Tailoring guanidyl-rich polymers for efficient cytosolic protein delivery

Journal Pre-proof Tailoring guanidyl-rich polymers for efficient cytosolic protein delivery

Jia Lv, Echuan Tan, Yuqing Wang, Qianqian Fan, Jingwen Yu, Yiyun Cheng PII:

S0168-3659(20)30076-6

DOI:

https://doi.org/10.1016/j.jconrel.2020.01.056

Reference:

COREL 10152

To appear in:

Journal of Controlled Release

Received date:

9 December 2019

Revised date:

17 January 2020

Accepted date:

30 January 2020

Please cite this article as: J. Lv, E. Tan, Y. Wang, et al., Tailoring guanidyl-rich polymers for efficient cytosolic protein delivery, Journal of Controlled Release (2020), https://doi.org/10.1016/j.jconrel.2020.01.056

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© 2020 Published by Elsevier.

Journal Pre-proof

Tailoring guanidyl-rich polymers for efficient cytosolic protein delivery Jia Lv1, 2 , Echuan Tan1 , Yuqing Wang2 , Qianqian Fan2 , Jingwen Yu2 , Yiyun Cheng1, 2, * 1

South China Advanced Institute for Soft Matter Science and Technology, School of Molecular

Science and Engineering, South China University of Technology, Guangzhou, 510641, China. 2 Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China.

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E-mail address of the corresponding author: [email protected]

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*

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Abstract

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Cytosolic protein delivery is important for the development of protein therapeutics towards intracellular targets. Guanidyl polymers exhibit high binding affinity with

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cargo proteins, and thus were designed as carriers for intracellular protein delivery. However, the structure-activity relationship and mechanism of these polymers in

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cytosolic protein delivery remained to be investigated. In this study, we synthesized a

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total number of eighteen guanidyl-rich polymers by grafting various guanidyl containing compounds onto a polyethylenimine scaffold. The investigated guanidyl

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analogues were consisted of a guanidyl group and a hydrophobic component including cyclohexane, benzene, and alkanes with various chain lengths. It is surprising that only the polymers with both benzene and guanidyl possessed high efficiency in cytosolic protein delivery. Further results showed that all the synthesized polymers have efficient protein binding in water and high cellular uptake, but these polymers except the benzene-guanidyl based one enter the cytosol of cells without carrying their cargo proteins, suggesting poor stability of the polymer/protein complexes in culture medium. Paired guanidinium-π interactions between the ligands on benzene-guanidyl polymers are critical to the stabilization of polymer/protein complexes. In addition, a lead polymer in the library exhibited robust delivery efficacy to various cargo proteins, while maintaining their bioactivity after cell internalization. The results suggest that complex stability is a critical factor in

Journal Pre-proof polymer- mediated intracellular protein delivery systems, and provide new insights to guide design of polymeric protein vehicles. Keywords: polymer, cytosolic protein delivery, protein, guanidyl, PEI

Introduction Intracellular protein delivery plays vital roles in protein-based therapeutics and biotechnologies[1–6]. Comparing with genetic and chemical medicines, protein therapeutics exhibit several benefits such as higher specificity, limited adverse effects,

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and low risk of off-targets[7–10]. However, most of proteins cannot passage cellular membrane owning to their hydrophilic and macromolecular characteristics, thus two

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strategies were usually adopted to facilitate the membrane transport of proteins acting on intracellular targets to exert their bio-functions. The first strategy was chemical

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modification of proteins with membrane permeable species such as cell penetrant

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peptides[11,12], amphiphilic ligands[13], polymers[14,15] and spherical nucleic acids[16] to increase their membrane affinity and internalization. Alternatively, carriers such as

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polymers[17–19], inorganic nanoparticles[20,21], liposomes and exosomes[22–24] are used to complex with cargo proteins and deliver them inside cells. Considering that

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cationic species are beneficial for efficient endocytosis, most of designed carriers for

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cytosolic protein delivery are positively charged. Proteins have relatively large size and limited number of binding sites (negative charges) on their surface, therefore, efficient binding of cargo proteins with the carriers is a challenging issue. To overcome this limitation, proteins were decorated with anionic molecules such as proteins[24], peptides[20], and anionic compounds[18,25] to increase their negative charge intensity, and thus increase their binding affinity with the cationic carriers. Besides, non-covalent

molecular

recognitions

such

as

biotin-avidin[26],

transcription

factor-DNA promoter[27], and nickel- Histidine tag[28] were introduced to enhance binding affinity between carriers and proteins. Though the strategies achieved high cytosolic protein delivery efficacy, most of them need genetically engineering or chemical modification on cargo proteins, which are usually related to lots of synthetic and purification work, and possibly altered protein function[29].

Journal Pre-proof Recently, polymeric and nanoparticle carriers for intracellular delivery of native proteins without the need of chemical modification were developed by increasing the binding affinity between carriers and cargo proteins [4]. For example, guanidinium-rich nanoparticles[30–35], fluoropolymers[36–40], coordinative polymers[41], boronate-rich polymers[42–44] and amphiphilic polymersomes[45–47] were reported with high efficacy in intracellular protein delivery by several research groups. Guanidyl groups can strongly bind with the residual groups of protein by a combination of salt bridge and hydrogen bonding interactions. When grafting guanidyl ligands onto nanoparticles or

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polymers at a high ligand density, the multivalent effect of guanidyl groups allows efficient protein binding, and guanidyl polymers were used as molecular glues for

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protein molecules[48,49]. Conjugation of guanidyl ligands are also beneficial for the endocytosis of the polymers[50–55].

et al.

synthesized

a

list of

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Rotello

guanidyl-conjugated gold nanoparticles for cytosolic delivery of proteins and Cas9

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ribonucleoproteins[56–59]. Tew et al. synthesized a series of polyoxanorbornene-based peptide mimics containing both guanidyl and hydrophobic groups by ring-opening

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metathesis polymerization[32–34]. Cheng et al. reported a cationic dendrimer grafted

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with a high density of guanidyl ligands for efficient cytosolic protein delivery[30,31,60]. These guanidyl-rich materials were able to transport proteins possess different

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isoelectric points (pIs) and molecular weights to cells with their bioactivity maintained. However, the structure-activity relationship and

mechanism of

guanidyl-rich materials in cytosolic protein delivery remained to be investigated. In this work, we synthesized a library of guanidyl- grafted polyethylenimine (PEI) with varying chemical structures to investigate their behaviors in cytosolic protein delivery. Guanidyl analogues with different hydrophobic components including cyclohexane, benzene, and alkanes conjugated to the guanidine group were constructed as candidates to reveal the structure-activity relationship (Figure 1). The results suggest that the benzene and guanidyl groups are critical for the stabilization of polymer/protein complexes and

efficient cytosolic protein delivery. Paired

guanidinium-π interactions between the grafted ligands form a knotted structure between the polymers, and thus increase the stability of complexes, especially in salt

Journal Pre-proof solutions. The aim of this study is to provide insights to guide the design of

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guanidyl-rich materials for efficient cytosolic protein delivery.

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Figure 1. Guanidyl-rich polymers in cytosolic protein delivery. (a) Synthesis of guanidyl-rich polymers and the structure of guanidyl ligands with different

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hydrophobic components. (b) The proposed mechanism for guanidyl-rich polymers in

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cytosolic protein delivery. Guanidinium groups on the polymer strongly interact with

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carboxyl groups on proteins via salt bridges and other interactions to bind cargo proteins. The polymers with a benzene ring adjacent to the guanidinium group can form paired guanidinium-π interactions between the conjugated ligands and thus form intermolecular crosslinking network between the polymer chains to stabilize the formed complexes. Guanidyl polymers without the benzene group failed to form stable complexes with proteins in buffer solutions, and thus failed to deliver the proteins into cytosols of living cells. (c) BSA-FITC delivery images of guanidyl-rich polymers modified with a similar number of guanidyl ligands with different hydrophobic components at their optimal delivery conditions on HeLa cells.

Materials and methods Materials. Branched PEI (25 kDa), bovine serum albumin (BSA), fluorescein isothiocyanate

Journal Pre-proof (FITC), rhodamine B isothiocyanate (RB), 4-guanidinobutyric acid (4GTA), N-hydroxysuccinimide

(NHS),

dicyclohexylcarbodiimide

(DCC),

methyl-β-cyclodextrin, genistein, wortmannin, and cytochalasin D were bought from Sigma-Aldrich (USA). Pulsin was bought from Polyplus Transfection (France). 3-guanidinopropionic acid (3GPA), 6-aminohexanoic acid, 8-aminooctanoic acid, 12-aminododecanoic acid, 3-aminobenzoic acid, 4- guanidinobenzoic acid (4GBA) hydrochloride, 4-aminocyclohexanecarboxylic acid and β- galactosidase (β-Gal) were bought from J&K Scientific (China). Trypan blue was obtained from Yesen (China).

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2-Methyl-2-thiopseudourea sulfate was purchased from Adamas (China). Horseradish peroxidase (HRP) was purchased from Yuanye Bio-Technology (China). Cyanamide,

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trypsin (Trp), chymotrypsin (Ctp), and 4-aminobenzoic acid were obtained from

Synthesis of guanidyl analogues

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(China).

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Macklin (China). Lyso-tracker red and hoechst 33342 were bought from Beyotime

For 6-guanidinohexanoic acid (6GHA), 262 mg 6-aminohexanoic acid (2 mmol)

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was reacted with 80 mg sodium hydroxide (2 mmol) for 3 h at room temperature (r.t.).

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S-methylisothiourea sulfate (278.5 mg/mL, 2 mmol) was dissolved in deionized water and dropped with the above solution slowly. White precipitates were obtained after 5

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hours reaction at r.t. The precipitates were collected and washed several times with drops of ethanol and water, and then lyophilized to obtain 6GHA as white powers. 8-Guanidinooctanoic acid (8GOA) and 12- guanidinododecanoic acid (12GDA) were synthesized by the same method. For 4- guanidinocyclohexanecarboxylic acid (4GCHA), 1g 4-aminocyclohexanecarboxylic acid (6.98 mmol) was dissolved in 5 mL ammonia and added with 862 mg 2-methyl-2-thiopseudourea sulfate (4.58 mmol). White precipitates were obtained after the mixture solution was stirred at r.t. for 24 hours. Precipitates were collected and washed with acetic acid and diethyl ether, respectively, then centrifuged to discard the supernatant. After that, the precipitates were

dried

and

recrystallized

in

acetic

acid

to

obtain

4-guanidinocyclohexanecarboxylic acid as white powers. For 3-guanidinobenzoic acid (3GBA), 274 mg 3-aminobenzoic acid (2 mmol) was dissolved in 10 mL

Journal Pre-proof anhydrous ethanol and added with 333 μL concentrated hydrochloric acid (4 mmol) and stirred to make it completely dissolved. The solution was added with 168 mg cyanamide (4 mmol) dissolved in 2 mL anhydrous ethanol, and white precipitates were obtained under reflux condition for 8 hours. The precipitates were collected and washed with drops of cold ethanol for several times and lyophilized to obtain 3GBA. 1

H NMR and liquid chromatograph- mass spectrometer (LC-MS) were used to analyze

the products, which were shown in supporting information. Synthesis and characterization of guanidyl-rich polymers DCC,

and

NHS

were

dissolved

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ligands,

in

anhydrous

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Guanidyl

N,N-dimethylformamide, and the mixtures were stirred at r.t. for 6 h followed by

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addition of PEI (The molar ratios of guanidyl ligands to PEI were listed in Table S1)

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and triethylamine (the molar ratio of guanidyl ligand: DCC: NHS: triethylamine was 1:1.3:1.2:1.5). The solutions were reacted at r.t. for 7 days, and then dialyzed against

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dimethyl sulfoxide and deionized water. The average number of guanidyl ligands conjugated on PEI was calculated by 1 H NMR or ninhydrin assay.

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For ninhydrin assay, a working solution was first prepared by dissolving

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hydrindantin (1.5 mg/mL) and ninhydrin (8.5 mg/mL) in ethylene glycol- monomethyl ether. The guanidyl-rich polymers were dissolved in 200 μL deionized water, followed

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by addition of 200 μL 0.2 M sodium acetate buffer (pH 5.4) and 200 μL working solution. The mixture were heated for 10 minutes in boiling water, followed by cooling to r.t. and added with 600 μL 60% ethanol solution. Absorbance value of the above mixture was tested by a UV-Vis spectrophotometer at 570 nm. Different concentrations of PEI were tested by the same way to obtain a standard curve to calculate the number of residual amino groups on each guanidyl-rich polymer. Synthesis of BSA-FITC and RB-labelled polymers 66 mg BSA (1 μmol) was dissolved in PBS, followed by addition of 1.17 mg FITC (3 μmol). The solution was reacted for 24 h at r.t. followed by dialyzed against PBS and distilled water, and lyophilized to obtain BSA-FITC. For the synthesis of RB- labelled polymers, the guanidyl-rich polymers were mixed with RB at different polymer to RB molar ratios (The polymer to RB molar ratio was set in the range of

Journal Pre-proof 1:3 to 1:5 to make sure the fluorescence intensity of each RB-conjugated polymer is similar to each other). The mixtures were stirred for 24 h at r.t. followed by dialyzed against distilled water, and lyophilized to obtain RB- labelled polymers. Fluorescence spectra of the RB-conjugated polymers were tested by a fluorescence spectrometer (F4500, HITACHI, Japan), (Ex: 520 nm, Em: 550-700 nm). Characterization of the polymer/BSA complexes Polymer was mixed with BSA and incubated at r.t. for 30 min, followed by diluted

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with deionized water or cell culture medium without phenol red (buffer). The

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complexes were characterized by dynamic light scattering (DLS, Malvern, UK), transmission electron microscope (TEM, Hitachi, Japan) and circular dichroism

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spectrometer (CD, Jasco, USA), respectively. For protein binding assay, the

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polymer/protein complexes were centrifuged at 12000 rpm for 20 min. After that, protein concentrations in supernatant were analyzed by fluorescence spectroscopy.

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Protein without addition of any polymer was tested as control. Protein binding ratio of a polymer was calculated as follows: (C control-Ccomplex )/Ccontrol×100%, in which C is

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protein concentration in the supernatant. For fluorescence resonance energy transfer

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(FRET) assay, polymer-RB was incubated with BSA-FITC for 30 min, then the

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complexes were added with different concentrations of heparin (0.05, 0.1, and 0.2 mg/mL, respectively). The fluorescence spectra of polymer-RB/BSA-FITC complexes were tested by a fluorescence spectrometer (Ex: 418 nm, Em: 480-700 nm). Dissociation constant (K d) calculation from the BSA-FITC binding experiments was conducted according to a method reported by Tew et al[34]. Briefly, polymer was mixed with BSA-FITC, then added with deionized water to make the total volume of the solution to 50 μL and incubated at r.t. for 30 min. After that, 950 μL deionized water was added to the above solution and fluorescence intensity of the polymer/BSA-FITC complexes were measured by a fluorescence spectrometer (Ex: 480 nm, Em: 518 nm). The fluorescence intensity of BSA-FITC without addition of any polymer was set as 1. Data are presented as mean ± SD (n = 3). The concentration of BSA-FITC was 100 nM, and the concentrations of the polymers were ranged from

Journal Pre-proof 0 nM to 60 nM. The fluorescence intensity was converted to fractional saturation according to the Equation 1. Fractional saturation = (𝐹x − 𝐹0 /𝐹S − 𝐹0 )

（1）

Fx is the fluorescence intensity of the polymer/BSA-FITC complexes measured at different polymer concentrations. F 0 is the fluorescence intensity of the complexes measured at a polymer concentration of 0 nM. Fs is the fluorescence intensity at saturation.

( 𝑥+𝑛𝐶𝑃 +𝐾𝑑 ) −√( 𝑥+𝑛𝐶𝑃 +𝐾𝑑 ) 2 −4𝑥𝑛𝐶𝑃 2𝑛𝐶𝑃

（2）

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y=

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K d was calculated according to the Equation 2.

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y is the fractional saturation calculated by Equation 1. x is the concentration of the polymer. n is equal to the number of binding sites which need to be calculated from

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this equation. C p is the concentration of BSA-FITC. K d is dissociation constant.

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OriginPro 2015 software was used to obtain the values of K d and n by a nonlinear curve module fitting with Equation 2 and the Levenberg−Marquardt iteration

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algorithm.

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Cell culture and cytosolic protein delivery HeLa cells were cultured at 37 °C under 5% CO 2 in DMEM medium (GIBCO)

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containing 10% fetal bovine serum (FBS, Gemini). For β-Gal enzymatic activity assay, 5 μg β-Gal was mixed with 2 μg 4GBA64 followed by addition of 50 μL cell culture media without FBS and incubation at r.t. for 30 min. After that, the mixture was further added with 450 μL cell culture media without FBS. HeLa cells incubated in 48 well plates were incubated with the above polymer/β-Gal complexes for 4 hours, then washed thrice with PBS and analyzed by a β-Gal staining kit and a β-Gal assay kit, respectively. For BSA-FITC delivery efficacy assay, HeLa cells incubated in 48 well plates or cell culture dish (15 mm) were added with the polymer/BSA-FITC complexes. After 4 h incubation, cells were washed thrice with PBS and added with trypan blue (0.2 mg/mL). BSA-FITC delivery efficacies of the polymers were analyzed by flow cytometry (BD FACSCalibur, San Jose), and laser scanning confocal microscopy

Journal Pre-proof (Leica SP5, Germany), respectively. For endosomal escape assay, Hela cells were treated with the polymer/BSA-FITC complexes for 1 h, 2 h, and 3 h, respectively. Lyso-tracker red (200 nM) and Hoechst 33342 were used to stain the endosomal and nuclei, respectively. The stained cells were observed by laser scanning confocal microscopy. For HRP enzymatic activity assay, HeLa cells incubated in cell culture dish (15 mm) or 48 well plates were washed thrice with PBS after incubated with the polymer/HRP complexes for 4 hours. After that, the cells were treated with Amplex Red (50 μM)

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and hydrogen peroxide (250 μM) for 30 min at 37 o C, followed by washed thrice with PBS and analyzed by laser scanning confocal microscopy. The HRP enzymatic

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activity was also tested by a tetramethylbenzidine (TMB) staining assay. After washed

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thrice with PBS, the treated cells were incubated with 200 μL acetate buffer (pH 5) containing TMB (6.24 mM) and hydrogen peroxide (3 mM) for 3 min at r.t. The

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yielded blue-colored products catalyzed by HPR in each well were captured by an optical camera. The concentrations of HRP was 20 μg/mL.

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For Trp and Ctp, HeLa cells incubated in 96 well plates were refreshed with 100 μL

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cell culture media containing 10% FBS after incubated with the polymer/protein complexes for 4 h, and further incubated for 20 h. The viabilities of treated cells were

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determined by a standard MTT assay. Endocytosis mechanism

HeLa cells were added with methyl-β-cyclodextrin (10 mM), genistein (700 μM), wortmannin (400 nM), and cytochalasin D (10 μM), respectively, or cultured at 4 °C. After 1 h incubation, the cells were washed with PBS and added with the polymer/BSA-FITC complexes. After 4 h incubation, cells were washed thrice with PBS and added with trypan blue (0.2 mg/mL). BSA-FITC delivery efficacies of the polymers were analyzed by flow cytometry.

Results and discussion Branched PEI with a molecular weight of 25 kDa was modified with 9 kinds of

Journal Pre-proof guanidyl ligands (Figure 2a, Figure S1- S5) via condensation reactions between amine groups on PEI and carboxyls on the guanidyl ligands. Various guanidyl to PEI feeding ratios were chosen to get PEI polymers modified with average numbers of 30 and 60 guanidyl ligands, respectively. The synthesized polymers were characterized by 1 H NMR (Figure S6-S9) or ninhydrin assay. The polymers were termed according to the name of guanidyl ligands and average conjugated numbers on each PEI. Take 6GHA-conjugated polymers for example, average numbers of 30 and 64 6GHA were conjugated on each PEI, respectively, and the products were named 6GHA30 and

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6GHA64, respectively. A total number of 18 guanidyl polymers (Table S1) were used as the library to investigate the structure-activity relationship of guanidyl polymers in

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cytosolic protein delivery. Unmodified PEI and the commercial reagent Pulsin were

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used as negative and positive controls, respectively. BSA-FITC was tested as a model protein. HeLa cells were treated with the polymer/BSA-FITC complexes at different

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polymer to protein weight ratios ranging from 0.15:1 to 0.76:1. Trypan blue was added to quench fluorescence of BSA-FITC absorbed on the outer surface of cellular

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membrane before measurement by flow cytometry. Efficiencies of the polymers in

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cytosolic delivery of BSA-FITC at optimal weight ratios (Table S1) were shown in Figure 2b. Among the investigated polymers, four polymers (4GBA64, 3GBA64,

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4GBA30, 3GBA30) showed superior efficiencies to the positive control Pulsin. All these polymers contain an aromatic ring in the conjugated guanidyl ligands. PEIs grafted with more GBA moieties exhibited much higher efficiency in deliver ing BSA-FITC into HeLa cells. 4GBA-grafted PEIs showed similar efficiency with 3GBA-conjugated ones at a fixed modification degree, suggesting that the position of guanidyl group on the benzene ring has little effect on the efficiency. It is surprising that all the other polymers without aromatic rings (PEIs modified with GA, 3GPA, 4GTA, 6GHA, 8GOA, 12GDA, and 4GCHA) showed poor performance in cytosolic BSA-FITC delivery. The efficiency of these polymers generally increase with the hydrophobicity of guanidyl ligands, however, the most e fficient polymer 12GDA60 is still far from 3GBA- and 4GBA-based polymers. It seems that the benzene ring adjacent to guanidyl group on PEIs is critical for efficient intracellular protein

Journal Pre-proof delivery. Therefore, we further investigated the reason accounts for this strange

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phenomenon.

Figure 2. Guanidyl- grafted PEI for cytosolic protein delivery. (a) Structures of the

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guanidyl analogues. (b) Efficiency of the synthesized polymers in BSA-FITC delivery on HeLa cells at their optimal polymer to protein weight ratios. BSA-FITC,

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unmodified PEI and commercial protein delivery regent Pulsin were tested as controls.

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Concentrations of BSA-FITC and PEI were 13.2 μg/mL and 4 μg/mL, respectively. The optimal weight ratios of guanidyl polymers to BSA-FITC were listed in Table S1.

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Data are presented as mean ± SD (n = 3). **p < 0.01 analyzed by Student’s t-test. We firstly investigated the protein binding ability of all synthesized guanidyl polymers in water. As shown in Figure 3a, all the polymers could form positively charged and uniform nanoparticles of 200-300 nm with BSA at different polymer to protein weight radios. The polymers showed high affinity to BSA-FITC with calculated K d [34] below 1 nM (Figure S10, 0.104±0.061 nM, 0.116±0.081 nM, 0.152± 0.099 nM, 0.142±0.255 nM, and 0.116±0.083 nM for 4GTA60, 6GHA64, 8GOA58, 4GCHA64 and 4GBA64, respectively). TEM images and CD spectra of the complexes confirmed the formation of complexes (Figure 3b and 3c). The quantitative data in Figure 3d showed that the guanidyl polymers have similar protein binding

Journal Pre-proof capability in aqueous solutions. We further modified the guanidyl polymers (4GTA60, 6GHA64, 8GOA58, 4GCHA64 and 4GBA64) with a red fluorescent dye RB to monitor the internalization of polymers and the polymer/protein complexes by HeLa cells (Figure S11). It is surprising to observe that all the polymers were efficiently internalized into cells after 4 h incubation, but only 4GBA64 was able to carry the cargo protein BSA-FITC into cells at the same time (Figure 4a). We also quantitatively tested the cellular uptake of guanidyl polymers labelled with RB at different time of incubation (Figure 4b). The results also showed that all the guanidyl

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polymers can efficiently penetrate across cell membranes. Therefore, the cellular uptake of polymers is not the decisive factor to influence the cytosolic delivery

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efficiency of polymers. Since all the investigated guanidyl polymers were capable of

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binding cargo proteins in aqueous solutions, but only the polymers themselves (except 4GBA64) were observed inside cells, we speculate that the difference between

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4GBA64 and other polymers is attributed to difference in the complex stability in culture medium. In this case, we investigated the stability of these polymer/protein

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complexes in the presence of salts. As shown in Figure 4c, all the guanidyl polymers

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except 4GBA64 showed significantly decreased amount of bound proteins in cell culture buffer solutions due to the competition effect of ions on the ionic interactions

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between polymers and cargo proteins. For 4GBA64, 80% of proteins complexed with polymer in deionized water were still remained in buffer, suggesting high stability of the formed complexes. Though all the guanidyl polymers form nanoscale complexes with BSA in water, only the 4GBA64-based one was detectable by DLS in cell culture medium under the same condition (Figure 4d). We further investigated the interactions of RB-conjugated guanidyl polymers with BSA-FITC by FRET. The polymers were complexed with BSA-FITC at an equal molar ratio, and the fluorescence spectra of the complex were measured. As shown in Figure 4e, the FITC-labelled BSA showed significantly reduced fluorescence intensity after the addition of RB-conjugated polymers, while FRET signals were observed at the same time, suggesting the combination of the polymers and proteins. Among the polymers, 4GBA64 showed the strongest FRET signal, which also proved that 4GBA64 can

Journal Pre-proof form more compact complex with BSA-FITC. The formed complexes were further added with different concentrations of heparin sodium to release the bound BSA-FITC. As expected, the 4GBA64-based complex is the most resistant to polyanion competition. All these results suggest that 4GBA64 can form the most stable complexes with cargo proteins among the investigated guanidyl polymers. It is known that guanidinium groups possess strong affinity with proteins by a synergy of salt bridge and hydrogen bonding formation, and a high density of guanidyl ligands on polymers could be an effective strategy to transport cargo proteins across cell

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membranes[48,61,62]. Here, the guanidyl analogues and polymers have the same number of guanidinium groups and the high stability of GBA-based polymer/protein

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complexes is not attributed to the number of guanidyl groups. In addition, differences

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in hydrophobicity of the guanidyl analogues should not be the reason accounts for the phenomenon. The hydrophobicity of 4GBA is higher than GA, 3GPA and 4GTA, but

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lower than 6GHA, 8GOA and 12GDA. Considering that the benzene ring in 3GBA and 4GBA could interact with guanidyl groups via a guanidinium-π interaction, which

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is widely discovered in protein structures and used to strengthen protein- ligand

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binding[63,64], we hypothesized that the paired guanidinium-π interactions between the conjugated ligands on PEI form crosslinking networks between the polymer chains,

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and reduce the charge repulsions between the cationic polymer chains within a congested space (Figure 4f). Therefore, guanidyl polymers with aromatic rings adjacent to the guanidyl group can form stable complexes with cargo proteins and the high complex stability ensures the high efficiency of GBA- modified polymers such as 4GBA64 in cytosolic protein delivery. The cellular uptake of 4GBA64/BSA-FITC complexes by HeLa cells were decreased at 4 °C or in the presence of methyl-β-cyclodextrin, wortmannin, genistein, and cytochalasin D (Figure S12a) , which are the inhibitors of lipid raft-, clathrin-, and caveolin- mediated endocytosis, and macropinocytosis, respectively. This result suggests that the endocytosis of 4GBA64/BSA-FITC complexes is energy-dependent and involved with multiple pathways. Most of proteins delivered by 4GBA64 were able to escape from acidic compartments after 2 h treatment (Figure S12b).

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Figure 3. Characterization of guanidyl-rich polymers in protein complexation. (a) DLS results of polymer/BSA complexes at weight ratios ranging from 0.15 to 1, and the characterizations of the polymer/protein complexes at their optimal polymer to protein weight ratios at which the polymer exhibited the highest BSA-FITC delivery efficacy and low cytotoxicity (cell viability above 85% (Figure S13)) were marked in red. TEM images (b) and CD spectra (c) of the polymer/protein complexes at their optimal polymer to protein weight ratios, the scale bar in (b) is 200 nm. (d) BSA-FITC binding capability of the guanidyl polymers in distilled water at their optimal conditions. Data are presented as mean ± SD (n = 3).

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Figure 4. Mechanism of the guanidyl-rich polymers in cytosolic protein delivery. (a)

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Fluorescence images of HeLa cells treated with polymer-RB/BSA-FITC complexes

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for 4 hours were obtained by laser scanning confocal microscopy. (b) HeLa cells treated with an equal concentration of RB- labelled polymers for 1 h, 2 h, and 4 h, respectively were analyzed by flow cytometry. The concentrations of polymer-RB and BSA-FITC were fixed at 0.1 and 0.2 μM, respectively. (c) Relative BSA-FITC binding capability of polymers in buffers at their optimal conditions. The protein binding ratio of each polymer in distilled water at optimal polymer to protein weight ratio was set as 100%, respectively. (d) Hydrodynamic size of the polymer/BSA complexes in cell culture media at their optimal conditions. (e) Fluorescence spectra of polymer-RB/BSA-FITC complexes at their optimal conditions before and after the addition of heparin (0.05, 0.1, and 0.2 mg/mL, respectively). (f) Paired guanidinium-π interactions between the conjugated GBA moieties on PEI in the complexes. We further tested the robust efficiency of the lead polymer 4GBA64 discovered in

Journal Pre-proof the library to deliver other cargo proteins. Four kinds of bioactive enzymes including β-galactosidase (β-Gal, 470 kDa, pI 5.1), horseradish peroxidase (HRP, 40 kDa, pI 7.2), chymotrypsin (Ctp, 25 kDa, pI 8.8), and trypsin (Trp, 24 kDa, pI 10.5) were used to test whether 4GBA64 can preserve bioactivities of cargo proteins during intracellular delivery processes. HeLa cells were incubated with the 4GBA64/protein for different times, then the bioactivity of cargo enzymes in the treated cells were tested. β-Gal is a negative charged enzyme that can catalyze X-Gal, a colorless substrate, into galactose and a blue dye (Figure 5a). As shown in Figure 5b,

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4GBA64/β-Gal treated HeLa cells were successfully stained with blue dyes after the addition of the substrate X-Gal. In comparison, free β-Gal treated cells were scarcely

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observed with blue pigments. Similarly, β-Gal can also catalyze colorless O-nitrophenyl-β-D-galactopyranoside (ONPG) into yellow colored onitrophenol

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(Figure 5a), which was used to quantitatively measure the activity of β-Gal in the

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treated cells. According to the measurement, about 80% of β-Gal activity in the catalysis of ONPG were maintained after intracellular delivery, and this efficiency

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was much superior to the commercial protein transduction regent Pulsin (Figure 5c).

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HRP is an enzyme that can catalyze a nonfluorescent substrate Amplex Red into a red fluorescent product in the presence of hydrogen peroxide, HRP can also catalyze a

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colorless substrate TMB into a blue dye in the presence of hydrogen peroxide (Figure 5d). After addition of substrate Amplex Red and hydrogen peroxide, cells treated with 4GBA64/HRP complex generated strong red fluorescence (Figure 5e). Similarly blue-colored products were observed in the cells treated with 4GBA64/HRP complex after treated with substrate TMB and hydrogen peroxide (Figure 5f). Both results confirmed the high efficiency of 4GBA64 in cytosolic delivery of HRP which was superior to the commercial protein delivery regent Pulsin. Trp and Ctp are two kinds of positively charged proteases that can degrade proteins into peptides. If these enzymes were delivered into cytosol, the proteolytic effect of these enzymes may cause toxicity on the treated cells. Both proteases showed minimal toxicity to treated cells at concentrations up to 50 μg/mL due to their membrane impermeable property (Figure 5g and 5h). However, significant cell death was observed when these proteins

Journal Pre-proof were complexed with 4GBA64. Control experiments suggest that the toxicity was not generated by the polymer. These results demonstrated that 4GBA64 could efficiently deliver a variety of cargo proteins into living cells and keep their bioactivity during

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the intracellular delivery process.

Figure 5. Intracellular delivery of enzymes by 4GBA64. (a) β-Gal catalyzes X-Gal and ONPG into a blue dye and a yellow colored product onitrophenol, respectively. β-Gal activities of HeLa cells treated with 4GBA64/β-Gal, Pulsin/β-Gal, PEI/β-Gal complexes and free β-Gal for 4 hours were tested by a β-Gal staining kit (b) and a β-Gal assay kit (n=3) (c). (d) HRP catalyzes Amplex Red and TMB into a fluorescent product and a blue dye, respectively, in the presence of hydrogen peroxide. HRP enzymatic activity of HeLa cells treated with 4GBA64/HRP, P ulsin/HRP, PEI/HRP complexes or free HRP for 4 hours were tested by the Amplex Red (e) and TMB (f) staining assay, respectively. The concentrations of β-Gal, HRP, 4GBA64 and PEI were 10, 20, 4, and 4 μg/mL, respectively. Cell viability of HeLa cells treated with

Journal Pre-proof 4GBA64/Trp (g) and 4GBA64/Ctp (h), respectively at different protein concentrations for 24 hours. Free proteins at equal concentrations were tested as negative controls. The polymer concentration was 4 μg/mL. In summary, we prepared a library of guanidyl-rich polymers with different kinds of hydrophobic ligands for cytosolic protein delivery. The structure-activity relationship of these polymers in protein delivery was investigated. It is found that PEI grafted with GBA ligands showed high protein delivery efficiency among the

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synthesized polymers and the benzene ring adjacent to the guanidyl group plays a critical role in stabilization of the polymer/protein complexes. The paired

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guanidinium-π interactions between the conjugated GBA moieties on PEI provides crosslinking network in the formed polymer/protein comple xes and reduces the

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charge repulsions between polymer chains in the nanoparticles. The lead polymer in

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the library was capable of delivering various cargo proteins into cells and maintaining their activity after intracellular delivery. The results in this study suggest that

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polymer/protein complex stability is a critical parameter that should be considered during material design. Supramolecular assembly and dynamic covalent linkages

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could be introduced into the polymer/protein complexes to strengthen the complex

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stability. This material design principle for cytosolic protein delivery will be investigated in our future work.

Acknowledgements

This work was financially supported by Guangdong Innovative and Entrepreneurial Research Team Program (2016ZT06C322), the National Natural Science Foundation of China (21725402), and Science and Technology Commission of Shanghai Municipality (17XD1401600). We thank the supports from the Flow Cytometry Core Facility and the Confocal Microscopy Facility at East China Normal University.

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Highlights 1. A guanidyl-rich polymer exhibited robust efficacy in the delivery of various cargo proteins into cytosol of living cells, while maintaining their bioactivity after intracellular release. 2. The polymer-protein complex stability is a critical factor for polymer-mediated cytosolic

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protein delivery systems.