ABSTRACT The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery,

bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability,

cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses

2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs

isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel

avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic

understanding of organic synthesis and cell biology techniques. KEY POINTS * Light-driven free radical and photoinduced electron transfer-reversible addition–fragmentation chain-transfer

polymerization synthesis of structurally defined cellular polymers from biocompatible monomers in the presence of light-sensitive catalysts can modulate cell function and is a potential

anticancer therapy. * The protocol includes guidelines for the design of biotin and/or histidine-tagged polymers and streptavidin- and/or immobilized metal affinity chromatography-based

strategies for their isolation from cell lysate for downstream analysis. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS NANOCOMPARTMENT-CONFINED POLYMERIZATION IN LIVING SYSTEMS Article Open access 26 August 2023

APPLICATIONS OF SYNTHETIC POLYMERS DIRECTED TOWARD LIVING CELLS Article 20 June 2024 INTRINSICALLY FLUORESCENT POLYUREAS TOWARD CONFORMATION-ASSISTED METAMORPHOSIS, DISCOLORATION AND

INTRACELLULAR DRUG DELIVERY Article Open access 05 August 2022 DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author upon reasonable

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peptides. _Anal. Biochem._ 34, 595–598 (1970). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank the National Natural Science Foundation of China

(22071263), the Guangdong Province Zhujiang Talents Program (2019QN01Y127) and the Shenzhen Fundamental Research Program (JCYJ20200109110215774). We thank M. Galluzzi for the AFM

measurement. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China Mohamed Abdelrahim, Quan Gao, Yichuan

Zhang, Qi Xing & Jin Geng * School of Pharmacy, Henan University, Kaifeng, China Yichuan Zhang * Center for Molecular Metabolism, School of Environmental and Biological Engineering,

Nanjing University of Science and Technology, Nanjing, China Weishuo Li * Precision Healthcare University Research Institute, Queen Mary University of London, London, UK Mark Bradley Authors

* Mohamed Abdelrahim View author publications You can also search for this author inPubMed Google Scholar * Quan Gao View author publications You can also search for this author inPubMed 

Google Scholar * Yichuan Zhang View author publications You can also search for this author inPubMed Google Scholar * Weishuo Li View author publications You can also search for this author

inPubMed Google Scholar * Qi Xing View author publications You can also search for this author inPubMed Google Scholar * Mark Bradley View author publications You can also search for this

author inPubMed Google Scholar * Jin Geng View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.A. and Q.G. contributed equally to the work.

All authors contributed extensively to the work presented and wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Mark Bradley or Jin Geng. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Protocols_ thanks Greg Qiao, Dayong Yang and the other, anonymous, reviewer(s) for their contribution

to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. RELATED LINKS KEY REFERENCES USING THIS PROTOCOL Geng, J. et al. _Nat. Chem_. 11, 578–586 (2019): https://doi.org/10.1038/s41557-019-0240-y Zhang, Y. et al. _JACS Au_ 2,

579–589 (2022): https://doi.org/10.1021/jacsau.1c00373 EXTENDED DATA EXTENDED DATA FIG. 1 CONCENTRATION CALIBRATIONS OF DMA, HPMA, CA-CTA, AND EOSIN Y AND 1H NMR OF ISOLATED POLYMER. A,

UV-vis spectral representation of DMA at concentrations of 1, 5, 10, 20, and 50 mM. B, UV-vis spectra showcasing HPMA at concentrations of 2, 5, 10, 20, and 50 mM. C, UV-vis spectra of

CA-CTA at concentrations of 0.02, 0.1, 0.2, 0.5, and 1.0 mM. D, UV-vis spectral plots of Eosin Y at concentrations of 0.002, 0.01, 0.02, 0.05, and 0.1 mM. E, The graphical depiction of

cellular uptake data. This data was quantified utilizing UV-vis spectroscopy. F, G, 1H NMR spectra (recorded in D2O) of isolated polymers. F, Displays the 1H NMR spectrum for His-PDMA. The

upper spectrum represents the polymer when polymerized in PBS, whereas the lower spectrum is for the polymerized variant in cells. G, Showcases the 1H NMR spectrum for His-PHPMA. As with

(f), the upper graph pertains to the polymer processed in PBS, and the lower represents the cell-polymerized version. For both (f) and (g), the polymer isolated from PBS underwent a dialysis

process against water with a molecular weight cut-off (MWCO) of 1,000. On the other hand, the polymers derived from cell lysates were isolated using magnetic beads and then released at a

temperature of 60 °C. Panels a–e reproduced with permission from ref. 20, American Chemical Society. EXTENDED DATA FIG. 2 CHARACTERIZATIONS OF HIS-POLYMERS ISOLATED FROM HELA CELLS. PET-RAFT

polymerization was conducted within cells utilizing a hexa-histidine-tagged CTA known as His-CTA (for detailed polymerization cocktail compositions, refer to Table 1). Polymers synthesized

intracellularly were procured through dialysis (with a molecular weight cut-off, MWCO, of 1,000 Da) and IMAC isolation. The following polymers were characterized using 1H NMR (in d6-DMSO):

A, His-PHPMA-1. B, His-PHPMA-2. C, His-PDMA-3. D, His-PDMA-1. E, His-PDMA-2. F, His-PDMA-3. G, GPC traces for isolated polymers: His-PHPMA-1, His-PHPMA-2, and His-PHPMA-3. H, GPC traces for

isolated polymers: His-PDMA-1, His-PDMA-2, and His-PDMA-3. Figure adapted with permission from ref. 20, American Chemical Society. EXTENDED DATA FIG. 3 EXPLORATION OF INTRACELLULAR RETENTION

TIME OF MONOMERS, CTAS, AND EOSIN Y. Cells were exposed to PC1, supplemented with YFMA (1.0 µM) and RF-CTA (100 µM), for a duration of 4 h. Then, cells were washed and subjected to either a

10-minute illumination (+hv) or left without illumination (-hv). Representative confocal microscopy images are presented here to show the cellular state immediately after (0 h) and 24 hours

post-illumination. The columns, from left to right, depict the cellular uptake and distribution of Eosin Y, monomers, CTAs, and a merged view, respectively. The top two rows showcase cells

without illumination (- hν) and the bottom two rows present cells with illumination (+ hν). Scale bar = 50 µm. Figure reproduced with permission from ref. 20, American Chemical Society.

EXTENDED DATA FIG. 4 INTRACELLULAR POLYMERIZATION INDUCES CELL DEATH. A, Viability assessment of HeLa, 4T1, and MDA-MB-231 cells post-intracellular polymerization using the CCK-8 assay.

Cells underwent treatment with PC1 for a duration of 4 hours, were subsequently washed, and then illuminated for 10 minutes. This was followed by an incubation period of 24 hours. The

results are expressed as the mean ± SD, based on 6 independent samples per group. For statistical analysis, one-way ANOVA with Dunnett post-test was utilized, comparing to the untreated

cells. Significance levels are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, with indicating non-significance. B, Evaluation of apoptosis and

necroptosis levels induced by intracellular polymerization, determined using flow cytometry. The cells undergoing apoptosis are Annexin-V-positive and PI-negative, while those undergoing

necroptosis are indicated in the graph. Numerical analysis regarding the number of cells evaluated and the absolute numbers or percentages of the relevant cell populations within post-sort

fractions is provided in the figure. C, Quantitative representation of apoptosis and necroptosis levels from flow cytometry data presented in panel b. Data are presented as percentages of

cells undergoing apoptosis and necroptosis. Significance levels are indicated as: *P < 0.05, **P < 0.01, ****P < 0.0001, ns (not significant). D, E, Analysis of crucial biological

markers using techniques such as immunoblotting and immunoprecipitation assays. The displayed bands correspond to markers like PARP, C-PARP, p53, BCL-2, BAX, AIF, and γ-H2AX. The molecular

weight (in kDa) of each protein is indicated on the right. Figure adapted with permission from ref. 20, American Chemical Society. Source data EXTENDED DATA FIG. 5 INTRACELLULAR

POLYMERIZATION INHIBITED PROLIFERATION. A, Colony growth assay depicting the impact of intracellular polymerization on the proliferative ability of HeLa cells. Left panel shows cells treated

with PBS in the absence of light, while the right panel displays cells under similar conditions but with light exposure. The scale bar is indicative of 1 cm. B, Representation of cell cycle

phases for HeLa cells subjected to treatments with or without intracellular polymerization. The four groups depicted are cells treated with PBS in the absence and presence of light, and

cells treated with PC1 in the absence and presence of light. The percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle are presented. Data were based on n = 6 samples. C,

Immunoblotting assays highlight the expression levels of key cell cycle regulators, specifically Cyclin B1 and Cyclin E1, in HeLa cells under different treatment conditions. GAPDH serves as

the loading control, and the molecular weight (in kDa) of each protein is denoted on the right. Statistical evaluations were executed using one-way ANOVA accompanied by a Dunnett post-test

in comparison with the untreated control groups. Levels of significance are denoted as: *P < 0.05, ***P < 0.001, ****P < 0.0001, ns (not significant). Figure reproduced with

permission from ref. 20, American Chemical Society. Source data EXTENDED DATA FIG. 6 ANALYSIS OF CELL MOBILITY AND PHYSIOLOGICAL CHANGES INDUCED BY INTRACELLULAR POLYMERIZATION. A,

Representative wound-healing assay images capture cell mobilities across different treatment conditions and time points. Each image denotes the percentage of the area not covered by cells in

the wound area at the indicated time post-treatment. B, Quantitative representation of the wound-healing assay in A, presenting the normalized gap percentage over the incubation periods of

0, 24, 48, and 72 hours. Data are from n = 3 samples per condition. C, Immunoblotting assays showing the expression levels of cell-motility-associated proteins E-cadherin, Snail, and

Vimentin. GAPDH is used as the loading control, with the molecular weight (in kDa) for each protein mentioned on the right. D, Illustration of the viscosity p’obe’s molecular structure,

which is activated at an excitation/emission wavelength (_λ_ex/em) of 640/660 nm. The increase in viscosity corresponds to a higher fluorescence intensity. E, Flow cytometric analysis

detailing cellular viscosity across various treatment groups. The measurements employ a Cy5-based viscosity probe, with the fluorescence intensities depicted on the x-axis. F, Atomic force

microscopy results indicating the stiffness of cells subjected to different treatments. In all figures, data points represent individual measurements with mean values shown. Statistical

analysis was performed using one-way ANOVA with a Dunnett post-test compared to untreated control groups, ****P < 0.0001, ns (not significant). Panels a–e reproduced with permission from

ref. 20, American Chemical Society. Source data EXTENDED DATA FIG. 7 IN VIVO EVALUATION OF INTRACELLULAR POLYMERIZATION FOR CANCER TREATMENT. A–C, Tumor size progression in BALB/c nude mice

bearing different tumor models: HeLa (a), MDA-MB-231 (b), and 4T1 (c) over a specified number of days. Mice were subjected to different treatment regimens, and data are representative of n =

5 mice per group. Error bars represent for standard deviations. D, Histological analyses of tumor sections stained with hematoxylin/eosin (H&E), TUNEL, and Ki67. Each staining method

illuminates different features within the tumor tissue, including cellular morphology, apoptosis, and proliferation, respectively. Red arrowheads highlight areas of interest. E, F,

Quantitative evaluations of the tumor sections. The number of TUNEL-positive cells (E) and Ki67-positive cells (F) are represented. Cell numbers were quantified from six random areas within

the tissue, with each experiment conducted with n = 6 samples. Statistical evaluations were executed using one-way ANOVA accompanied by a Dunnett post-test in comparison with the untreated

control groups. Levels of significance are denoted as: *P < 0.05, ***P < 0.001, ****P < 0.0001, ns (not significant). G, Visual representation of the xenografted tumors at day 14

post various treatments, showcasing the size reduction in response to intracellular polymerization. H, Comprehensive hematoxylin and eosin (H&E) stained lung sections taken from mice on

day 19 post-treatment. These illustrations offer insights into potential metastatic spread, with metastatic sites demarcated by black dashed lines. Scale notations: For H&E images in

(d), the scale bar corresponds to 1 mm, whereas for the TUNEL and Ki67 images, the scale bar represents 200 μm and 2 mm in (h). Panels b–h reproduced with permission from ref. 20, American

Chemical Society. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1 and 2 and Table 1. REPORTING SUMMARY SOURCE DATA SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed

western blots. SOURCE DATA EXTENDED DATA FIG. 5 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots. RIGHTS AND PERMISSIONS Springer Nature or its licensor

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manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Abdelrahim,

M., Gao, Q., Zhang, Y. _et al._ Light-mediated intracellular polymerization. _Nat Protoc_ 19, 1984–2025 (2024). https://doi.org/10.1038/s41596-024-00970-8 Download citation * Received: 20

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