Membrane Translocation and Organelle-Selective Delivery Steered

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Membrane translocation and organelle-selective delivery steered by polymeric zwitterionic nanospheres Nobuyuki Morimoto, Masaru Wakamura, Kanna Muramatsu, Sayaka Toita, Masafumi Nakayama, Wataru Shoji, Makoto Suzuki, and Francoise M Winnik Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00172 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 16, 2016

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Biomacromolecules

Membrane translocation and organelle-selective delivery

steered

by

polymeric

zwitterionic

nanospheres Nobuyuki Morimoto,*,† Masaru Wakamura,† Kanna Muramatsu,† Sayaka Toita,‡ Masafumi Nakayama,§ Wataru Shoji,§ Makoto Suzuki,† and Françoise M. Winnik*,‡,|| †

Department of Materials Processing, Graduate School of Engineering, Tohoku University, 6-6-

02 Aramaki-aza Aoba, Aoba-ku, Sendai, 980-8579, Japan ‡

Department of Chemistry and Faculty of Pharmacy, University of Montreal, CP6128

Succursale Center Ville, Montreal, QC, H3C 3J7, Canada §

Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Aramaki

aza Aoba 6-3, Aoba-ku, Sendai 980-8578, Japan ||

National Institute for Materials Science, WPI International Center for Materials

Nanoarchitectonics (MANA), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan

KEYWORDS: polysulfobetaines, guide delivery, membrane translocation, organelle-selective delivery, anticancer drug conjugate

ACS Paragon Plus Environment

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ABSTRACT

The majority of nanoparticles designed for cellular delivery of drugs and imaging agents enter the cell via endocytotic pathways leading to their entrapment in endosomes that present a robust barrier to further trafficking of the nanoparticles within the cells. A few materials, such as the cell penetrating peptides (CPPs), are known to enter cells not only via endocytosis but also via translocation through the cell membrane into the cytoplasm, successfully bypassing the endosomes.

We

report

here

dimethyl(methacryloyloxyethyl)ammonium

that propane

random

copolymers

sulfonate

and

of

3-

poly(ethyleneglycol)

methacrylate, p(DMAPS-ran-PEGMA), are internalized in cells primarily via translocation through the cell membrane rather than endocytosis. The properties of the polymers and their modes of uptake were investigated systematically by dynamic light scattering, confocal fluorescence microscopy, and flow cytometry. Using specific inhibitors of the cellular uptake machinery in a human cervical carcinoma cell line (HeLa), we show that these non-toxic synthetic polyzwitterions exist in cell media as self-assembled nanospheres that unravel as they adsorb on the plasma membrane and translocate through it. Conjugates of p(DMAPS-ranPEGMA) with rhodamine B were delivered selectively to the mitochondria whereas doxorubicin (Dox)-p(DMAPS-ran-PEGMA) conjugates were accumulated in both the nucleus and the mitochondria and effectively inducing apoptosis in HeLa cells. These findings suggest that the non-cytotoxic and readily synthesized p(DMAPS-ran-PEGMA) can find applications as bioimaging tools and drug nanocarriers.


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Introduction Nanoparticulate drug delivery systems offer many advantages over standard pharmaceutical formulations, including improved drug solubility, pharmacokinetic and pharmacodynamic profiles, decreased premature drug elimination or degradation during transit, and preferential drug localization in targeted disease sites, such as cancerous tumors.1-3 The design of nanoparticles for drug delivery has evolved over the years, leading to a set of preferred strategies concerning parameters, such as their composition, size, and surface chemistry. Most current nanocarriers are internalized in cells by endocytotic pathways, such as phagocytosis and pinocytosis, that leads them in the cell entrapped in endosomes.4,5 Eventually they reach lysosomal compartments where they are subjected to enzymatic degradation.

It is now

recognized that only a small fraction of endocytosed drug reaches the cell cytoplasm. The efficacy of nano drug delivery systems is enhanced significantly when they are able to reach the cytoplasm passively, bypassing endocytotic pathways altogether.

Much effort has been spent

recently towards the design of polymers and nanoparticles able to translocate passively through the cell membrane. A major breakthrough occurred when it was discovered that many short peptide sequences, commonly known as cell penetrating peptides (CPPs), effectively translocate through the cell membrane.5-8 CPP sequences vary greatly, however most of them contain arginine and lysine residues, which give them a net positive charge allowing them to interact electrostatically with anionic phospholipids of the cell membrane.9 Current research thrusts address issues related to CPPs cytotoxicity, stability, and cost, all of which need to be addressed prior to using CPPdriven drug carriers clinically.


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A number of studies on model lipid bilayers have brought convincing evidence that amphiphilic polymers perturb lipid bilayers and may translocate passively through them. Krylova et al. observed that block copolymers of oxyethylene and oxypropylene units (Pluronics) accelerate the flip-flop of lipids and increase the permeability of the bilayer to doxorubicin (Dox).10 Similar effects were reported earlier by Ringsdorf and his coworkers in the case of poly(N-isopropylacrylamide).11 Tribet et al. established that amphiphilic copolymers bound to giant lipid vesicles formed well-defined pores stabilized by macromolecules,12 in much the same way as pores formed by amphiphilic peptides that act as venoms in vivo.13 Passive diffusion of polymers through model lipid membranes has been demonstrated also for poly(ethylene glycol) (PEG)-based polymeric surfactants.14 More recently, the group of Ishihara demonstrated that amphiphilic copolymers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA) translocate without energy consumption through the membrane of mammalian cells.15 Both the MPC units and the phospholipid head groups are highly hydrated, which may facilitate polymer/lipid membrane fusion. Like poly(phosphorylcholines), polysulfobetaines belong to the family of polyzwitterions which possess both positively and negatively charged groups on each repeat unit. They have been shown to be biocompatible and to have anti-fouling properties.16-19 Well-defined polysulfobetaines homo- and co-polymers are readily accessible by controlled radical polymerization at a much lower cost than MPC polymers.19 They are used increasingly in drug delivery systems in the form of block copolymers with cationic blocks,20 pH-responsive blocks21 or hydrophobic moieties.22 Some polysulfobetaines present an upper critical solution temperature (UCST) in water. This property was exploited in formulations of thermoresponsive delivery systems for hydrophobic drugs.21


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We report here that a group of polysulfobetaines recently prepared in our laboratory, namely random copolymers of (3-dimethyl(methacryloyloxyethyl)-ammonium propane sulfanate) and poly(ethylene glycol) monomethacrylate, p(DMAPS-ran-PEGMA), translocate efficiently through the cell membrane. This unexpected observation was confirmed by uptake experiments with several cell lines, including a human cervical carcinoma cell line (HeLa). The extent of copolymer uptake and its distribution in HeLa cells were evaluated using Fluorescein-end labeled copolymers. To probe the specific modes of copolymer entry in cells, experiments were carried out in the presence and absence of pharmacological inhibitors. Finally, the targetability of the copolymers and their possible use as drug carrier were assessed by monitoring the intracellular localization of copolymers labeled with the dye rhodamine B (Rho), known to target the mitochondrion or with the cancer drug Dox.

Materials and Methods Materials 2-(1-Isobutyl)sulfanylthiocarbonylsulfanyl-2-methyl propionic acid was prepared following a procedure reported previously.3 2,2’-Azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride (VA-044) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM) were purchased from Wako Pure Chemical Industries (Tokyo, Japan). The cell counting kit-8 and calcein-AM were purchased from Dojindo Laboratories (Kumamoto, Japan). Inhibitors and Poly(ethylene glycol)methyl ether methacrylate (PEGMA) samples of average Mn = 500, 950, and 2,000 g/mol were purchased from Sigma-Aldrich Co (St. Louis, MO). All other reagents were purchased from Sigma-Aldrich and used without further purification.


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Preparation and characterization of p(DMAPS-ran-PEGMA) All p(DMAPS-ran-PEGMA) samples were prepared by reversible addition fragmentation transfer

(RAFT)

copolymerization

of

PEGMA

and

DMAPS

using

2-(1-isobutyl)

sulfanylthiocarbonylsulfanyl-2-methyl propionic acid as chain transfer agent. The monomers DMAPS and PEGMA (total concentration 0.1 mol/L) were dissolved in cold phosphate buffer saline (PBS, containing 137 mM NaCl, pH 7.4). To this solution kept at 4 ºC were added, first, the

initiator

VA-044

(0.31

mmol/L)

and,

second,

a

solution

of

2-(1-isobutyl)

sulfanylthiocarbonylsulfanyl-2-methyl propionic acid (1.03 mmol/L) in methanol. The mixture was purged with N2 for 30 min at 4 ºC. It was heated to 50 ºC to induce polymerization and kept at this temperature for 3 to 6 h. The mixture was cooled to 4 ºC to stop the polymerization. The polymerization mixture was purified by dialysis against water for 7 days using a membrane of 14,000 MWCO to remove residual monomers and salts. The polymer was recovered by freezedrying. The composition of the polymer was determined by analysis of the copolymers 1H NMR spectra recorded on a ECA-600 spectrometer (JASCO Co. Tokyo, Japan) for copolymer solutions in D2O containing 1 M sodium chloride. The molecular weight of the polymers was determined by gel permeation chromatography (GPC) with a JASCO GPC system equipped with TSKgel G3000PWXL and G4000PWXL columns (Tosoh Co. Tokyo, Japan) eluted with aqueous NaNO3 (100 mM) calibrated with PEG standards. The compositions of the polymerization solutions and of the resulting polymers are listed in Table 1.

Preparation of p(MATMA-ran-PEGMA)


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Poly[(2-methacryloyloxyethyl)trimethylammonium chloride-ran-PEGMA], p(MATMA-ranPEGMA) was prepared by RAFT copolymerization of MATMA and PEGMA. The monomers MATMA and PEGMA (total concentration 0.2 mol/L) were dissolved in pure water. To this solution kept at 4 ºC were added, first, the initiator VA-044 (14.8 mol/L) and, second, a solution of 2-(1-isobutyl) sulfanylthiocarbonylsulfanyl-2-methyl propionic acid (49.4 µmol/L) in methanol. The mixture was purged with N2 for 30 min at 4 ºC. It was heated to 50 ºC to induce polymerization and kept at this temperature for 3 h. The mixture was cooled to 4 ºC to stop the polymerization. The polymerization mixture was purified by dialysis against water for 7 days using a membrane of 3,500 MWCO to remove residual monomers. The molecular weight and the composition were characterized by using the same conditions with p(DMAPS-ran-PEGMA).

Preparation of p(SPMA-ran-PEGMA) Poly(3-sulfopropyl methacrylate-ran-PEGMA), p(MATMA-ran-PEGMA) was prepared by RAFT copolymerization of SPMA and PEGMA. The monomers MATMA and PEGMA (total concentration 0.2 mol/L) were dissolved in pure water. To this solution kept at 4 ºC were added, first, the initiator VA-044 (12.5 µmol/L) and, second, a solution of 2-(1-isobutyl) sulfanylthiocarbonylsulfanyl-2-methyl propionic acid (41.6 µmol/L) in methanol. The mixture was purged with N2 for 30 min at 4 ºC. It was heated to 50 ºC to induce polymerization and kept at this temperature for 3 h. The mixture was cooled to 4 ºC to stop the polymerization. The polymerization mixture was purified by dialysis against water for 7 days using a membrane of 3,500 MWCO to remove residual monomers. The molecular weight and the composition were characterized by using the same conditions with p(DMAPS-ran-PEGMA).


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Preparation and characterization of the p(DMAPS-ran-PEGMA) nanospheres The p(DMAPS-ran-PEGMA) samples (1 mg/mL) recovered were dispersed in PBS (pH 7.4) yielding self-assembled nanoparticles. The resulting solution was filtered through a PVDF membrane (0.45 µm) to remove aggregates before analysis or use in cellular studies. The hydrodynamic diameter and zeta-potential of nanospheres were measured using a nanoPartica SZ-100 system (Horiba, Kyoto, Japan) equipped with a Peltier temperature controller. Measurements were carried out on dispersions heated to 37 ºC.

Transmission electron

microscopy (TEM) (JEM-1011, JEOL, Tokyo, Japan) observation was performed at an accelerating voltage of 75 kV. p(DMAPS-ran-PEGMA) nanospheres (1 mg/mL) were dispersed in PBS and absorbed onto copper grids. phosphotungstic acid before observation.

The specimens were negatively stained with The surface tension of polymers in water was

evaluated by the pendant drop method using a Drop Master DM-301 tensiometer (Kyowa Interface Science, Saitama, Japan). Each solution was prepared by dilution of a concentrated polymer stock solution (30 mg/mL) and kept at 25 ºC prior to measurements. Measurements were carried out on samples kept at room temperature. Data were analyzed by the YoungLaplace method using FAMAS software version 3.7.2. Fluorescence measurements 8-Anilino-1naphthalenesulfonic acid (ANS, 1.0 µM) in PBS was performed on a F-2500 fluorescence spectrometer (Hitachi, Tokyo, Japan) with an excitation wavelength of 350 nm.

Emission

spectra were recorded from 400 nm to 650 nm. A stock solution ((2k-2.5) 30K, 30 mg/mL) was diluted to concentrations ranging from 0.003 mg/mL to 10 mg/mL with a solution of ANS in PBS (1.0 µM). All samples were kept in dark at 25 ˚C for 30 min prior to measurements.


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Labeling of p(DMAPS-ran-PEGMA) with fluorophores Fluorescein was modified the ω-terminus of p(DMAPS-ran-PEGMA). The p(DMAPS-ranPEGMA) (7.60 µmol, 1.0 equiv.) was treated with buthylamine (0.76 mmol, 100 equiv.) in 1 M sodium chloride aqueous solution containing small amount of tris(2-carboxyethyl)phosphine hydrochloride at room temperature for 2 h. The polymer was purified by dialysis for 7 days and isolated by freeze-drying. The obtained polymer (6.08 µmol, 1.0 equiv.) was dissolved in PBS with small amount of tris(2-carboxyethyl)phosphine hydrochloride, mixed with a solution of fluorescein O-acrylate (18.2 µmol, 3.0 equiv) in dimethylsulfoxide, and react in the dark at room temperature for 10 h. The polymer was purified by dialysis for 7 days and isolated by freezedrying. The degree of labeling was estimated by UV-Vis spectrometry analysis of polymer solutions in PBS using the absorbance at 492 nm of Fluorescein (ε = 67,100 [M-1 cm-1]). Rhodamine B isothiocyanate (Rho) was linked to one end of p(DMAPS-ran-PEGMA) via a 2step modification of the carboxyl group linked to the α-terminus. p(DMAPS-ran-PEGMA) (21.1 µmol, 1.0 equiv) was dissolved in MilliQ water (20 mL). DMT-MM (25.3 µmol, 1.2 equiv.) was added to the solution and the mixture was stirred for 1 h at room temperature. A large excess of ethylenediamine (2.1 mmol, 100 equiv.) was added to the mixture. The solution was kept at room temperature for 15 hr. The resulting polymer, purified by dialysis (MWCO = 14,000) against MilliQ water, was treated with a solution of the Rho (42.2 µmol, 2.0 equiv) in dimethylsulfoxide.

The reaction was allowed to proceed for 10 h in the dark at room

temperature. The polymer was purified by dialysis for 7 days and isolated by freeze-drying. The


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degree of labeling was estimated by UV-Vis spectrometry analysis of polymer solutions in PBS using the absorbance at 552 nm for Rho (ε = [98,500 M-1 cm-1]).

Conjugation of doxorubicine to p(DMAPS-ran-PEGMA) DMT-MM (8.6 µmol, 1.2 equiv.) was added to a solution of p(DMAPS-ran-PEGMA) (2k2.5)19K (7.1 µmol, 1.0 equiv.) in MilliQ water (20 mL). The mixture was stirred for 1 h at room temperature. Doxorubicine hydrochloride (Dox: 7.85 µmol, 1.1 equiv.) and triethylamine were added to the solution, which was then kept at room temperature in the dark for 15 h. The resulting polymer was purified by dialysis for 7 days and isolated by freeze-drying. The degree of Dox substitution (33.4 %) was estimated by UV-Vis spectrometry analysis of a solution of the polymer in PBS using the absorbance at 495 nm of Dox (ε = 10,800 [M-1 cm-1]). Yield: 88.0 %

Cell culture HeLa, a human hepatoma cell line (Hep G2), Human promyelocytic leukemia (HL-60), and a Chinese hamster ovary cell line (CHO-K1) cells were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University and pgs A-745 cells were purchased from the American Type Culture Collection (ATCC). HeLa and HepG2 cells were cultured in Dulbecco’s modified eagle medium supplemented with 10 % (v/v) fetal bovine serum. HL-60 cells were cultured in Roswell Park Memorial Institute (RPMI)1640 supplemented with 10 % (v/v) fetal bovine serum. Wild type CHO-K1 and pgs A-745 cells, CHO-K1 mutant deficient in proteoglycan biosynthesis, were cultured in F-12 nutrient mixture


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(Ham’s F-12) supplemented with 10 % (v/v) fetal bovine serum. The cells were incubated at 37 ºC in a humidified atmosphere of 5% CO2. Dispersions of fluorescently modified p(DMAPSran-PEGMA) samples were added to the culture medium in amounts such that their final concentration was 1 mg/mL.

Cytotoxicity test HeLa cells were seeded at 5,000 cells per 96 well microplates and cultured for 24 h. Polymer dispersions were added to HeLa cells in amounts such that with their final concentration ranged from 0 to 1.0 mg/mL. Treated cells were incubated for 24 h. The cytotoxicity of the polymers was evaluated using the cell counting kit-8 (CCK-8), which is based on the dehydrogenase activity detection using 2-(2-methoxy-4-nitrophenyl)- 3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8).

After a 4h incubation of the

microplates at 37 ºC, the reduced WST-8 formazan was detected by UV-Vis spectroscopy at 450 nm.

Observation of the polymers internalization by confocal laser scanning fluorescence microscopy (CLSFM) Cells were seeded at a concentration of 200,000 cells per 35 mm glass-bottomed dish and cultured for 24 h. They were washed with cold PBS and treated with 10 % FBS containing medium. Dispersions of fluorescently modified p(DMAPS-ran-PEGMA) samples were added to the culture medium in amounts such that their final concentration was 1 mg/mL. The treated


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cells were incubated for 1 h at 4 ºC or at 37 ºC. They were washed with PBS twice, retreated with PBS and observed by CLSFM (LSM 5 Pascal, Carl Zeiss, Germany) upon excitation with an argon laser (488 nm for Fluorescein) or a He-Ne laser (543 nm for Rho and Dox) and detection through a 503–530 nm bandpass filter (for Fluorescein) or a 543 nm long-pass filter for Rho and Dox).

Flow cytometry HeLa cells were seeded at 50,000 cells per 24-well microplates and cultured for 24 h. After incubation for periods of constant duration, the cells were washed twice with cold PBS and trypsinized. The cells were harvested and centrifuged at 5,000 rpm for 2 min. The cell pellet was suspended and washed twice with cold PBS. Flow cytometry was performed on a BD AccuriTM C6 flow cytometer equipped with a 488 nm argon laser. emission (530/30) was used for Fluorescein.

Signals from the FL1 bandpass

For Rho and Doxorubicine a FL2 bandpass

emission (585/40) was employed. For each sample 5,000 events were analyzed.

Inhibition of cellular uptake with various inhibitors HeLa cells were plated at 50,000 cells/mL in 24-well microplates and cultured for 24 h. For the assay performed at 4 ºC, a dispersion of Fluorescein-labeled polymers in cold PBS was added to the cells in amounts such that the final polymer concentration was 1 mg/mL. Cells were incubated for 1 h at 4 ºC. The inhibitors, cytochalasin D (3 µM and 10 µM for 15 min),24 methyl-β-cyclodextrin, (M-β-CD, 5 mM and 10 mM for 45 min)25, nystatin (11 µM and 27 µM


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for 30min)26 and sucrose (300 mM and 450 mM for 45 min)27 were added and incubated with cells at 37 oC. Then Fluorescein-labeled polymers (final concentration = 1.0 mg/mL) were added and the cells were incubated for another 1 h. The cells were washed with PBS, trypsinized, harvested, and centrifuged at 5,000 rpm for 2 min. The cells were resuspended in cold PBS and evaluated by flow cytometry using the intensity of fluorescence of Fluorescein linked to the polymer.

Calcein AM leakage HeLa cells were plated in 24-well microplates at 50,000 cells/mL and cultured for 24 h. HeLa cells were incubated with Calcein-AM for 30 min with the final concentration of 100 nM. The cells were washed with PBS twice, treated with DMEM containing 10% FBS, then with copolymer dispersions(final concentration: 1.0 mg/mL) and incubated for another 1 h. The cells were washed with PBS, trypsinized, harvested, and centrifuged at 5,000 rpm for 2 min.

They were resuspended in cold PBS and evaluated by flow cytometry using the

fluorescence intensity originating from the calcein that remained in the HeLa cells. For comparison, Tween 20 (final concentration: 1.0 mg/mL) was added to HeLa cells. The amount of calcein remaining in the cells was calculated from the fluorescence intensity of cells using the following equation: Remaining calcein (%) = (I(a)-I(c)) / (I(b)-I(c)) x 100 where I(b) is initial fluorescent intensity of cell before polymer addition, I(a) is that after polymer treatment, and I(c) is the autofluorescence of cell.


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Interaction of giant liposomes and nanospheres Giant liposomes were prepared from a mixed solution of 1,2-dioleioyl–sn-glycero-3phosphatidylcholine

(DOPC),

N-(Texas

Red

sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-

phosphoethanolamine (Texas Red-DHPE) and cholesterol in chloroform.

The mixture was

placed in a glass tube and chloroform was evaporated by blowing nitrogen gas flow, followed by vacuum drying overnight at room temperature to form a thin film on the glass tube inner surface. The thin film was hydrated with HEPES buffer (10 mM including 10 mM MgCl2, pH 7.5) at 30 ºC for 1h. The final concentration of DOPC, Texas Red-DHPE and cholesterol were 1 mM, 1 µM, and 0.1 mM.

Fluorescein-labeled p(DMAPS-ran-PEGMA) was dissolved in HEPES

buffer and mixed with giant liposome at r.t. and then observed by CLSFM.

Evaluation of Dox conjugated polymer nanospheres Cellular uptake and localization of Dox-conjugated p(DMAPS-ran-PEGMA), (2k-2.5)19K, in HeLa cells were evaluated after a 1h incubation of the cells with the copolymer at 37 ˚C. The same concentration of Dox was used in experiments using free Dox and Dox-conjugated copolymer: a Dox concentration of 10 µg/mL corresponds to a Dox-conjugated p(DMAPS-ranPEGMA) concentration of 1 mg/mL. The cytotoxicity of Dox-conjugated p(DMAPS-ranPEGMA) was evaluated using the cell counting kit-8 (CCK-8) after 24 h incubation as a function of concentration of Dox or conjugated Dox. Apoptosis was examined by staining the nucleus. Hoechst 33342 (1.0 µg/mL) was added to HeLa cells in a 6-well plate and incubated for 15 min


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at 37 ºC.

After washing with PBS twice, the 6-well plate was filled with fresh DMEM

containing 10% FBS, followed by the addition of Dox-conjugated p(DMAPS-ran-PEGMA). After a 24-h incubation and two consecutive washes with PBS, the residual fluorescence of Hoechst 33342 in the nucleus of HeLa cells was observed by fluorescence microscopy.

Results and Discussion Polymer synthesis, characterization and self-assembly in aqueous electrolyte solutions Six p(DMAPS-ran-PEGMA) samples (Figure 1a, Table 1) containing from 2.5 to 10 mol % PEGMA units were prepared by RAFT free radical polymerization of DMAPS and PEGMA bearing PEG chains of molecular weight (Mn) 500, 1,000 or 2,000 g/mol with VA-044 as initiator and 2-(1-isobutyl) sulfanylthiocarbonylsulfanyl-2-methyl propionic acid as chain transfer agent.23 The polymerizations were conducted at 50 ºC in methanol-PBS mixture (1/5 by volume). Methanol was added in an amount sufficient to ensure complete solubilization of the chain transfer agent in the polymerization mixture. Salt was added to enhance the solubility of the copolymers formed in the polymerization mixture. The copolymers composition was estimated from their 1H NMR spectra, presented in Figure S1, using the signals at 3.75 ppm for PEGMA, 3.66 ppm and 3.88 ppm for PDMAPS. It corresponds well to the monomers feed ratio.(Table 1) Their molecular weight, determined by GPC and based on PEG molecular weight standards, ranged from 19,000 to 87,000 g/mol. A summary of the polymerization conditions as

Table 1 Synthetic condition and characterization of p(DMAPS-ran PEGMA)s


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in feed [mol%]

in copolymer 1

H NMR [mol%]

p(DMAPS-ran-PEGMA) DMAPS

PEGMA

DMAPS

GPC

PEGMA Mn

Mw/Mn

[×104 g/mol]

(0.5k-10)35K

90

10

90.5

9.5

3.49

1.42

(1k-5)36K

95

5

94.3

5.7

3.63

1.54

(2k-2.5)19K

97.5

2.5

97.5

2.5

1.92

1.34

(2k-2.5)30K

97.5

2.5

97.4

2.6

2.98

1.76

(2k-2.5)51K

97.5

2.5

97.6

2.4

5.14

1.66

(2k-2.5)87K

97.5

2.5

97.4

2.6

8.71

1.66

p(SPMA-ran-PEGMA)*1

97.5

2.5

97.4

2.6

2.43

1.39

p(MATMA-ran-PEGMA)*1

97.5

2.5

97.4

2.6

2.26

1.22

*1

The molecular weight of PEGMA was 2k.

well as the copolymers composition and molecular weight is given in Table 1, where copolymers are designated by their acronym followed by the molecular weight of the PEG chain and the PEGMA content (in brackets) and, last, the molecular weight of the copolymer e.g. (2k-2.5)30K. A cationic and an anionic copolymer, p(MATMA-ran-PEGMA) and p(SPMA-ran-PEGMA), respectively, were prepared under the same RAFT polymerization conditions as p(DMAPS-ranPEGMA)(2k-2.5)30K, using PEGMA of Mn 2,000 g/mol as macro-comonomer. Their composition (Table 1) was determined by 1H NMR spectroscopy using the signals at 4.21 ppm for p(SPMA-ran-PEGMA) (Figure S2) and the signals at 3.91 ppm for MATMA-ran-PEGMA), Figure S3). All copolymers bear a carboxylic acid terminal group that was used as a handle for fluorophore tagging or drug conjugation.


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The copolymers dissolved readily in PBS at all concentrations up to 30 mg/mL, as anticipated in view of their zwitterionic nature. Further analysis of the copolymer solutions by DLS and TEM (Figures 1c, 1d) uncovered the presence of nanoparticles with a hydrodynamic diameter ranging from ~ 20 to 40 nm (Figure S4, Table S1) depending on the copolymer molar mass and concentration. The nanoparticles are electrically neutral (ξ ~ 0 mV). Polymer dispersions in PBS were slightly surface active (γ ~ 65 mN/m) for concentrations above 1.0 mg/mL (Figure S5) suggesting that the copolymers behave as amphiphiles.

The amphiphilic nature of the

copolymers and their self-assemblies was confirmed by fluorescence probe experiments using 8anilino-1-naphthalene sulfonate (ANS). ANS has a low fluorescence yield in polar environments, which is greatly enhanced and exhibits a blue shift when the probe experiences a hydrophobic environment. Changes in the emission of ANS in PBS dispersions of p(DMAPS-ran-PEGMA) (2k-2.5)30K of increasing concentration up to 1 mg/mL are presented in Figure 1e. The ANS emission intensity increases sharply and shifts from 510.5 nm to 489.0 nm with increasing the copolymer concentration. The critical aggregation concentration of (2k-2.5)30K was estimated ~ 0.61 mg/mL, indicating the formation of hydrophobic domains and consistent with the core-shell architecture also suggested by surface tension measurements and DLS studies. The micelles increase in size as the copolymer molecular weight increases (Figure S4). The length of the PEG chain of the PEGMA units has no detectable effect on the size of the nanoparticles. Moreover, the size and size distribution of the micelles remain constant upon changes in concentration, from 1 and 10 mg/mL, and upon storage for at least a week. Since both PDMAPS and PEGMA homopolymers dissolve molecularly in PBS, it is surprising that p(DMAPS-ran-PEGMA) samples self-assemble in PBS in the absence of an external trigger. In salt-containing aqueous media, such as PBS, the sulfobetaine moieties of the


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DMAPS units undergo dipole-dipole interactions. In principle, the betaine groups of the DMAPS units can assume six-membered rings due to internal salt bridges. Such ion-paired rings with mutually-neutralized positive and negative charges could exhibit enhanced thermodynamic stability compared to linear chains. Similar effects were reported by Menger et al., in the case of polybetaines bearing ammonium and carboxylates linked by a propyl chain to explain their enhanced affinity for lipid bilayers, compared to homologous carboxybetaines.28 The ion-paired rings are attracted to each other in PBS via hydrophobic interactions and may form transiently in PDMAS solutions. The colloidal stability of self-assembled p(DMAPS-ran-PEGMA) is attributed to the formation of a PEG corona surrounding the assembled sulfobetaines core, which points to the critical stabilizing role of the PEG chains.

A schematic representation of a

p(DMAPS-ran-PEGMA) micelle is presented in Figure 1b. Unlike the p(DMAPS-ran-PEGMA) samples, the strong polyelectrolytes copolymers p(MATMA-ran-PEGMA) and p(SPMA-ran-PEGMA) dissolve molecularly in PBS, with no sign of association up to a concentration of 1.0 mg/mL.

Intracellular uptake of p(DMAPS-ran-PEGMA) Prior to undertaking internalization studies, we examined the cytotoxicity of the copolymers towards HeLa cells by the WST-8 calorimetric assay. The p(DMAPS-ran-PEGMA) showed little or no cytotoxicity towards HeLa cells upon 24 h incubation for concentrations up to 1.0 mg/mL (Figure 2a), as anticipated given the reported non-cytotoxicity of both PDMAPS29 and PEGMA.30 The cationic copolymer p(MATMA-ran-PEGMA) exhibited significant cytotoxicity


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towards HeLa cells, triggering important loss in cell viability as its concentration exceeded 0.03 mg/mL.(Figure S6) The uptake by HeLa cells of Fluorescein-labeled p(DMAPS-ran-PEGMA) (2k-2.5)30K and (2k-2.5)51K was assessed by fluorescence optical microscopy observation of cells incubated with the copolymers (1 mg/mL) for 1 h at 37 ºC, in the presence or absence of serum. In all cases, the Fluorescein green emission was observed throughout the cell, indicating that the copolymers were taken up by the cells and diffused through the cytoplasm(Figure 2b). It is generally recognized that serum interferes with nanoparticles cellular uptake.

For

instance, in the case of the artificial CPP (R12), composed of 12 arginine residues, the cellular uptake in the presence of serum was 7 ~ 8 times lower than in serum-free conditions.31 It was suggested that serum proteins may adsorb on the cationic peptide, which may lead them to adhere to the cell membrane and prevent them from penetrating in the cell. In our case, the fluorescence intensity of HeLa cells treated with Fluorescein-p(DMAPS-ran-PEGMA), was ~ 3 times lower in presence of serum than in serum-free conditions (Figure 2c). Given the nonfouling properties of the PEG corona of the copolymer nanoparticles, protein adsorption is expected to be less prominent, which may account for the surprisingly high level of internalization of this polymer in the presence of serum. The molecular weight of the copolymer has a mild effect on the internalization efficiency, shorter copolymers being internalized more efficiently than longer ones.

For example,

quantitative flow cytometry experiments (Figure 2c) indicate that the cellular uptake of Fluorescein-(2k-2.5)51K was lower than that of Fluorescein-(2k-2.5)30K by factors of ~49 % and ~36 % in the presence and in the absence of serum, respectively.

In contrast, neither the


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level of PEGMA incorporation along the copolymer chain, nor the length of the PEG chain exert a significant effect on the copolymers internalization efficiency (Figure S7). The intracellular distribution of the Fluorescein-p(DMAPS-ran-PEGMA) nanoparticles internalized in HeLa cells upon incubation for 1 h in the presence of serum was assessed by confocal laser scanning fluorescence microscopy (CLSFM). The copolymer fluorescence was detected throughout the HeLa cells including the nucleus, but the fluorescence intensity was not uniform throughout. (Figure 3a, Figure S8, S9 and S10). Using HeLa cells co-stained with Mitotracker®, a fluorophore known to stain mitochondria were incubated with Fluoresceinp(DMAPS-ran-PEGMA)-(2k-2.5)30K for 1 h at 37 ºC in the presence of serum, we established that the areas of high fluorescein intensity were localized in mitochondria (Figure 3a). An enhancement of fluorescein emission in the mitochondria is expected due to know increase of fluorescein emission intensity when it experiences a change in pH from 7.4 (cytosol) to ~8.0 (mitochondria). In addition, it may indicate preferential incorporation of the copolymer in mitochondria. Experiments carried out using Hep G2 and HL-60 cells treated with p(DMAPSran-PEGMA) (2k-2.5)30K in the presence of serum gave similar results (Figure S11). The cationic copolymer Fluorescein-p(MATMA-ran-PEGMA) was also taken up by HeLa cells. In this case, however, fluorescence was confined to granules within the cytosol suggesting that, like most cationic carriers, Fluorescein-p(MATMA-ran-PEGMA) was internalized via endocytotic pathways32 (Figure S12).

Biological assays probing the mechanism of p(DMAPS-ran-PEGMA) internalization in HeLa cells.


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The distribution of p(DMAPS-ran-PEGMA) throughout the cytosol suggests that the copolymer does not enter the cell via endocytosis. To gain insight into the mechanisms responsible for the transport of p(DMAPS-ran-PEGMA) through the cell membrane, we examined the effect of temperature (Figure 3b), of pharmaceutical inhibitors that block specific endocytotic pathways (Figures 3c and 3d), and of heparan sulfate proteoglycans known to assist the internalization of oligopeptides (Figure 3e). The cellular uptake of fluorescein labeled p(DMAPS-ran-PEGMA) samples was determined experimentally by flow cytometry using two copolymers differing in molecular weight, Fluorescein-(2k-2.5)30K and Fluorescein-(2k2.5)51K. As endocytotic pathways are energy-dependent processes, they are affected by changes in temperature. We performed internalization assays after incubation of HeLa cells at 37 ºC and at 4 ºC in the presence of serum (Figure 3b). The uptake efficiencies of the two polymers in cells incubated at 37 ºC were similar, the uptake of the shorter polymer being slightly larger. When the cells were kept at 4 ºC rather than at 37 ºC, the uptake of the longer polymer decreased by ~ 50 %. Incubation of cells at 4 ºC has several effects: it leads to a reduction of the synthesis of ATP needed for the function of active transport through the cell membrane,33 it decreases the fluidity of the membrane leading to a tighter packing of cholesterol-rich domains of the membrane,34 and inhibit pinocytosis.35 The observed decrease in internalization of Fluorescein(2k-2.5)51K implies that this copolymer has two modes of entry into cells: translocation though the membrane and endocytosis, depending on the conditions, as was observed also in the case of CPPs.31,36 Remarkably, the uptake of the shorter polymer, Fluorescein-(2k-2.5)30K was enhanced by two orders of magnitude (Figure 3b) upon incubation at 4 ºC, compared to incubation at 37 ºC.


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The internalized polymer was localized in the cytosol, nucleus and mitochondria (Figure 3a). Enhanced cellular uptake at 4˚C was observed also in the case of three other cell lines: Hep G2 cells, HL-60 cells and CHO-K1 cells (Figure S11), which suggests that the uptake mechanism of (2k-2.5)30K studied here extensively in case of HeLa cells is also effective in other cell lines. The enhancement of the overall fluorescence emission may reflect the change in pH experienced by fluorescein as it enters mitochondria in the cytosol.37 Oligo-arginine and HIV-1 Tat peptides exhibited the same tendency of enhancement uptake at 4 ºC than that at 37 ºC31,36,38, in agreement with the membrane translocation mechanism. We take it as a indication that entry of Fluorescein-(2k-2.5)30K at 4 ºC occurs preferentially via translocation through the membrane. This may also be the case at 37 ºC. However, as in the case of CPP, several other factors may affect the uptake mechanism such as concentration,39 membrane potential,40 membrane-induced membrane curvature,41 etc. Based on these results, we undertook next a systematic study of various active transport mechanisms available for the transport of p(DMAPS-ran-PEGMA) through the membrane. Cells were treated with specific inhibitors prior to incubation with each of the two copolymers Fluorescein-(2k-2.5)30K and Fluorescein-(2k-2.5)51K.

The resulting internalization levels,

determined quantitatively by flow cytometry are presented in Figures 3c and 3d. The pathways affected by each inhibitor are represented schematically in Figure 4a.42,43 Pretreatment of cells with Cytochalasin D, known to disrupt the actin organization necessary to form phagosomes or macropinosomes,44 had no effect on the internalization of either of the two copolymers at 37 ºC. This result implies that the copolymers enter cells via processes other than phagocytosis, caveolae formation, and macropinocytosis. Similarly, pre-incubation of HeLa cells with nystatin, known to inhibit caveolae-mediated endocytosis by binding to cholesterol and disrupting the


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functions of cholesterol, did not affect the copolymers internalization, independently of their molar mass. The hypertonic sucrose medium, known to inhibit specifically clathrin-mediated endocytosis,27,45 had no effect on the internalization of Fluorescein-(2k-2.5)51K, but it enhanced the cellular uptake of the shorter copolymer Fluorescein-(2k-2.5)30K. To examine the role of lipid rafts in copolymer uptake, cells were treated with methyl-βcyclodextrin (M-β-CD) for 45 min and thereafter incubated with the copolymers. M-β-CD depletes the membrane cholesterol, disrupts lipid rafts, and increases membrane fluidity. The Mβ-CD pre-treatment led to a significant inhibition of cellular uptake of Fluorescein-(2k-2.5)51K in Hela cells, compared with cells treated with the copolymer in the absence of M-β-CD. The reduction in the amount of cellular uptake after pre-treatment with 5 mM and 10 mM M-β-CD was ~15 % and ~40 %, respectively. This result indicates that lipid rafts may be involved in the cellular uptake of Fluorescein-(2k-2.5)51K, but other mechanisms of entry are effective as well, as suggested also by the effect of temperature described above. As for the shorter copolymer Fluorescein-(2k-2.5)30K, pre-incubation of HeLa cells with Mβ-CD resulted in an increase of uptake by HeLa cell of ~ 20 % and ~ 470 %, when HeLa cells were pre-incubated with of 5 mM and 10 mM M-β-CD, respectively.

The effect of the

copolymer molar mass on cellular uptake post M-β-CD treatment concurs remarkably well with the effect of temperature on cellular uptake presented above. The fact that M-β-CD enhances the entry of the shorter polymer may be attributed to the increased flexibility and permeability of the cell membrane depleted of cholesterol, which favors translocation of p(DMAPS-ran-PEGMA) through the membrane. In studies of the internalization of oligo-arginine (R8, R9, R12) peptides and Tat peptide, a similar enhancement of the internalization was observed in the presence of Mβ-CD. It was taken as an indication of the implication of lipid rafts in the CPP entry in the


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cell.39,46 Interestingly, the CPP membrane translocation was increasingly more effective as the peptides concentration was increased, which the authors attributed to the formation on the cell membrane of zones of high peptide concentration that served as translocation nucleation sites. In the case of Fluorescein-(2k-2.5)30K, such zones may form even more readily, since they are delivered as nanospheres that upon contact with the cell membranes release a large number of polymer chains in close vicinity. Heparan sulfate proteoglycans present on the cell surface (HSPG) are known to assist the internalization of oligo-arginine peptides and TAT peptide (> 5 µM).47,48 They are considered to act as multivalent low affinity receptors of cationic CPPs. Whether or not HSPGs are important also in the internalization of p(DMAPS-ran-PEGMA)s, was evaluated here by comparing the uptake of p(DMAPS-ran-PEGMA) (Fluorescein -(2k-2.5)30K) by CHO-K1 cells and a CHO-K1 cell mutant lacking heparan sulfate and chondroitin sulfate (A-745). As seen in Figure 3e, internalization of Fluorescein -(2k-2.5)30K (33.3 µM) proceeded similarly for both cell lines, indicating that an HSPG-facilitated internalization cannot be invoked in the case of p(DMAPSran-PEGMA)s. Negatively charged HSPGs can undergo electrostatic interactions with cationic CPPs and, as such, enable intimate contact between CPPs and the cell membrane which may facilitate the CPP translocation.

The sulfobetaine groups of p(DMAPS-ran-PEGMA)s are

electrostatically neutral and do not interact with the sulfate functions of HSPGs. The fact that p(DMAPS-ran-PEGMA)s are taken up by A-745 cells suggests that the copolymers interact directly with the phospholipid bilayer. To test this hypothesis, we monitored by CLSFM the interactions between Fluorescein -labeled p(DMAPS-ran-PEGMA)s and giant multilamellar liposomes of DOPC containing Texas Red-labeled DHPE, fluorescence phospholipid analogue. Fluorescence imaging in real time provided evidence that p(DMAPS-ran-PEGMA)( Fluorescein


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-2k-2.5)30K interacts with the lipid bilayer immediately upon mixing and adheres onto the lipid bilayer (Figure S13). One could envisage that sulfobetaine moieties of the copolymer form ion pairs with phosphatidyl choline (PC) groups of the phospholipidic components of the cell membrane. However, several reports indicate that such interactions are unlikely, given the difference in the hydration levels and hydrophilicity of the two zwitterions. Phosphoryl choline groups are highly hydrated and hydrophilic, whereas sulfobetaines have a low hydration level, especially in the case of DMAPS in which the ammonium moiety is linked to the end sulfonate by a propyl (C3) linker. Moreover, it has been reported that the sulfobetaines with a C3 linker, which can adopt a stable cyclic conformation (see Figure 1b) are able to penetrate deeply into the lipid bilayer, as depicted in Figure 4b, top. This step may initialize the translocation of the copolymer through the membrane. Unfortunately, given the multilamellar nature of the giant liposomes, we could not obtain reliable data on the copolymer internalization within the aqueous interior of the liposomes. Having established that p(DMAPS-ran-PEGMA)s are taken up readily by HeLa cells, it was important to ensure that their internalization proceeds without permanent damage to the cell membrane. To address this point, we performed internalization assays with HeLa cells loaded with calcein. No calcein leakage was detected from cells treated with either (2k-2.5)30K or (2k2.5)51K, vouching for the integrity of the cell membrane. Under similar conditions, the addition of Tween 20 (1 g/L) caused calcein leakage on the order of ~ 50 % (Figure S14). Next, we carried out a study of the effect of copolymer concentration on the efficiency of internalization.

We know from surface tension measurements (see Figure S5) that the

copolymers are molecularly dissolved as isolated copolymer chains in aqueous solutions of [p(DMAPS-ran-PEGMA)] < 0.1 g/L, whereas in more concentrated solutions the copolymers


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assemble into nanospheres (multichain aggregates). By testing the extent of cell internalization as a function of polymer concentration ranging from 0.01 to 1.0 g/L, we should be able to determine if the copolymers transit through the membrane as isolated chains or as nanospheres. For the experiment, we used the labeled copolymer Fluorescein -(2k-2.5)30K and measured the mean fluorescence intensity of HeLa cells treated with the copolymer of increasing concentration. As shown in Figure 3f, the mean fluorescence intensity of the cells increased monotonously with increasing polymer concentration from concentrations well below the aggregation threshold, implying that isolated copolymer chains are able to transit through the membrane.

Proposed mechanism of the cellular internalization of p(DMAPS-ran-PEGMA)s The data presented above, led us to devise the multistep mechanism depicted in Figure 4 to account for the cell internalization of p(DMAPS-ran-PEGMA)s. To initiate the process, polymer nanospheres or individual chains residing in the medium must dock to the cell surface. This step is believed to involve ion pairing between sulfobetaine units along the polymer chain and zwitterionic, or possibly anionic, headgroups of the outer leaflet of the cell membrane. Initially nanospheres bind to the cell surface by just a few points of attachment.

Progressively, the

nanospheres spread on the cell surface allowing each chain to interact with numerous individual lipids. This process, which requires lateral phospholipid diffusion, proceeds efficiently in the case of fluid membranes.

The accelerating effect of M-β-CD on polymer internalization

described above may be related to the increased fluidity of cholesterol-depleted membranes. The resulting polymer/lipid complexes will then be able to translocate directly through the membrane


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in the form of reverse micelles, a process akin to the direct translocation of CPPs though the membrane. As revealed by the temperature-dependent experiments, direct membrane translocation occurs to the exclusion of endocytosis in the case of low molecular weight p(DMAPS-ran-PEGMA)s, but for high molecular weight p(DMAPS-ran-PEGMA)s the occurrence of competitive endocytosis cannot be excluded.

P(DMAPS-ran-PEGMA)-driven organelle specific drug delivery Triphenyl phosphonium and Rho have been used in the past as directional antennae to deliver small molecules selectively to mitochondria.49 Subsequently, triphenyl phosphonium or Rho were conjugated to polymers and shown to enhance mitochondria delivery of the polymers. 15, 50, 51

In particular, Rho-labeled amphiphilic MPC copolymers, known to have cell-penetrating

properties, were shown to target mitochondria selectively, which was attributed to the presence of conjugated Rho.15 The putative mechanism of cell-penetration of amphiphilic MPC copolymers invoked by Ishihara et al. involved micellar copolymer assemblies. Although Rho may reside preferentially in the hydrophobic core of the micelles, it retained its mitochondrial targeting activity upon cell internalization. The situation is different in the case of p(DMAPSran-PEGMA) nanospheres. Experimental evidence presented here points to the fact that the nanospheres unravel as they interact with the cell membrane and the copolymers translocate the cellular membrane as single chains. Reassembly of nanospheres in the cytosol is unlikely since the polymer concentration in the cell will be low, possibly below the critical aggregation concentration, and the chains will be surrounded by biomolecules that may interact with the polymers.

These considerations, together with prior reports on the mitochondrial targeting


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efficiency of polymer-tethered Rho, lead us to conclude that Rho modified p(DMAPS-ranPEGMA) will be localized preferentially into mitochondria. The exceptionally facile translocation of non-toxic p(DMAPS-ran-PEGMA)s suggests that this class of polymers could act as vehicles for the delivery of drugs active in specific organelles. To test this hypothesis, we linked to one end of the polymers either a drug, Dox or Rho, a fluorescent dye known to target the mitochondria. The copolymers Rho-p(DMAPS-ran-PEGMA)(2K-2.5)19K, 51K and 87 K (1.0 mg/mL) were incubated with HeLa cells for 1 h in the presence of FBS at 37 ºC. In each case, the bright red emission of Rho was observed in defined areas around the nucleus typical of mitochondria (Figure S15).

The internalization efficiency of Rho-p(DMAPS-ran-PEGMA)

decreased with increasing copolymers molecular weight. Dox is known to stabilize the topoisomerase-II-DNA cleavable complex and to induce apoptosis through direct oxidative DNA damage, although details of the apoptosis mechanism remain unclear.52,53

HeLa cells were incubated for 1 h with either Dox-p(DMAPS-ran-

PEGMA)(2K-2.5)19K or free Dox in the presence of serum at 37 ºC. Fluorescence microscopy observation of treated cells (Figure 5a) revealed preferential nuclear and mitochondria localization of Dox and the Dox conjugated polymer. The mean fluorescence intensity of treated cells was slightly stronger emission in the case of free Dox. Cytotoxicity data recorded for HeLa cells treated for 24 h with Dox or Dox-p(DMAPS-ran-PEGMA) are presented in Figure 5c as a function of Dox concentration.

In both cases, cell viability decreased significantly with

increasing Dox concentration, an important observation that establishes that the pharmacological activity of Dox is maintained in the conjugate. The cytotoxicity of the Dox conjugated was slightly lower than that of the free drug: a Dox concentration of ~ 10 µg/mL was needed to reduce cell viability to < 10 %, whereas a concentration of ~ 3 µg/mL of free drug was sufficient


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to reach < 10 % cell viability. Damage inflicted to cells can often be detected by changes in the nucleus morphology.54 Micrographs of Hoechst 33342 stained HeLa cells after a 24-h treatment with p(DMAPS-ran-PEGMA), Dox, and Dox-p(DMAPS-ran-PEGMA) are shown in Figure 5d. Nuclei of HeLa cells treated with Dox and the Dox conjugate are smaller and less spherical, compared to untreated cells indicative of the onset of apoptosis. The nuclei of cells treated with p(DMAPS-ran-PEGMA) were spherical with no sign of impairment, confirming the non-toxicity of the polymer.

CONCLUSIONS The availability of readily available non-toxic nanocarriers able to transport cargo into cells via non-endocytotic pathways would be useful across a range of applications from in-vitro studies to drug delivery.

We have demonstrated that P(DMAPS-ran-PEGMA) nanospheres,

particularly those of low molecular weight copolymers, are internalized in cells primarily by translocation through the cell membrane. Of particular importance is the ability to deliver Rholabeled p(DMAPS-ran-PEGMA)s preferentially to the mitochondria and the observation that Dox-conjugated to p(DMAPS-ran-PEGMA) was able to induce apoptosis in HeLa cells. The p(DMAPS-ran-PEGMA) copolymers owe their exceptional characteristics to the combination of the mildly hydrophobic characteristics of the ammonium-propyl-sulfonate moieties and the nonfouling hydrophilic PEG chains.

Further refinements of the copolymers composition may

provide a variety of intracellular nanocarriers tools useful for live cell imaging, analysis of pharmacological function for drug discovery research, and possibly as drug delivery systems.


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ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on the ACS Publications web site. 1

H NMR  spectra of polymers, characterization of nanospheres, and additional in vitro

evaluation. (PDF)

AUTHOR INFORMATION Corresponding Author *N. M., E-mail: [email protected] *F. M. W., E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. Alexandre Moquin (McGill University) for his help for the evaluation of the molecular weight of copolymers and Prof. Ikuhiko Nakase (Osaka Prefecture University) for his helpful advice on cellular uptake mechanisms. N. M. acknowledges financial support from Grants-in-Aid for Scientific Research (#25350549) from the Japan Society for the Promotion of Science (JSPS). F.M.W gratefully acknowledges support from the World Premier International Research Center Initiative (WPI) MEXT, Japan.


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Polymers on Glass Slides. Langmuir 2006, 22, 10072-10077. 20. Dai, F.; Liu, W.; Enhanced Gene Transfection and Serum Stability of Polyplexes by PDMAEMA-Polysulfobetaine diblock copolymers. Biomaterials 2011, 32, 628-638. 21. Chen, C-Y.; Wang, H-L. Dual Thermo- and pH-Responsive Zwitterionic Sulfobetane Copolymers for Oral Delivery System. Macromol. Rapid Commun. 2014, 35, 1534-1540. 22. Zhai, S.; Ma, Y.; Chen, Y.; Li, D.; Cao, J.; Liu, Y.; Cai, M.; Xie, X.; Chen, Y.; Luo, X. Synthesis of an Amphiphilic Block Copolymer Containing Zwitterionic Sulfobetaine as a Novel pH-Sensitive Drug Carrier. Polym. Chem. 2014, 5, 1285-1297. 23. Qiu, X. –P.; Winnik, F. M. Synthesis of α,ω-Dimercapto Poly(N-isopropylacrylamide) by RAFT

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Grinberg, V. Ya.; Menger, F. M. Contrasting Behavior of Zwitterionic and Cationic Polymers Bound to Anionic Liposomes. Langmuir 2007, 23, 7539-7544. 29. Hu, H.; Wang, X. B.; Xu, S. L.; Yang, W. T.; Xu, F. J.; Shen, J.; Mao, C. Preparation and Evaluation of Well-defined Hemocompatible Layered Double Hydroxide-poly(sulfobetaine) nanohybrids. J. Mater. Chem. 2012, 22, 15362-15369. 30. Pissuwan, D.; Boyer, C.; Gunasekaran, K.; Dais, T. P.; Bulmus, V. In Vitro Cytotoxicity of RAFT Polymers. Biomacromolecules 2010, 11, 412-420. 31. Kosuge, M.; Takeuchi, T.; Nakase, I.; Jones, A. T.; Futaki, S. Bioconjugate Chem. 2008, 19, 656-664. 32. Chu, Z.; Miu, K.; Lung, P.; Zhang, S.; Zhao, S.; Chang, H-C.; Lin, G.; Li, Q. Rapid Endosomal Escape of Prickly Nanodiamonds: Implications for Gene Delivery. Sci. Rep. 2015, 5, 11661. 33. Verna, A.; Uzun, O.; Hu, Y.; Hu Y.; Han, H-S.; Watson, N.; Chen, S.; Irvine, D. J.; Stellacci, F.

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Monolayer-protected

Nanoparticles. Nat. Mater. 2008, 7, 588-595. 34. van Dooremalen, C.; Koekkoek, J.; Ellers, J. Temperature-induced Plasticity in Membrane and Strage Lipid Composition: Thermal Reaction Norms Across Five Different Temperatures. J. Insect Physiol. 2011, 57, 285-291. 35. Banerji, S. K.; Hayes, M. A. Examination of Nonendocytotic Bulk Transport of Nanoparticles Across Phospholipid Membranes. Langmuir 2007, 23, 3305-3313. 36. Jiao, C-Y.; Delaroche, D.; Burlina, F.; Alves, I. D.; Chassaing, G.; Sagan, S. Translocation and Endocytosis for Cell-penetrating Peptide Internalization. J. Biol. Chem. 2009, 284, 33957-33965.


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Jones, A. T. Temperature-, Concentration- and Cholesterol-dependent Translocation of L- and D-octa-arginine Across the Plasma and Nuclear Membrane of CD34(+) Leukaemia Cells. Biochem. J. 2007, 403, 335-342. 47. Tayagi, M.; Rusnati, M.; Presta, M.; Giacca, M. Internalizatio of HIV-Tat Requires Cell Surface Heparan Sulfate Proteoglycans. J. Biol. Chem. 2001, 276, 3254-3261. 48. Nakase, I.; Tadokoro, A.; Kawabata, N.; Takeuchi, T.; Katoh, H.; Hiramoto, K.; Negishi, M.; Nomizu, M.; Sugiura, Y.; Futaki, S. Interaction of Arginine-rich Peptides with Membraneassociated

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Doxorubicin Loaded Star-shaped Micelles with Hydrophilic Zwitterionic Sulfobetaine Segments. Biomaterials 2014, 35, 4517-4524.

Figure captions Figure 1. a) Chemical structure of p(DMAPS-ran-PEGMA) b) Schematic illustration of p(DMAPS-ranPEGMA) nanosphere. c) Hydrodynamic diameter and the distribution of (2k-2.5) 30K nanospheres in PBS at 37ºC. d) Negative-stained TEM image of (2k-2.5) 30K nanospheres. The nanospheres was dispersed in PBS (10 mg/mL) and dropped onto cupper grid, then stained with 2% phosphotungstic acid solution. e) Fluorescence of 8-anilino-1-naphthalene sulfonate (ANS) in p(DMAPS-ran-PEGMA) solution.

The polymer was dispersed in PBS with the

concentration of 0 ~10 mg/mL. [ANS] = 1.0 µM. Figure 2. a) Cell viability of HeLa cells treated with p(DMAPS-ran-PEGMA)s. HeLa cells were incubated with nanospheres in the presence of FBS at 37ºC. Values represent mean ± s.d., n= 6. b) Fluorescence microscopic images of Fluorescein-labeled (2k-2.5)30K (upper) and (2k2.5)51K (lower) nanospheres to HeLa cells in the presence (left) or absence (right) of FBS. c) Flow cytometry data for cellular uptake of Fluorescein-labeled p(DMAPS-ran-PEGMA) (2k2.5)30K and (2k-2.5)51K nanospheres in the presence or absence of FBS. Values represent mean ± s.d., n= 6. Figure 3. a) (left) CLSM images with the horizontal and vertical section image of Fluorescein(2k-2.5)30K nanospheres and the colocalization of nanospheres (green) and MitoTracker (red) in HeLa cells at 37 ºC (upper) and 4 ºC (lower). b) Flow cytometry data for cellular uptake of


37

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 44

Fluorescein-labeled p(DMAPS-ran-PEGMA) nanospheres ((2k-2.5)30K and (2k-2.5)51K) at 4 ºC and 37 ºC. Values represent mean ± s.d., n= 6. Effects of endocytosis inhibitors on c) (2k2.5)30K, and d) (2k-2.5)51K nanospheres in the presence of FBS at 37 ºC. None: No inhibitors. Values represent mean ± s.d., n= 6. e) Differential interference contrast microscope and CLSM image of CHO-K1 cells (upper) and the mutant pgsA-745 cells (lower). The schematics represent their cellular surfaces with or without oligosaccharides. f) Concentration dependence of nanospheres on cellular uptake. HeLa cells were co-incubated with (2k-2.5)30K for 1 h at 37 ºC. (inset) The flow cytometry data. Figure 4. a) Schematic illustration of the translocation of (2k-2.5)30K nanospheres in the presence of various endocytotic inhibitors. b) Proposed mechanism of intracellular uptake of p(DMAPS-ran-PEGMA) nanospheres. Figure 5. a) Localization of Free Dox (Left, 10 µg/mL) or Dox-conjugated p(DMAPS-ranPEGMA) (Right, 1 mg/mL: containing 10 µg/mL conjugated Dox) in HeLa cells after 24 h incubation at 37 ºC. b) Flow cytometry data for cellular uptake of free Dox and Dox-conjugated p(DMAPS-ran-PEGMA). Values represent mean ± s.d., n= 6. c) Relative cell viability in the presence of free Dox or Dox-conjugated p(DMAPS-ran-PEGMA). Values represent mean ± s.d., n= 6. The concentration was based on free Dox or the conjugated Dox estimated by UV-Vis spectroscopy. d) Morphological nuclear changes of HeLa cells after 24 h incubation. (1) Nontreatment. (2) p(DMAPS-ran-PEGMA) (1 mg/mL). (3) free Dox (10 µg/mL). (4) Doxconjugated p(DMAPS-ran-PEGMA) (1 mg/mL: containing 10 µg/mL conjugated Dox).


38

Page 39 of 44

Table of Contents graphic

Zwitterionic copolymer nanosphere

37 ºC

O

O

4 ºC O O

Cellular membrane

N

O SO O

O

O

N O SO O

O O

N O S O O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

N

O SO O

Translocate


43

Biomacromolecules

Morimoto et al. Figure 1

a)

O x

HO

O

O O S O

O

N+

O

S

y

S S O

O

z

b) O S O

O

N

CH3 CH3 CH2

Sulfobetaine units

PEG chains

c) Frequency [%]

15

10

5

0

1

10

100

1000

Hydrodynamic diameter [nm]

d)

e) Relative fluorescent intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 44

250 10 mg/mL 3 mg/mL

200

1 mg/mL 0.3 mg/mL 0.1 mg/mL

150

0.03 mg/mL 0.01 mg/mL

100

0.003 mg/mL 0 mg/mL

50 0 400

450

500

550

600

650

Emission wavelength (nm)


Page 41 of 44


a) 100

Relative Cell Viability [%]

(0.5k-10)35K (1k-5)36K (2k-2.5)19K

80

(2k-2.5)30K (2k-2.5)51K

60

(2k-2.5)87K

40

20

0 0

0.0003 0.001 0.003 0.01

0.03

0.1

0.3

1

Concentration of P(DMAPS-ran-PEGMA) [g/L]

b)

FBS (+)

FBS (-)

(2k-2.5)30K 50 µm

(2k-2.5)51K

c)

10000006 10

Mean F. I. [a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1000005 10

10000 104

1000 103

2 10 100

FBS (+) FBS (-) FBS (+) FBS (-)

Cell only

(2k-2.5)30K (2k-2.5)51K


Biomacromolecules


a) (2k-2.5)30K

MitoTracker®

Merge

b) 108

100000000

4 °C 37 °C

107

37 C

Mean F.I. [a.u.]

10000000

50 µm 50 µm

106

1000000

105

100000

104

10000

103

1000

102

100

10110 100

1

Cell only

50 µm

d) (2k-2.5)51K

c) (2k-2.5)30K 200 600

Mean F. I. [%]

200

150 550

100

50

0

(2k-2.5)30K (2k-2.5)51K

150

100

50

0

None 3µM10µM 5mM10mM11µM 27µM 0.3M 0.45M

None 3µM10µM 5mM10mM11µM 27µM 0.3M 0.45M

Cyto D M-β-CD Nystatin Sucrose

e)

Cyto D M-β-CD Nystatin Sucrose

CHO-K1

50 µm

pgsA-745

1000000 106

Mean F.I. [a.u.]

f)

100000 105

10 10000

R² = 0.9999

10 1000

200

4

3

102

100

－－－－－－

Count

4C

Mean F. I. [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 44

100

101

10

100

0

103

104

F. I.

0 mg/mL 0.01 mg/mL 0.03 mg/mL 0.1 mg/mL 0.3 mg/mL 1 mg/mL

105

106

1

0.01


0.1

Concentration of (2k-2.5)30K [mg/mL]

1

Page 43 of 44


a)

Nanosphere

Pathway

Clathrin-mediated Caveolae-mediated endocytosis endocytosis

Phagocytosis

Macropinocytosis

Inhibitor Cytochalasin D

b)

Hypertonic Methyl-β-cyclodextrin medium Nystatin

O

O

O O

O

O

O

O

N O

O

O

Loosely packed nanosphere

S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

N

O SO O

N O SO O

N

O SO O

Outer leaflet

~ 5 nm

Interaction between phospholipid and sulfobetaine unit in p(DMAPS-ran-PEGMA) nanosphere

Loosen nanosphere by side-diffusion of phospholipids

Formation of inverted micelle-like complex between phospholipids and p(DMAPS-ran-PEGMA)

p(DMAPS-ran-PEGMA)


Biomacromolecules


b)

100000 105

Mean F.I. [a.u.]

a)

10000 104

1000 103

2 100 10

1010 1

50 µm Dox

101 0

Dox-(2k-2.5)19K

Dox

d)

c) Relative Cell viability [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 44

Dox Dox-(2k-2.5)19K

120

Dox-(2k-2.5)19K

(2k-2.5)19K

Control (1)

(2)

(3)

(4)

100 80 60 40 20 0 0

0.003 0.01 0.03 0.1

0.3

1

Doxorubicin [µg/mL]

3

10

100#µm

Dox


Dox-(2k-2.5)19K

Membrane Translocation and Organelle-Selective Delivery Steered

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