Phase separation enhanced drug delivery system for anti-cancer therapy

Phase separation enhanced drug delivery system for anti-cancer therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Phase separation enhanced drug delivery system for anti-cancer therapy Xun Bai, Pengfei Pei, Guangfei Duan, Long Chen, Song Xue, Shi-Zhong Luo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1788552/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The cellular phase-separated condensates compartmentalize and concentrate biomolecules with distinct physicochemical properties, which has great potential for therapy purposes. However, the discovered phase separation phenomena in living organisms were restricted intracellularly, which limits the biomedical application of phase separation. Here, we designed a phase separation enhanced delivery system to specifically condensate molecules on the cell surface for efficient drug delivery. As a proof of concept, we demonstrated that an anti-cancer drug conjugate can selectively co-phase separate with the targeting component on cancer cell surface and efficiently kill the cells after internalization. Both cellular and in vivo assays showed more potency with this system than traditional antibody conjugates. The method provides insights in the application of phase separation as a powerful tool for therapeutic purpose. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Liquid-liquid phase separation (LLPS) has drawn increasing attention in understanding biological processes. Condensates formed via LLPS provide specific local environments for broad cellular activities 1 , 2 . With the elucidation of the function of LLPS within cells, utilizing this phenomenon to intervene cell activities has great potential for biomedical applications. Klein et al. reported that selective recruiting and concentration of small molecules into nuclear condensates contributes to drug pharmacodynamics 3 , demonstrating the possible application of LLPS in disease therapies. Until recently the discovered phase separation phenomena in living organisms were restricted intracellularly, as crowded environment inside of cells is critical for LLPS. This limits the prospect of LLPS in drug development. Our recent work demonstrated that cytokines like bFGF could undergo LLPS on cell surface with its receptor, which plays important role for effective signal transduction 4 . Inspired by the discovery, we realized that condensing functioning molecules on specific cells was possible by utilizing LLPS, which can be developed as a powerful biological tool for targeted delivery of massive number of molecules. In this report, we developed a P hase separation E nhanced drug D elivery S ystem (PEDS) for targeted, high-efficient delivery. As a proof of concept, we chose an anticancer drug as the payload and evaluated its phase separation and anticancer efficacy. The antibody drug conjugate (ADC), which conjugates monoclonal antibodies with antitumor cytotoxic molecules for specific cancer cell delivery, has been an emerging technique for cancer therapy 5 . ADCs have achieved great successes in clinical and several drugs have been approved by FDA 6 . However, one major challenge is the limitation of drug to antibody ratio (DAR) 7 , 8 due to the limited number of thiol- or amine- groups available on the antibody surface 9 . Increasing copies of drugs can also lead to heterogeneity or aggregation, which are problems for reagent preparation and efficacy evaluation 10 . The requirement of low DAR restricts the choice of the payloads within extremely toxic drugs in this technique, leading to a narrow therapeutic window and safety concerns 11 . Some groups have tried to develop new hydrophilic linkers with multivalency to reach a higher DAR and better efficacy, such as the Fleximer platform 12 . Alternatively, instead of developing a large complex ADC molecule with high DAR, we designed PEDS by utilizing LLPS of simple units, which can condensate into homogeneous coacervates containing mounts of payloads. (Fig. 1 A). PEDS contains two components, a targeting component (TC) and a drug component (DC), which can co-phase separate. Once the TC binds to its target on cell surface, the DC will be recruited and condensate with it via LLPS, possibly with other TC as “glue”. Since LLPS is a macroscopical phenomenon, the condensates are usually in around micrometer size, composed of millions of molecules, which results in great increase for local concentration of the payloads. The system is highly modular and each part of the components can be modified for the delivery of various molecules into different tissues, including the co-phase separation pair, linkers, the targeting molecule and the payload. In this report, for the anticancer drug delivery we fused an the single-chain variable fragment (scFv) of cetuximab, which targets epidermal growth factor receptor (EGFR) 13 , and a negatively charged peptide, poly glutamate (polyE) as TC. For DC, we used positive charged peptide, repeated glycine-lysine (polyGK), and chemically conjugated on the linkers with monomethyl auristatin F (MMAF), which is a comely used ADC payload 14 and blocks the polymerization of tubulin (Fig. 1 B). PolyE and polyGK are proved able to phase separate when mixed together, driven by the electrostatic interactions 15 . Once administrated, TC binds to the target protein on cell surface and DC phase separates with TC to condensate, and finally the condensates are spontaneously internalized into targeted cells mediated by EGFR, causing cell death. Results And Discussion The TC as a protein was expressed with the E.coli system and purified by metal affinity resin. It was chemically stained by Cyanine 3 (Cy3) monosuccinimidyl ester for imaging. To verify if phase separation occurs, in DC we firstly used enhanced yellow fluorescent protein (EYFP) as the pseudo payload for imaging purpose. This pseudo-DC as a protein was expressed and purified the same as above. To confirm the two components can co-phase separate, we mixed 10 µM TC-Cy3 and polyGK-EYFP in vitro (Fig. 2 ). Typical condensates emerged in the mixture, indicating LLPS occurs (Fig. 2 A). As comparisons, TC-Cy3 alone remains homogenous in one phase and polyGK-EYFP showed minimum phase separation. The turbidity assays of the samples were consistent with the images (Fig. 2 B). The fluorescence of the condensates recovered over time in fluorescence recovery after photobleaching (FRAP) experiment (Fig. 2 C), indicating their fluidity. Since the two components were designed to phase separate by electrostatic interactions, we confirmed this mechanism by using different salt concentrations (Fig. 2 D&E). The phase separation was inhibited by high concentration of salt, demonstrated by both turbidity and fluorescent imaging. These results indicated that electrostatic interactions from polyE and polyGK of each component do drive the phase separation. Next, we examined whether the phase separation occurs on cell surface. The epidermoid carcinoma cell line A431, which expresses high level of EGFR was used 16 . The two fluorescence labeled components were added together or separately in cell culture medium and the condensate formation was observed under confocal microscope (Fig. 3 A). Each component alone did not show phase separation. However, when mixed together the condensates formed on the cell surface and both were nicely colocalized. Notably, since the critical concentration of LLPS is lowered on membrane 17 , only 1 µM of each component demonstrated significant LLPS on cell surface while in solution the condensates were barely observed. To test its specificity, two other cell lines, the HeLa cell line expressing medium level of EGFR 17 , and HEK-293 cell line expressing no EGFR were used. Less condensates were observed on the surface of HeLa cells and no condensates formed on HEK-293. The results clearly demonstrated that the two components phase separated in the presence of EFGR, indicating that they can selectively target to and condensate around EGFR. The FRAP experiment also confirmed the liquid-like properties of the condensates (Fig. 3 B). The payload needs to be internalized to take effects. Thus, we monitored the internalization of the condensates at different time points. Those condensates appeared on the cell surface immediately after adding to the medium and after 4 hours they were observed inside of the cells (Fig. 3 C). By using the EYFP as a pseudo payload, we are confident that the two-component delivery system can successfully phase separate on cell surface with selectively and get internalized into the cells. Next, the real payload was added and examine their abilities to induce cancer cell death. The cysteines on the surface of EYFP were used for drug conjugation. Maleimidocaproyl-MMAF (Mc-MMAF) was used to react with the thiol of the cysteine, affording the product. The copy number of MMAF was determined using Ellman's Reagent (5,5'-dithio-bis-[2-nitrobenzoic acid], DTNB) with free cysteine as the standard (Extended Data Fig. 2 ). One MMAF was conjugated to polyGK-EYFP, affording polyGK-EYFP-MMAF. Fluorescent imaging and FRAP results demonstrated that the drug conjugation did not change the phase separation behavior (Fig. 4 A&B). The cytotoxicity was evaluated by MTT assays. Firstly, A431 cells were treated with each component alone or together with 1:1 ratio. The mixture had significantly better efficacy of cytotoxicity than each component alone (Fig. 4 C). To confirm that the improvement is the synergistic effect causing by phase separation rather than the simply sum of each, we used isobologram to analyze their synergies (Fig. 4 D) 18 . By plotting the concentrations of each component at different ratios when cells have the 50% survival rate, we noticed that the combinations fell below the line connecting the IC50s of each component, implying positive synergistic effects. At the condition in which the molar ratio of TC: polyGK-EGFP-MMAF = 10: 3 in solution, the strongest synergistic effect occurs. The EYFG above is merely for visualization. In real drug development, a fluorescent protein is not required. Thus, the EYFP was substituted with a variable linker for drug conjugation. Here we chose the commonly used (GGGS) 3 as the linker and mutated one or two S to C for cysteine conjugation, generating two DCs, polyGK-MMAF and polyGK-MMAF 2 . The copy numbers were determined by Ellman's Reagent as well (Extended Data Fig. 2 ). The efficacy of the two versions of the DCs were examined by the MTT assay. The polyGK-EYFP-MMAF above is added for comparison. Moreover, a traditional ADC version by directly conjugating MMAF to the scFv of cetuximab as a bench marker. Ellman's Reagent assay showed that two MMAFs were conjugated, affording scFv-MMAF 2 . The polyGK-MMAF showed similar efficacy as polyGK-EYFP-MMAF, suggesting that EYFP does not affect the activity (Fig. 4 E). With two drug molecules conjugated, polyGK-MMAF 2 demonstrated higher potency than the single drug version, implying that the payload number is an important parameter for drug efficacy. However, even with two drugs conjugated, the traditional scFv-MMAF 2 is less active than any phase separation version of the drugs at the same payload concentrations. This is strong evidence that by involving LLPS in the system, the payloads can effectively condensate on the cell surface and increase the local concentration of drugs, resulting in better efficacy. Meanwhile, the toxicity of the delivery system in A431 and HEK-293 cells was tested. It demonstrated good selectivity between the two cell lines (Fig. 4 F). Finally, we examined the efficacy of the PEDS on an A431 cell-derived mouse xenograft model. A431 cells were transplanted under the skin of CD-1 nude mice. Since the phase separation enhanced system demonstrated tremendous efficacy in cell assays, a very low dose was chosen in the experiment. The mice were administrated with 0.5 mg/kg TC separately or in combination with 3/10 molar quantity of DCs, every four days (Fig. 5 A). The same dose of scFv-MMAF 2 as TC was used as a control representing current ADC modality. The tumor volume was recorded over time for 20 days. The dose of all samples was doubled at day 12 to better evaluate the efficacy of single MMAF conjugated TC. Without TC, the polyGK-MMAF 2 alone did not show inhibition of tumor growth compared with PBS. At such a low dose, scFv-MMAF 2 demonstrated some effects to prevent the tumor growing but with no significant statistical difference from the control group. However, the groups of getting PEDS treatment showed markedly reduced volume of the tumors (Fig. 5 B). The TC + polyGK-MMAF 2 demonstrated best potency. Notably, the molar numbers of MMAF dosed by PEDS is less than scFv-MMAF 2 , indicating that local concentration of the drug played critical role in the efficacy. All the groups showed no significant weight loss, indicating good safety of the system (Fig. 5 D). After the experiment, the tumors and normal tissues were fixed, paraffin-embedded, and sectioned and analyzed with HE or immunofluorescence staining (Fig. 5 D). HE stained tumor slices showed that the cell morphologies in PBS group and polyGK-MMAF 2 group are complete and full, but there are more dead cells in the other three groups, especially around the blood vessels. TUNLE immunofluorescence staining indicates that more apoptosis cells exist in the tumors getting PEDS treatment than scFv-MMAF 2 while almost no apoptosis occurred in control and DC alone groups. Those cells are around blood vessels as well. Ki67 staining revealed the malignant proliferation of the cancer cells. With the PEDS treatment, the proliferation was completely halted while scFv-MMAF 2 only showed partial inhibition. Moreover, routine blood test and HE staining of other organs were performed for the safety test of the system. The WBC (white blood cell number) of polyGK-MMAF 2 and scFv-MMAF 2 groups decreased, but without significant difference (Fig. S5A). Blood biochemical tests showed no significant difference of liver and kidney function indicators (Fig. S5B). The HE stained slices of major organs didn’t show damage (Fig. S5C). The results clearly demonstrated that the PEDS can minimize the side effects while remains the high efficiency in vivo . Conclusion In conclusion, we designed a two-component delivery system based on cell surface phase separation. The main scaffold is a pair of peptides which can undergo LLPS when mixed, conjugated with targeting molecules or payloads respectively. We demonstrated the efficacy of the system by delivering an anticancer drug. In this case, the TC contains an antibody derivative to selectively target the cancer cell and DC has a linker conjugated with cytotoxic drugs. Each component is relatively stable alone. After dosing, TC recognizes the receptors on cancer cell surface first and DC can phase separate with it, forming condensates with high concentrated drugs locally. Both in vitro assays and in vivo animal models demonstrated good potency and selectivity of the system. Comparing with the delivery system directly conjugating antibodies with drugs, the PEDS breaks through the limitation of DAR by separating the drugs from antibodies and inducing LLPS, which significantly increases the efficacy. Additionally, for the payload selection, super-toxic compounds are not necessary in this method since copy number is no longer a problem. As original designed, the system can be a universal deliver tool but not limited for anti-cancer drug delivery. The payload can be variable, from drugs to probes. The targeting part is subject to change to other binders even chemical compounds, only if they can selectively target on a certain type of cells, for the delivery to different tissues. Currently the discovered protein or peptide pairs which can undergo LLPS under physiological conditions are limited but as more discoveries in this area are made, more phase separation scaffold will be available. In summary, we provided a novel method by utilizing phase separation for selective delivery and demonstrated that the system has high efficacy and selectivity. We believe that the method has great potential in drug development as well as a promising tool in biological research. Methods Protein expression and purification The scFv-polyE (sequence see Supplementary Materials Extended Data Table 1) protein was expressed in E.coli origami2 (DE3) strain for better folding. The gene encoded His 6 -scFv-polyE was synthesized by General Biology (China) and cloned into pET-28a vector (Miao Ling Plasmid, China). The vector was then transformed into to E.coli (Origami2(DE3)) for expression. Cells were grown to the optical density of 0.7-0.9 at 37 ℃ and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for expression at 37℃ for 6 to 8 h. The cells were collected and lysed and protein was purified with His-Trap chelating column (GE Healthcare). The purified recombinant proteins were analyzed by Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) and desalted into the final storage buffer (ddH 2 O) by ÄKTA pure (General Electric, USA). Proteins were concentrated to 1 mg/ml using Ultra centrifugal filters. Aliquots were taken and stored at -80 ℃. PolyGK-EYFP was expressed in E.coli (NiCo21). The gene of polyGK-EYFP-His 6 was synthesized and cloned into pET-28a vector as described above. Cells were then grown to an optical density of 0.7-0.9 at 37 ℃ and induced with 0.5 mM IPTG, letting the protein express at 16℃ for 16 to 20 h. The protein was purified as above. Proteins were concentrated to 2 mg/ml using Ultra centrifugal filters. Aliquots were taken and stored at -80 ℃. The polyGK-linker proteins, including polyGK-(GGGS) 2 -GGGC and polyGK-GGGS-(GGGC) 2 , was expressed using the 6P-1 vector (Miao Ling Plasmid, China) with a glutathione S-transferase (GST) tag. The proteins were expressed in E.coli (NiCo21) and purified with GST Sefinose beads (GE Healthcare, USA). After washing, the PreScission Protease was used to cleave GST tags from the proteins, affording tag-free proteins. Proteins were concentrated to 2 mg/ml using Ultra centrifugal filters. Aliquots were taken and stored at -80 ℃. Cy3 staining of TC TC was stained with Cy3 monosuccinimidyl ester (ATT Bioquest, USA). The protein (scFv-polyE) was concentrated to 6 mg/mL using Ultra centrifugal filters. Cy3-NHS ester was mixed with scFv-polyE to a final concentration of 0.5 mg/ mL, with pH adjusted to 9 with 1M NaHCO 3 . The solution was incubated with shaking at 37℃ for 1 h, and then dialyzed with 10 kDa dialysis tube in ddH 2 O, affording Cy3 stained protein seed. The volume ratio of protein to seed was 1:200 - 1:100 when used. Phase separation imaging in vitro TC and polyGK-EYFP was mixed in PBS with the final concentration of 10 μM each. The mixed protein solution was immediately loaded into a 96-well plate and incubated at room temperature before imaging. The images were captured by Leica SP8 confocal microscopy with 100 x objective (oil immersion). Turbidity assay The preparation of the proteins is the same as imaging. Turbidity is measured by absorption at 620 nm in the 96-well plate using the Spectra ax M2 microplate reader (molecular device). All samples had three replicates (N=3). Cell culture Human embryonic kidney cells (HEK-293) and Human skin cancer cells (A431) and HeLa cells were cultured in complete medium containing 44.5% Dulbecco’s modified Eagle’s medium (DMEM) 1× with glucose (4.5 g/L), 44.5% Minimum Essential Medium (MEM) Alpha 1×, 10% fetal bovine serum (FBS), and 1% antibiotics (penicillin/streptomycin). Imaging of phase separation on the cell surface The cells were plated onto an 8-well Lab-Tek chamber glass (Thermos Fisher Science) and let grow to the density about 70%. Before imaging, the culture medium was discarded and the cells were washed with PBS twice. Then, 1 μM TC and 1 μM polyGK-EYFP (or polyGK-EYFP-MMAF) was added. The images were taken by Leica SP8 inverted microscope with 100 x objective lens, and the laser excitation wavelength was 405nm, 514nm and 552 nm, respectively. FRAP The cell samples were prepared as above and observed under an inverted microscope (LSM 780, Carl Zeiss, Germany) equipped with a confocal spinning disk unit (CSU-X1; Yokogawa, Tokyo, Japan) and a Zeiss 100× oil immersion lens. A field (approx. 0.06 μm for the formed condensate in vitro and 0.04 μm for the punctate of cell surface) and bleached for 5 ns with 512 nm and 405 lasers (1AU) at 100% laser power, respectively. After being photobleached, images were acquired at a rate of 0.97s (in solution) or 1.26s (on cell surface) for 500s. The fluorescence intensity of the bleached area was calculated with Zeiss Zen software. The signal is normalized with pre-bleached as 100% and 0 s after bleach as 0. At least three samples were analyzed and averaged to obtain FRAP curves by GraphPad Prism 8.0. MMAF conjugation Proteins, including polyGK-EYFP, polyGK-(GGGS) 2 -GGGC and polyGK-GGGS-(GGGC) 2 , are desalted into water to the concentration of 50 μM. 200 μM Mc-MMAF (Shanghai Rechemscience, China) stock in ddH 2 O was mixed with the protein solutions and incubated for 50 min. Then the mixture was dialyzed in ddH 2 O for 10 min twice. Ellman's Reagent assay 300 μL 10 μM of free cysteine, polyGK-EYFP, polyGK-(GGGS) 2 -GGGC, polyGK-GGGS-(GGGC) 2 or their MMAF conjugated products was mixed with 20 μL 5m M Ellman's Reagent in the working solution (0.1 M sodium phosphate, 1 mM EDTA, pH=8.0) and immediately analyzed by the plate reader. The absorbance at 412 nm was recorded every 1 min. The slopes were calculated using Graphpad prism 8.0 to indicate available free thiols used during the conjugation. Cytotoxicity test by MTT assay Cells were plated in 96-well plate (5000 cell/well) in DMEM with 10% FBS and allowed to adhere for 12 h at 37 ℃. The medium was changed to serum-free medium containing different concentrations (2, 4, 8, 16, 32, 62.5, 125, 250, 500 and 1000 nM) of TC and DCs. After 48 hours, the supernatant was removed, and 100 μL of MTT working solution was added to each well for another 4 h at 37℃, then 100 μL 10% SDS was added to each well to dissolve the purple crystals. The optical density (OD) at 570 nm was measured using SpectraMax M2 microplate reader (Molecular Devices, USA). Each group was performed with 5 replicates. The MTT working solution was prepared with MTT stock solution (5 mg/mL) and DMEM at a 1:9 ratio. DMEM was used as blank control. Synergy test by isobologram Based on the IC 50 of A431 cells treated by TC or polyGK-EYFP-MMAF alone, different concentrations of TC (25, 50, 100, 200 and 400 nM) and polyGK- EYFP-MMAF (15, 30, 60, 120 and 240 nM) were added to A431 cells respectively and analyzed by MTT assay. Each group was repeated for 3 times. DMEM was used as blank control. The concentrations of different combinations of TC and polyGK-EYFP-MMAF when the cells were at the survival rate of 50 % were plotted. The synergy of TC and polyGK-EYFP-MMAF was determined by the following equation, where A and B is the IC 50 of TC and polyGK-EYFP-MMAF respectively and a and b are the concentrations when they were dosed together for 50 % of cell survival. The numerical value of C classifies the combination as follows: C=1, additivity; C 1: subadditivity. Selectivity assay The selectivity evaluation was performed by treating A431 and HEK-293 cells with the ratio of TC: polyGK- MMAF=10:3, as described above. A431 was treated with scFv-MMAF 2 containing different concentrations (10, 20, 40 nM), as described above. Mouse xenograft models CD-1 nude mice were injected with cell suspension subcutaneously on the right back. Each mouse was inoculated with A431 cells (5×10 6 ). When the tumor volume grew to about 160 mm3, drug administration started. The drug was injected via tail vein (TC: 0.5 mg/kg or DC: 0.0175 mg/kg or a mixture of both or scFv-MMAF 2 : 0.5 mg/kg, doubled after 12 days) and PBS was the control group. Every four days for a total of 20 days, 3 animals per group. The weight, and the long diameter (A) and short diameter (B) of the tumor were measured by Vernier calipers every two days, and the tumor volume was approximated by the formula V= (A×B 2 )/2. After 20 days of administration, the mice were sacrificed by overdose anesthesia, and the tumors and normal tissues were dissected out and photographed. The dissected organs and tumors were fixed overnight with tissue fixative, and paraffin sections were prepared. HE staining, Ki67 and TUNLE immunofluorescence staining were performed on the tumor and organ sections, and the images were obtained with a fluorescence microscope. All animal care and experimental procedures were reported in accordance with the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences & Peking Union Medical College and complied with NIH Guidelines for the Care and Use of Laboratory Animals (approval number: SYXK 2017-0020). Routine blood and blood biochemical tests At the end of the treatment, blood of the mice was collected in EDTA anticoagulant tubes. Routine blood tests were taken immediately with a hematology analyzer (TEK-II MINI). Then another 1 mL blood was centrifuged at 3500 rpm for 10 min, the supernatant serum was collected for blood biochemistry test with an automatic biochemistry analyzer (Hitachi 7600). Declarations Acknowledgments: We thank Dr. Meng Qin for the help of animal experiments. The illustrations of mechanism were created with BioRender.com, and we thank Dr. Mingjia Yu for helping to preparing the illustrations. Funding: National Key R&D Program of China (2021YFC2103900), National Natural Science Foundation of China (22077010, 21907007) Author contributions Project administration: SZL, SX, LC Supervision: SZL, SX Concept conceiving: SX Experiment: XB, PP, GD Data analysis: SX, XB, PP, SZL Funding acquisition: SZL, SX Writing – original draft: XB, PP, SX, SZL Writing – review & editing: SX, SZL Competing interests The authors declare that they have no competing interests. Data and materials availability All data are available in the main text or the supplementary materials. Supplementary Materials Extended Data Figs. 1 to 3 Extended Data Table 1 References Su, X. et al. Phase separation of signaling molecules promotes T cell receptor signal transduction. 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Life Sci 58 , PL 23–28, doi: 10.1016/0024-3205(95)02271-6 (1996). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedData.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1788552","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":119326242,"identity":"6b1ea389-921b-4e92-8cfb-7641bc7cc589","order_by":0,"name":"Xun Bai","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xun","middleName":"","lastName":"Bai","suffix":""},{"id":119326243,"identity":"a30b6b41-155d-4a21-9fd2-75215535910e","order_by":1,"name":"Pengfei Pei","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Pei","suffix":""},{"id":119326244,"identity":"a720ac94-56f7-47ff-b8e2-9b1b28962da6","order_by":2,"name":"Guangfei Duan","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guangfei","middleName":"","lastName":"Duan","suffix":""},{"id":119326245,"identity":"e6a9e7c6-e7c4-415c-8e5d-db49f2484397","order_by":3,"name":"Long Chen","email":"","orcid":"","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Chen","suffix":""},{"id":119326246,"identity":"d8405edb-fff5-4487-b72c-2191e8f3408c","order_by":4,"name":"Song Xue","email":"","orcid":"https://orcid.org/0000-0002-7955-4529","institution":"Beijing University of Chemical Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Song","middleName":"","lastName":"Xue","suffix":""},{"id":119326247,"identity":"b28b7f1d-e5d3-41c5-b3a3-9e3622f12080","order_by":5,"name":"Shi-Zhong Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACPjBZwcDAxs7ABhVLwK8FouwMkMEMZhsQqYWxDUgQr0Ui+fDHn/O2yfMBtTzmqfnDwM+eY8Dwcwc+LWlpEpLbbhu2MTOwG/McM2CQ7HljwNh7Bp+WHDMGw223GYFa2KR5GwwYDG7kGDCDnYpbi/GHxDm37eFa7InQYiBxsOF2IsIWCUJaeJ6lSTYcu53cxszYbjjnmDGPxJlnBQd78WjhZweG2I+a27bz25uPPXhTIyfH35688cFPPFqQAGMDiOQBEQeI0jAKRsEoGAWjACcAAA06QmKZvRnBAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4880-5962","institution":"Beijing University of Chemical Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shi-Zhong","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2022-06-23 13:56:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1788552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1788552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23599890,"identity":"02e74657-fa43-41c3-a501-57f22df0ba8b","added_by":"auto","created_at":"2022-07-07 17:41:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":808278,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe schematic illustration of the mechanism of phase separation enhanced drug delivery system (PEDS).\u003c/strong\u003e\u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) The general design of PEDS. (B) The mechanism of delivering MMAF to cancer cells as a proof of concept of PEDS.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/26547133a6fa1fe873e2227c.png"},{"id":23599892,"identity":"b1b898d7-dabd-4dc7-96c7-677375697e52","added_by":"auto","created_at":"2022-07-07 17:41:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1482463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTC and DC can co-phase separate in vitro by electrostatic interactions.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Fluorescent images of TC-Cy3, pseudo DC with EYFP as the “payload” and their mixture, demonstrating that TC and DC can form condensates when mixed. Scale bar = 5 μm. (\u003cstrong\u003eB\u003c/strong\u003e) FRAP of the condensates showed the recovery. n=3 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eC\u003c/strong\u003e) Turbidity of the TC-Cy3, pseudo DC and their mixture. n=3 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eD\u003c/strong\u003e) Turbidity of the mixture with different concentrations of salt. n=3 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eE\u003c/strong\u003e) Fluorescent images of the mixture with different concentrations of salt. Scale bar = 5 μm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/491f28819ac9fc978fcd040a.png"},{"id":23600271,"identity":"223797e5-8d1d-4ca0-bbd5-d814abf24104","added_by":"auto","created_at":"2022-07-07 17:46:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2887953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe two-component delivery system can selectively phase separate on cell membrane.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Fluorescent images of TC-Cy3, pseudo DC and their mixture with A431 cells, HeLa and HEK-293 cells used as comparison. Scale bar = 5 μm. (\u003cstrong\u003eB\u003c/strong\u003e) FRAP of the condensates on cell surface. n=3 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eC\u003c/strong\u003e) Fluorescent images at 0 or 4 h after TC-Cy3 and pseudo DC treatment, showing condenses internalization.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/b3ba1621bc67432b6cc4a062.png"},{"id":23599894,"identity":"449da3cc-9449-41d5-9ccd-146d7aac26f1","added_by":"auto","created_at":"2022-07-07 17:41:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1175686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phase separation and efficacy in cells of the delivery system with MMAF as the payload.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Phase separation of TC and polyGK-EYFP-MMAF on cell surface. Scale bar = 5 μm. (\u003cstrong\u003eB\u003c/strong\u003e) FRAP of the condensates on cell surface, showing recovery. n=3 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eC\u003c/strong\u003e) The cytotoxicity of different concentrations of TC, polyGK-EYFP-MMAF and their 1:1 mixture to A431 cells, with free Mc-MMAF as the comparison. n=5 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eD\u003c/strong\u003e) Isobologram of the concentration combinations of the two components when half of the cells were killed, demonstrating positive synergy. (\u003cstrong\u003eE\u003c/strong\u003e) Dose dependent curves of cytotoxicity to A431 cells for different MMAF conjugates, with TC : MMAF conjugates =10:3 in molar ratio if TC is used. n=5 biologically independent samples, data are presented as mean values +/- SEM. (\u003cstrong\u003eF\u003c/strong\u003e) Dose dependent curves of TC and polyGK-MMAF treatment for A431 and HEK-293 cells, demonstrating the selectivity. n=5 biologically independent samples, data are presented as mean values +/- SEM.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/f0b395b5c52490e75b3987cc.png"},{"id":23599891,"identity":"ee54f191-0003-4e98-80ad-94bb11fb386a","added_by":"auto","created_at":"2022-07-07 17:41:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2469496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMouse xenograft models testing the efficacy of PEDS.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) The schematic illustration of xenograft experiment. (\u003cstrong\u003eB\u003c/strong\u003e) Tumor volume from 0 to 20 days of treatment. n=3 randomized samples, data are presented as mean values +/- SEM. Comparisons among groups were performed using Two-Way ANOVA. Statistics: PBS vs. TC+polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e, p= 0.0196, 0.0080, 0.0055, 0.0042, 0.0003 and \u0026lt;0.0001 on day 10, 12, 14, 16, 18 and 20; PBS vs. TC+polyGK-MMAF, p=0.0348, 0.1660, 0.1263, 0.0245 and 0.0013 on day 12, 14, 16, 18 and 20; polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e vs. TC+polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e, p=0.0173, 0.0424, \u0026lt;0.0001, 0.0011, 0.0002 on day 12, 14, 16, 18 and 20;\u0026nbsp;scFv-MMAF\u003csub\u003e2\u003c/sub\u003e vs. TC+polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e, p=0.0153, 0.1964, 0.0086, 0.0070 on day 14, 16, 18 and 20. (\u003cstrong\u003eC\u003c/strong\u003e) Tumor images after sacrificing the mice on day 20. n=3 randomized samples. (\u003cstrong\u003eD\u003c/strong\u003e) Body weights of the mice from 0 to 20 days of treatment. n=3 randomized samples, data are presented as mean values +/- SEM. Comparisons among groups were performed using Two-Way ANOVA. No significance between the groups. (\u003cstrong\u003eE\u003c/strong\u003e) Staining of the tumor slides. HE staining showed the cell morphologies indicating cell damage. Ki67 demonstrated the cancer cell proliferation. TUNEL indicates cell apoptosis. Scale bar=50 μm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/fdd4ed566bfe8fe644298f43.png"},{"id":25032537,"identity":"3f235817-b85f-47bc-ab44-28063cde51e8","added_by":"auto","created_at":"2022-08-10 12:32:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3452196,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/2b34d588-4e1e-44e0-b5ed-a1fb6cf3aaa2.pdf"},{"id":23600272,"identity":"25c63542-8c2e-4950-a2cb-8a08d99e439a","added_by":"auto","created_at":"2022-07-07 17:46:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2413810,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-1788552/v1/b3759b6212a4aa90fc4b23c3.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Phase separation enhanced drug delivery system for anti-cancer therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiquid-liquid phase separation (LLPS) has drawn increasing attention in understanding biological processes. Condensates formed via LLPS provide specific local environments for broad cellular activities\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. With the elucidation of the function of LLPS within cells, utilizing this phenomenon to intervene cell activities has great potential for biomedical applications. Klein \u003cem\u003eet al.\u003c/em\u003e reported that selective recruiting and concentration of small molecules into nuclear condensates contributes to drug pharmacodynamics\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, demonstrating the possible application of LLPS in disease therapies. Until recently the discovered phase separation phenomena in living organisms were restricted intracellularly, as crowded environment inside of cells is critical for LLPS. This limits the prospect of LLPS in drug development. Our recent work demonstrated that cytokines like bFGF could undergo LLPS on cell surface with its receptor, which plays important role for effective signal transduction\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Inspired by the discovery, we realized that condensing functioning molecules on specific cells was possible by utilizing LLPS, which can be developed as a powerful biological tool for targeted delivery of massive number of molecules. In this report, we developed a \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eP\u003c/span\u003ehase separation \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eE\u003c/span\u003enhanced drug \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eD\u003c/span\u003eelivery \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eystem (PEDS) for targeted, high-efficient delivery. As a proof of concept, we chose an anticancer drug as the payload and evaluated its phase separation and anticancer efficacy.\u003c/p\u003e \u003cp\u003eThe antibody drug conjugate (ADC), which conjugates monoclonal antibodies with antitumor cytotoxic molecules for specific cancer cell delivery, has been an emerging technique for cancer therapy\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. ADCs have achieved great successes in clinical and several drugs have been approved by FDA\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, one major challenge is the limitation of drug to antibody ratio (DAR)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e due to the limited number of thiol- or amine- groups available on the antibody surface\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Increasing copies of drugs can also lead to heterogeneity or aggregation, which are problems for reagent preparation and efficacy evaluation\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The requirement of low DAR restricts the choice of the payloads within extremely toxic drugs in this technique, leading to a narrow therapeutic window and safety concerns\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Some groups have tried to develop new hydrophilic linkers with multivalency to reach a higher DAR and better efficacy, such as the Fleximer platform\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Alternatively, instead of developing a large complex ADC molecule with high DAR, we designed PEDS by utilizing LLPS of simple units, which can condensate into homogeneous coacervates containing mounts of payloads. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). PEDS contains two components, a targeting component (TC) and a drug component (DC), which can co-phase separate. Once the TC binds to its target on cell surface, the DC will be recruited and condensate with it via LLPS, possibly with other TC as \u0026ldquo;glue\u0026rdquo;. Since LLPS is a macroscopical phenomenon, the condensates are usually in around micrometer size, composed of millions of molecules, which results in great increase for local concentration of the payloads.\u003c/p\u003e \u003cp\u003eThe system is highly modular and each part of the components can be modified for the delivery of various molecules into different tissues, including the co-phase separation pair, linkers, the targeting molecule and the payload. In this report, for the anticancer drug delivery we fused an the single-chain variable fragment (scFv) of cetuximab, which targets epidermal growth factor receptor (EGFR)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and a negatively charged peptide, poly glutamate (polyE) as TC. For DC, we used positive charged peptide, repeated glycine-lysine (polyGK), and chemically conjugated on the linkers with monomethyl auristatin F (MMAF), which is a comely used ADC payload\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and blocks the polymerization of tubulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). PolyE and polyGK are proved able to phase separate when mixed together, driven by the electrostatic interactions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Once administrated, TC binds to the target protein on cell surface and DC phase separates with TC to condensate, and finally the condensates are spontaneously internalized into targeted cells mediated by EGFR, causing cell death.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eThe TC as a protein was expressed with the \u003cem\u003eE.coli\u003c/em\u003e system and purified by metal affinity resin. It was chemically stained by Cyanine 3 (Cy3) monosuccinimidyl ester for imaging. To verify if phase separation occurs, in DC we firstly used enhanced yellow fluorescent protein (EYFP) as the pseudo payload for imaging purpose. This pseudo-DC as a protein was expressed and purified the same as above.\u003c/p\u003e \u003cp\u003eTo confirm the two components can co-phase separate, we mixed 10 \u0026micro;M TC-Cy3 and polyGK-EYFP \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Typical condensates emerged in the mixture, indicating LLPS occurs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As comparisons, TC-Cy3 alone remains homogenous in one phase and polyGK-EYFP showed minimum phase separation. The turbidity assays of the samples were consistent with the images (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The fluorescence of the condensates recovered over time in fluorescence recovery after photobleaching (FRAP) experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating their fluidity. Since the two components were designed to phase separate by electrostatic interactions, we confirmed this mechanism by using different salt concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026amp;E). The phase separation was inhibited by high concentration of salt, demonstrated by both turbidity and fluorescent imaging. These results indicated that electrostatic interactions from polyE and polyGK of each component do drive the phase separation.\u003c/p\u003e \u003cp\u003eNext, we examined whether the phase separation occurs on cell surface. The epidermoid carcinoma cell line A431, which expresses high level of EGFR was used\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The two fluorescence labeled components were added together or separately in cell culture medium and the condensate formation was observed under confocal microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Each component alone did not show phase separation. However, when mixed together the condensates formed on the cell surface and both were nicely colocalized. Notably, since the critical concentration of LLPS is lowered on membrane\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, only 1 \u0026micro;M of each component demonstrated significant LLPS on cell surface while in solution the condensates were barely observed. To test its specificity, two other cell lines, the HeLa cell line expressing medium level of EGFR\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and HEK-293 cell line expressing no EGFR were used. Less condensates were observed on the surface of HeLa cells and no condensates formed on HEK-293. The results clearly demonstrated that the two components phase separated in the presence of EFGR, indicating that they can selectively target to and condensate around EGFR. The FRAP experiment also confirmed the liquid-like properties of the condensates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The payload needs to be internalized to take effects. Thus, we monitored the internalization of the condensates at different time points. Those condensates appeared on the cell surface immediately after adding to the medium and after 4 hours they were observed inside of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eBy using the EYFP as a pseudo payload, we are confident that the two-component delivery system can successfully phase separate on cell surface with selectively and get internalized into the cells. Next, the real payload was added and examine their abilities to induce cancer cell death. The cysteines on the surface of EYFP were used for drug conjugation. Maleimidocaproyl-MMAF (Mc-MMAF) was used to react with the thiol of the cysteine, affording the product. The copy number of MMAF was determined using Ellman's Reagent (5,5'-dithio-bis-[2-nitrobenzoic acid], DTNB) with free cysteine as the standard (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). One MMAF was conjugated to polyGK-EYFP, affording polyGK-EYFP-MMAF. Fluorescent imaging and FRAP results demonstrated that the drug conjugation did not change the phase separation behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026amp;B). The cytotoxicity was evaluated by MTT assays. Firstly, A431 cells were treated with each component alone or together with 1:1 ratio. The mixture had significantly better efficacy of cytotoxicity than each component alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To confirm that the improvement is the synergistic effect causing by phase separation rather than the simply sum of each, we used isobologram to analyze their synergies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. By plotting the concentrations of each component at different ratios when cells have the 50% survival rate, we noticed that the combinations fell below the line connecting the IC50s of each component, implying positive synergistic effects. At the condition in which the molar ratio of TC: polyGK-EGFP-MMAF\u0026thinsp;=\u0026thinsp;10: 3 in solution, the strongest synergistic effect occurs.\u003c/p\u003e \u003cp\u003eThe EYFG above is merely for visualization. In real drug development, a fluorescent protein is not required. Thus, the EYFP was substituted with a variable linker for drug conjugation. Here we chose the commonly used (GGGS)\u003csub\u003e3\u003c/sub\u003e as the linker and mutated one or two S to C for cysteine conjugation, generating two DCs, polyGK-MMAF and polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e. The copy numbers were determined by Ellman's Reagent as well (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The efficacy of the two versions of the DCs were examined by the MTT assay. The polyGK-EYFP-MMAF above is added for comparison. Moreover, a traditional ADC version by directly conjugating MMAF to the scFv of cetuximab as a bench marker. Ellman's Reagent assay showed that two MMAFs were conjugated, affording scFv-MMAF\u003csub\u003e2\u003c/sub\u003e. The polyGK-MMAF showed similar efficacy as polyGK-EYFP-MMAF, suggesting that EYFP does not affect the activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). With two drug molecules conjugated, polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e demonstrated higher potency than the single drug version, implying that the payload number is an important parameter for drug efficacy. However, even with two drugs conjugated, the traditional scFv-MMAF\u003csub\u003e2\u003c/sub\u003e is less active than any phase separation version of the drugs at the same payload concentrations. This is strong evidence that by involving LLPS in the system, the payloads can effectively condensate on the cell surface and increase the local concentration of drugs, resulting in better efficacy. Meanwhile, the toxicity of the delivery system in A431 and HEK-293 cells was tested. It demonstrated good selectivity between the two cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eFinally, we examined the efficacy of the PEDS on an A431 cell-derived mouse xenograft model. A431 cells were transplanted under the skin of CD-1 nude mice. Since the phase separation enhanced system demonstrated tremendous efficacy in cell assays, a very low dose was chosen in the experiment. The mice were administrated with 0.5 mg/kg TC separately or in combination with 3/10 molar quantity of DCs, every four days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The same dose of scFv-MMAF\u003csub\u003e2\u003c/sub\u003e as TC was used as a control representing current ADC modality. The tumor volume was recorded over time for 20 days. The dose of all samples was doubled at day 12 to better evaluate the efficacy of single MMAF conjugated TC. Without TC, the polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e alone did not show inhibition of tumor growth compared with PBS. At such a low dose, scFv-MMAF\u003csub\u003e2\u003c/sub\u003e demonstrated some effects to prevent the tumor growing but with no significant statistical difference from the control group. However, the groups of getting PEDS treatment showed markedly reduced volume of the tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The TC\u0026thinsp;+\u0026thinsp;polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e demonstrated best potency. Notably, the molar numbers of MMAF dosed by PEDS is less than scFv-MMAF\u003csub\u003e2\u003c/sub\u003e, indicating that local concentration of the drug played critical role in the efficacy. All the groups showed no significant weight loss, indicating good safety of the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). After the experiment, the tumors and normal tissues were fixed, paraffin-embedded, and sectioned and analyzed with HE or immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). HE stained tumor slices showed that the cell morphologies in PBS group and polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e group are complete and full, but there are more dead cells in the other three groups, especially around the blood vessels. TUNLE immunofluorescence staining indicates that more apoptosis cells exist in the tumors getting PEDS treatment than scFv-MMAF\u003csub\u003e2\u003c/sub\u003e while almost no apoptosis occurred in control and DC alone groups. Those cells are around blood vessels as well. Ki67 staining revealed the malignant proliferation of the cancer cells. With the PEDS treatment, the proliferation was completely halted while scFv-MMAF\u003csub\u003e2\u003c/sub\u003e only showed partial inhibition. Moreover, routine blood test and HE staining of other organs were performed for the safety test of the system. The WBC (white blood cell number) of polyGK-MMAF\u003csub\u003e2\u003c/sub\u003e and scFv-MMAF\u003csub\u003e2\u003c/sub\u003e groups decreased, but without significant difference (Fig. S5A). Blood biochemical tests showed no significant difference of liver and kidney function indicators (Fig. S5B). The HE stained slices of major organs didn\u0026rsquo;t show damage (Fig. S5C). The results clearly demonstrated that the PEDS can minimize the side effects while remains the high efficiency \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we designed a two-component delivery system based on\u0026nbsp;cell surface\u0026nbsp;phase separation. The main scaffold is a pair of peptides which can undergo LLPS when mixed, conjugated with targeting molecules or payloads respectively. We demonstrated the efficacy of the system by delivering an anticancer drug. In this case, the TC contains an antibody derivative to selectively target the cancer cell and DC has a linker conjugated with cytotoxic drugs. Each component is relatively stable alone. After dosing, TC recognizes the receptors on cancer cell surface first and DC can phase separate with it, forming condensates with high concentrated drugs locally. Both \u003cem\u003ein vitro\u003c/em\u003e assays and \u003cem\u003ein vivo\u003c/em\u003e animal models demonstrated good potency and selectivity of the system. Comparing with the delivery system directly conjugating antibodies with drugs, the PEDS breaks through the limitation of DAR by separating the drugs from antibodies and inducing LLPS, which significantly increases the efficacy. Additionally, for the payload selection, super-toxic compounds are not necessary in this method since copy number is no longer a problem. As original designed, the system can be a universal deliver tool but not limited for anti-cancer drug delivery. The payload can be variable, from drugs to probes. The targeting part is subject to change to other binders even chemical compounds, only if they can selectively target on a certain type of cells, for the delivery to different tissues. Currently the discovered protein or peptide pairs which can undergo LLPS under physiological conditions are limited but as more discoveries in this area are made, more phase separation scaffold will be available. In summary, we provided a novel method by utilizing phase separation for selective delivery and demonstrated that the system has high efficacy and selectivity. We believe that the method has great potential in drug development as well as a promising tool in biological research.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein expression and purification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scFv-polyE (sequence see Supplementary Materials Extended Data Table 1) protein was expressed in \u003cem\u003eE.coli\u003c/em\u003e origami2 (DE3) strain for better folding. The gene encoded His\u003csub\u003e6\u003c/sub\u003e-scFv-polyE\u0026nbsp;was synthesized by General Biology (China) and cloned into pET-28a vector (Miao Ling Plasmid, China). The vector was then transformed into to \u003cem\u003eE.coli\u003c/em\u003e (Origami2(DE3)) for expression. Cells were grown to the optical density of 0.7-0.9 at 37 ℃ and induced with 0.5 mM isopropyl-\u0026beta;-d-thiogalactopyranoside (IPTG) for expression at 37℃ for 6 to 8 h. The cells were collected and lysed and protein was purified with His-Trap chelating column (GE Healthcare). The purified recombinant proteins were analyzed by Coomassie-stained SDS\u0026ndash;polyacrylamide gel electrophoresis (PAGE) and desalted into the final storage buffer (ddH\u003csub\u003e2\u003c/sub\u003eO) by \u0026Auml;KTA pure (General Electric, USA). Proteins were concentrated to 1 mg/ml using Ultra centrifugal filters. Aliquots were taken and stored at -80 ℃.\u003c/p\u003e\n\u003cp\u003ePolyGK-EYFP was expressed in \u003cem\u003eE.coli\u003c/em\u003e (NiCo21). The gene of polyGK-EYFP-His\u003csub\u003e6\u003c/sub\u003e was\u0026nbsp;synthesized and cloned into pET-28a vector as described above. Cells were then grown to an optical density of 0.7-0.9 at 37 ℃ and induced with 0.5 mM IPTG, letting the protein express at 16℃ for 16 to 20 h. The protein was purified as above. Proteins were concentrated to 2 mg/ml using Ultra centrifugal filters. Aliquots were taken and stored at -80 ℃.\u003c/p\u003e\n\u003cp\u003eThe polyGK-linker proteins, including polyGK-(GGGS)\u003csub\u003e2\u003c/sub\u003e-GGGC and polyGK-GGGS-(GGGC)\u003csub\u003e2\u003c/sub\u003e, was expressed using the 6P-1 vector (Miao Ling Plasmid, China) with a glutathione S-transferase (GST) tag. The proteins were expressed in \u003cem\u003eE.coli\u0026nbsp;\u003c/em\u003e(NiCo21) and purified with GST Sefinose beads (GE Healthcare, USA). After washing, the PreScission Protease was used to cleave GST tags from the proteins, affording tag-free proteins. Proteins were concentrated to 2 mg/ml using Ultra centrifugal filters. Aliquots were taken and stored at -80 ℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCy3 staining of TC\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTC was stained with Cy3 monosuccinimidyl ester (ATT Bioquest, USA). The protein (scFv-polyE) was concentrated to 6 mg/mL using Ultra centrifugal filters. Cy3-NHS ester was mixed with scFv-polyE to a final concentration of 0.5 mg/ mL, with pH adjusted to 9 with 1M NaHCO\u003csub\u003e3\u003c/sub\u003e. The solution was incubated with shaking at 37℃ for 1 h, and then dialyzed with 10 kDa dialysis tube in ddH\u003csub\u003e2\u003c/sub\u003eO, affording Cy3 stained protein seed. The volume ratio of protein to seed was 1:200 - 1:100 when used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhase separation imaging in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTC and polyGK-EYFP was mixed in PBS with the final concentration of 10 \u0026mu;M each. The mixed protein solution was immediately loaded into a 96-well plate and incubated at room temperature before imaging. The images were captured by Leica SP8 confocal microscopy with 100 x objective (oil immersion).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTurbidity assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of the proteins is the same as imaging. Turbidity is measured by absorption at 620 nm in the 96-well plate using the Spectra ax M2 microplate reader (molecular device). All samples had three replicates (N=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman embryonic kidney cells (HEK-293) and Human skin cancer cells (A431) and HeLa cells were cultured in complete medium containing 44.5% Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) 1\u0026times; with glucose (4.5 g/L), 44.5% Minimum Essential Medium (MEM) Alpha 1\u0026times;, 10% fetal bovine serum (FBS), and 1% antibiotics (penicillin/streptomycin).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImaging of phase separation on the cell surface\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were plated onto an 8-well Lab-Tek chamber glass (Thermos Fisher Science) and let grow to the density about 70%. Before imaging, the culture medium was discarded and the cells were washed with PBS twice. Then, 1 \u0026mu;M TC and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e1 \u0026mu;M polyGK-EYFP (or polyGK-EYFP-MMAF) was added. The images were taken by Leica SP8 inverted microscope with 100 x objective lens, and the laser excitation wavelength was 405nm, 514nm and 552 nm, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFRAP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell samples were prepared as above and observed under an inverted microscope (LSM 780, Carl Zeiss, Germany) equipped with a confocal spinning disk unit (CSU-X1; Yokogawa, Tokyo, Japan) and a Zeiss 100\u0026times; oil immersion lens. A field (approx. 0.06 \u0026mu;m for the formed condensate in vitro and 0.04 \u0026mu;m for the punctate of cell surface) and bleached for 5 ns with 512 nm and 405 lasers (1AU) at 100% laser power, respectively. After being photobleached, images were acquired at a rate of 0.97s (in solution) or 1.26s (on cell surface) for 500s. The fluorescence intensity of the bleached area was calculated with Zeiss Zen software. The signal is normalized with pre-bleached as 100% and 0 s after bleach as 0. At least three samples were analyzed and averaged to obtain FRAP curves by GraphPad Prism 8.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMMAF conjugation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins, including polyGK-EYFP,\u0026nbsp;polyGK-(GGGS)\u003csub\u003e2\u003c/sub\u003e-GGGC and polyGK-GGGS-(GGGC)\u003csub\u003e2\u003c/sub\u003e, are desalted into water\u0026nbsp;to the concentration of 50\u0026nbsp;\u0026mu;M.\u0026nbsp;200\u0026nbsp;\u0026mu;M Mc-MMAF (Shanghai Rechemscience, China)\u0026nbsp;stock\u0026nbsp;in ddH\u003csub\u003e2\u003c/sub\u003eO\u0026nbsp;was mixed with the protein solutions and incubated for 50 min. Then the mixture was dialyzed in ddH\u003csub\u003e2\u003c/sub\u003eO for 10 min twice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEllman\u0026apos;s Reagent assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e300 \u0026mu;L 10 \u0026mu;M of free cysteine,\u0026nbsp;polyGK-EYFP,\u0026nbsp;polyGK-(GGGS)\u003csub\u003e2\u003c/sub\u003e-GGGC, polyGK-GGGS-(GGGC)\u003csub\u003e2\u003c/sub\u003e or their MMAF conjugated products was mixed with 20 \u0026mu;L 5m M Ellman\u0026apos;s Reagent\u0026nbsp;in the working solution (0.1 M sodium phosphate, 1 mM EDTA, pH=8.0)\u0026nbsp;and immediately analyzed by the plate reader. The absorbance at 412 nm was recorded every 1 min. The slopes were calculated using Graphpad prism 8.0 to indicate available free thiols used during the conjugation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCytotoxicity test by MTT assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated in 96-well plate (5000 cell/well) in DMEM with 10% FBS and allowed to adhere for 12 h at 37 ℃. The medium was changed to serum-free medium containing different concentrations (2, 4, 8, 16, 32, 62.5, 125, 250, 500 and 1000 nM) of TC and DCs. After 48 hours, the supernatant was removed, and 100 \u0026mu;L of MTT working solution was added to each well for another 4 h at 37℃, then 100 \u0026mu;L 10% SDS was added to each well to dissolve the purple crystals. The optical density (OD) at 570 nm was measured using SpectraMax M2 microplate reader (Molecular Devices, USA). Each group was performed with 5 replicates. The MTT working solution was prepared with MTT stock solution (5 mg/mL) and DMEM at a 1:9 ratio. DMEM was used as blank control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSynergy test by isobologram\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the IC\u003csub\u003e50\u003c/sub\u003e of A431 cells treated by TC or polyGK-EYFP-MMAF alone, different concentrations of TC (25, 50, 100, 200 and 400 nM) and polyGK- EYFP-MMAF (15, 30, 60, 120 and 240 nM) were added to A431 cells respectively and analyzed by MTT assay. Each group was repeated for 3 times. DMEM was used as blank control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concentrations of different combinations of TC and polyGK-EYFP-MMAF when the cells were at the survival rate of 50 % were plotted. The synergy of TC and polyGK-EYFP-MMAF was determined by the following equation, where A and B is the IC\u003csub\u003e50\u003c/sub\u003e of TC and polyGK-EYFP-MMAF respectively and a and b are the concentrations when they were dosed together for 50 % of cell survival.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eThe numerical value of C classifies the combination as follows: C=1, additivity; C\u0026lt; 1, superadditivity; C\u0026gt; 1: subadditivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSelectivity assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selectivity evaluation was performed by treating A431 and HEK-293 cells with the ratio of TC: polyGK- MMAF=10:3, as described above.\u003c/p\u003e\n\u003cp\u003eA431 was treated with scFv-MMAF\u003csub\u003e2\u003c/sub\u003e containing different concentrations (10, 20, 40 nM), as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMouse xenograft models\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD-1 nude mice were injected with cell suspension subcutaneously on the right back. Each mouse was inoculated with A431 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e). When the tumor volume grew to about 160 mm3, drug administration started.\u0026nbsp;The drug was injected via tail vein (TC: 0.5 mg/kg or DC: 0.0175 mg/kg or a mixture of both or scFv-MMAF\u003csub\u003e2\u003c/sub\u003e: 0.5 mg/kg, doubled after 12 days) and PBS was the control group. Every four days for a total of 20 days, 3 animals per group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe weight, and the long diameter (A) and short diameter (B) of the tumor were measured by Vernier calipers every two days, and the tumor volume was approximated by the formula V= (A\u0026times;B\u003csup\u003e2\u003c/sup\u003e)/2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter 20 days of administration, the mice were sacrificed by overdose anesthesia, and the tumors and normal tissues were dissected out and photographed. The dissected organs and tumors were fixed overnight with tissue fixative, and paraffin sections were prepared. HE staining, Ki67 and TUNLE immunofluorescence staining were performed on the tumor and organ sections, and the images were obtained with a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003eAll animal care and experimental procedures were reported in accordance with the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College and complied with NIH Guidelines for the Care and Use of Laboratory Animals (approval number: SYXK 2017-0020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRoutine blood and blood biochemical tests\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the treatment, blood of the mice was collected in EDTA anticoagulant tubes. Routine blood tests were taken immediately with a hematology analyzer (TEK-II MINI). Then another 1 mL blood was centrifuged at 3500 rpm for 10 min, the supernatant serum was collected for blood biochemistry test with an automatic biochemistry analyzer (Hitachi 7600).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Dr. Meng Qin for the help of animal experiments. The illustrations of mechanism were created with BioRender.com, and we thank Dr. Mingjia Yu for helping to preparing the illustrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Key R\u0026amp;D Program of China (2021YFC2103900), National Natural Science Foundation of China (22077010,\u0026nbsp;21907007)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProject administration: SZL, SX, LC\u003c/p\u003e\n\u003cp\u003eSupervision: SZL, SX\u003c/p\u003e\n\u003cp\u003eConcept conceiving: SX\u003c/p\u003e\n\u003cp\u003eExperiment: XB, PP, GD\u003c/p\u003e\n\u003cp\u003eData analysis: SX, XB, PP, SZL\u003c/p\u003e\n\u003cp\u003eFunding acquisition: SZL, SX\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: XB, PP, SX, SZL\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: SX, SZL\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtended Data\u0026nbsp;Figs. 1 to 3\u003c/p\u003e\n\u003cp\u003eExtended Data Table 1\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSu, X. \u003cem\u003eet al.\u003c/em\u003e Phase separation of signaling molecules promotes T cell receptor signal transduction. 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Testing for synergism over a range of fixed ratio drug combinations: replacing the isobologram. \u003cem\u003eLife Sci\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e, PL 23\u0026ndash;28, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0024-3205(95)02271-6\u003c/span\u003e\u003cspan address=\"10.1016/0024-3205(95)02271-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1996).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1788552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1788552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The cellular phase-separated condensates compartmentalize and concentrate biomolecules with distinct physicochemical properties, which has great potential for therapy purposes. However, the discovered phase separation phenomena in living organisms were restricted intracellularly, which limits the biomedical application of phase separation. Here, we designed a phase separation enhanced delivery system to specifically condensate molecules on the cell surface for efficient drug delivery. As a proof of concept, we demonstrated that an anti-cancer drug conjugate can selectively co-phase separate with the targeting component on cancer cell surface and efficiently kill the cells after internalization. Both cellular and in vivo assays showed more potency with this system than traditional antibody conjugates. The method provides insights in the application of phase separation as a powerful tool for therapeutic purpose.","manuscriptTitle":"Phase separation enhanced drug delivery system for anti-cancer therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-07 17:41:40","doi":"10.21203/rs.3.rs-1788552/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70731bdf-dd48-4175-a2f2-1469f7852b09","owner":[],"postedDate":"July 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-10T12:32:10+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-07 17:41:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1788552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1788552","identity":"rs-1788552","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}