441.43, respectively. These results show that the cellular uptake of the polymer micelles, which is represented by the fluorescence intensity, increased significantly with an increase of the cell spreading area [Figs. 5(a) and 5(b)].Lung carcinoma A549 cells show that the fluorescence intensities per unit area for 20-, 30-, and 40-μm micropatterns were 28.03 ± 8.61, 38.50 ± 10.56, and 15.31 ± 4.60, respectively. The cells cultured on the 30-μm micropattern had a significantly higher cellular uptake than the 20- or 40-μm micropatterned cells. However, the total fluorescence intensities per cell were 6163.48 ± 3287.20, 9564.00 ± 4150.21, and 11 221.09 ± 6095.79 on micropatterns with 20–40 μm circles and the cellular uptake of A549 cells increased as the degree of cell spreading was enhanced [Figs. 6(a) and 6(b) and Fig. S2 in the supplementary material].5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1. While the fluorescence intensity per unit area represents an average of bright and dim pixels within the ROI, the integrated intensity can capture very bright and very dim pixels in the same object more accurately in accordance with their contribution to the biological phenomenon. It was previously reported that cell spreading favors cellular uptake due to large contact areas between cells and nanoparticles, which facilitated total cellular uptake.2121. X. Wang, X. Hu, J. Li, A. C. M. Russe, N. Kawazoe, Y. Yang, and G. Chen, Biomater. Sci. 4, 970 (2016). https://doi.org/10.1039/C6BM00171H This result is consistent with the other report in which flow cytometry measurements were carried out to identify the correlation between the size of the MDA-MB 231 breast cancer cells and their uptake of 100-nm nanoparticles.3737. J. Khetan, M. Shahinuzzaman, S. Barua, and D. Barua, Biophys. J. 116, 347 (2019). https://doi.org/10.1016/j.bpj.2018.11.3134 Although the trend of the cellular uptake of A549 cells was similar to that of MCF-7 cells, the former displayed higher cellular uptake than A549 cells. It is well understood that nanoparticle properties such as size, shape, stiffness, and surface chemistry play a crucial role in directing their interactions with cells.38−4038. J. Zhao and M. H. Stenzel, Polym. Chem. 9, 259 (2018). https://doi.org/10.1039/C7PY01603D39. P. Foroozandeh and A. A. Aziz, Nanoscale Res. Lett. 13, 339 (2018). https://doi.org/10.1186/s11671-018-2728-640. N. Ma, C. Ma, C. Li, T. Wang, Y. Tang, H. Wang, X. Moul, Z. Chen, and N. Hel, J. Nanosci. Nanotechnol. 13, 6485 (2013). https://doi.org/10.1166/jnn.2013.7525 Additionally, cell type is of importance in the endocytosis and fate of nanoparticles. Santos et al. suggested that the same nanoparticles might exploit different endocytic pathways to enter different cell types.4141. T. dos Santos, J. Varela, I. Lynch, A. Salvati, and K. A. Dawson, PLOS One 6, e24438 (2011). https://doi.org/10.1371/journal.pone.0024438 Likewise, in a comprehensive comparison of numerous cell lines, Lai et al. suggested that nanoparticle uptake is highly dependent on the cell type.4242. Y. Lai, P.-C. Chiang, J. D. Blom, N. Li, K. Shevlin, T. G. Brayman, Y. Hu, J. G. Selbo, and L. Hu, Nanoscale Res. Lett. 3, 321 (2008). https://doi.org/10.1007/s11671-008-9160-2 Differences in uptake efficiency can be explained by variations in metabolic activity and cell membrane composition of the tested cells. A previous study of maghemite–rhodium citrate nanoparticle uptake in breast cancer cells suggested that the high metabolic activity of tumor cells can lead to overexpression of surface receptors such as clathrin, which can contribute to an increase in nanoparticle uptake in MCF-7 and MDA-MB-231 cells.4343. N. L. Chaves, I. Estrela-Lopis, J. Böttner, C. A. Lopes, B. C. Guido, A. R. de Sousa, and S. N. Báo, Int. J. Nanomed. 12, 5511 (2017). https://doi.org/10.2147/IJN.S141582 This variation in nanoparticle uptake among the different cell lines has important implications for designing nanoparticle-based drug delivery systems.Besides cancerous cells, Hs-27 and MRC-5 fibroblasts were also used to compare the effect of different spreading areas on the cellular uptake of normal cells. Due to the larger size of the fibroblasts compared with that of MCF-7 and A549 cells, 30-, 40-, and 50-μm micropatterns were used to control the spreading of fibroblasts. The quantitative analysis of the fluorescence images of HS-27 cells showed that the total cellular uptake of micelles significantly decreased an the increase of the spreading area. The fluorescence intensities per unit area were 29.57 ± 10.89, 15.11 ± 5.69, and 7.09 ± 3.02 for 30-, 40-, and 50-μm micropatterned cells, respectively. Likewise, the total fluorescence intensities per cell of 30-, 40-, and 50-μm micropatterns were 12 040.72 ± 7129.78, 7060.66 ± 4537.71, and 5291.70 ± 3239.16, as shown in Figs. 6(c) and 6(d) and Fig. S3 in the supplementary material.5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1. Consequently, small cells had a significantly higher cellular uptake than cells with large sizes. The results indicated that the size of a single HS-27 cell could affect the cellular uptake of micelles.Similar uptake profiles have been obtained for MRC-5 cells, as the result indicates that cell fluorescence decreased as the degree of cell spreading was increased. The mean fluorescence intensities were 86.12 ± 30.16, 48.90 ± 16.12, and 46.02 ± 14.96 for the 30, 40, and 50 micropatterns, respectively [Fig. 6(e) and Fig. S4 in the supplementary material].5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1. The trend of the mean fluorescence intensity of this cell line was similar to that of HS-27 cells. However, the fluorescence intensity per cell of MRC-5 were 17 570.16 ± 9786.60, 20 753.19 ± 9250.81 and 20 295.62 ± 11 922.73 for 30, 40 and 50 μm micropatterns, respectively [Fig. 6(f)]. These results were in good agreement with previous studies on the cellular uptake of PEG-AuNPs in which a large hMSC spreading area resulted in high membrane tension at the cell periphery region. That, in turn, required high membrane deformation energy for engulfing nanoparticles and hence reduced the cellular uptake of nanoparticles.21,4421. X. Wang, X. Hu, J. Li, A. C. M. Russe, N. Kawazoe, Y. Yang, and G. Chen, Biomater. Sci. 4, 970 (2016). https://doi.org/10.1039/C6BM00171H44. S. Zhang, H. Gao, and G. Bao, ACS Nano 9, 8655 (2015). https://doi.org/10.1021/acsnano.5b03184 In comparison with HS-27, the cellular uptake obtained with MCR-5 cells was higher than that obtained with HS-27 cells. This comparable cellular uptake between both cell lines suggested a difference in sensitivity of cells toward the same nanoparticles. These results were consistent with the findings of Sahu et al., who also reported differential sensitivity of human lung epithelial (L-132) and human monocyte (THP-1) cell lines toward particles of the same composition.4545. D. Sahu, G. M. Kannan, M. Tailang, and R. Vijayaraghavan, J. Nanosci. 2016, 4023852. https://doi.org/10.1155/2016/4023852The data of four cell lines indicated that the cellular uptake of HPMA-based micelles was positively correlated with the cell size or spreading area in both MCF-7 and A549 cell lines but negatively correlated with cell size in HS-27 and MRC-5 cell lines. Plasma membrane tension plays a key role in the endocytic process. Thottacherry et al. previously reported that decreasing tension by the stimulation of secretion or addition of amphiphilic compounds increases endocytosis, while an increase in membrane tension results in a decrease in endocytic processes.4646. J. J. Thottacherry et al., Nat. Commun. 9, 4217 (2018). https://doi.org/10.1038/s41467-018-06738-5 Our results indicated that the membrane tension was increased by increasing the cell spreading area in normal cells, which in turn resulted in decreased cellular uptake of nanoparticles. On the other hand, Elkin et al. assumed that many components of the endocytic machinery are altered in cancer cells to enhance their proliferative and metastatic potential.4747. S. R. Elkin, N. Bendris, C. R. Reis, Y. Zhou, Y. Xie, K. E. Huffman, J. D. Minna, and S. L. Schmid, Cancer Res. 75, 4640 (2015). https://doi.org/10.1158/0008-5472.CAN-15-0939 Consequently, these findings could explain the two opposite uptake behaviors in normal and cancer cells. Moreover, the results would shed light on the influence of the cell spreading on nanoparticle uptake behavior.

F. Influence of cell shape on micelle uptake

Besides cell spreading, the effect of cell shape on the uptake of micelles was also investigated in this work. To manipulate cell shapes, three types of micropatterns with geometries of round, square, and triangles with the same spreading area of 1256 μm2 were prepared. MCF-7, A549, HS-27, and MRC-5 cells were adhered and spread following the underlying geometric micropatterns. In MCF-7 cells, the fluorescence intensities per unit area for round, triangle, and square micropatterns were 55.037 ± 12.313, 22.50 ± 6.61, and 10.72 ± 3.50, respectively, while the fluorescence intensities per cell were 52 022.6 ± 23 393.4, 21 738.14 ± 9917.16, and 10 667.38 ± 4346.69, respectively. As shown in Figs. 7(a) and 7(b), the circular micropattern favors micelle uptake in MCF-7.The effect of different cell shapes on micelle uptake in A549 cells is shown in Figs. 8(a) and 8(b) and Fig. S5 in the supplementary material.5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1. The fluorescence intensity per unit area shows that the cellular uptake of the tringle micropattern was 19.20 ± 8.06 significantly higher than round (15.30 ± 4.59) and square (12.77 ± 4.39) micropatterns. However, the fluorescence intensity per cell of the round shape( 111 221.09 ± 6095.79) showed high uptake relative to the cellular uptake of triangle (8608.08 ± 4901.14) and square (8618.30 ± 4750.79) shapes, which was consistent with MCF-7 cellular uptake behavior. The results suggest that shape can modulate the uptake of nanoparticles into MCF-7 and A549 cells, and round was the shape with the most efficient cellular uptake.Next, the influence of cell shape on micelle uptake of noncancerous cells was also investigated in HS-27 and MRC-5 cells. In HS-27 cells, the fluorescence intensities per unit area for round, triangle, and square micropatterns were 15.10 ± 5.68, 17.97 ± 6.23, and 22.92 ± 9.52, respectively, while the fluorescence intensities per cell for these three shapes were 7060.66 ± 4537.71, 10 002.25 ± 3409.46, and 15 776.95 ± 8988.03, respectively. The efficiency of cellular uptake of nanoparticles was found to rank in the following order from the lowest to the highest: round, triangle, and square [Figs. 8(c) and 8(d) and Fig. S6 in the supplementary material].5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1.Moreover, the effect of cell shapes on the cellular uptake in MRC-5 cells is shown in Figs. 8(e) and 8(f) and Fig. S7 in the supplementary material.5050. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001012 for Figures S1-S7 and Table S1. The fluorescence intensities per unit area for round, triangle, and square micropatterns were 48.90 ± 16.12, 46.91 ± 20.90, and 73.11 ± 30.85, respectively, while the fluorescence intensities per cell were 7060.66 ± 4537.71, 21 809.32 ± 10 040.9, and 33 445.38 ± 25 544.98, respectively. The square micropattern has a significantly higher cellular uptake than round and triangle micropatterns in MRC-5 cells.The data obtained from four types of cells, both cancerous and noncancerous cells, clearly indicated that the cell shape plays an important role in micelle uptake. The three types of geometric micropatterns (round, triangle, and square) have varying degrees of roundness. However, the round shape was shown to be beneficial for the cellular uptake in MCF-7 and A549 cells. This could be due to changes in the cellular tension, which depends on the cytoskeletal organization, in addition to the weakness of membrane–cytoskeleton linkages in round cells. On the other hand, HS-27 and MRC-5 fibroblasts cultured on square micropatterns showed higher micelle uptake in comparison with round and triangle micropatterns. The plasma membrane tension of mammalian cells is coordinated and maintained through the actin network, and this strongly influences endocytosis. He et al. highlighted that disruption of actin in some cell types promotes cellular uptake of specific molecules while having no effects or even inhibitory effects on others.4848. L. He, E. J. Sayers, P. Watson, and A. T. Jones, Sci. Rep. 8, 7318 (2018). https://doi.org/10.1038/s41598-018-25600-8 It was found that the reduced cortical tension resulting from actin disruption in A431 cells may favor the formation of invaginations and tubulation to allow entry of macromolecules.4848. L. He, E. J. Sayers, P. Watson, and A. T. Jones, Sci. Rep. 8, 7318 (2018). https://doi.org/10.1038/s41598-018-25600-8 Besides cell spreading area, cell shape could also modulate the uptake of nanoparticles into cancer and normal cells through various endocytic pathways. Another possible reason that induced the uptake difference among different cell types may be related to the metabolism. It has been reported that cell lines (which are derived from a tumor) display a high metabolic rate, a process that can respond to a higher cellular uptake of nanoparticles.4949. J. P. Peñaloza et al., J. Nanobiotechnol. 15, 1 (2017). https://doi.org/10.1186/s12951-016-0241-6