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PEGylated Albumin based Polyion Complex Micelles for protein delivery Yanyan Jiang, Hongxu Lu, Fan Chen, Manuela Callari, Mohammad Pourgholami, David L. Morris, and Martina Heide Stenzel Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01537 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 31, 2016

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PEGylated Albumin based Polyion Complex Micelles for protein delivery Yanyan Jiang,a Hongxu Lu,a Fan Chen,a Manuela Callari,a,c Mohammad Pourgholami,b David L. Morris,b Martina. H. Stenzel*,a a

Centre for Advanced Macromolecular Design, School of Chemistry and School of Chemical

Engineering, University of New South Wales UNSW, Kensington NSW 2052, Australia Email: [email protected]. b

Cancer Research Laboratory, Department of Surgery, St George Hospital, Sydney, NSW 2217, Australia. c

Liverpool Hospital Clinical School, and Molecular Medicine Research Group, University of Western Sydney, Sydney NSW 2170, Australia

Abstract An increasing amount of therapeutic agents are based on proteins. However, proteins as drug have intrinsic problems such as their low hydrolytic stability. Delivery of proteins using nanoparticles has increasingly been of the focus of interest with polyion complex micelles, prepared from charged amphiphilic block copolymer and the oppositely charged protein, as an example of an attractive carrier for proteins. Inspired by this approach, a more biocompatible pathway has been developed here, which replaces the charged synthetic polymer with an abundant protein such as albumin. Although bovine serum albumin (BSA) was observed to form complexes with positive charged proteins directly, the resulting protein nanoparticle were not stable and aggregated to large precipitates over the course of a day. Therefore, maleimide functionalized poly(oligo (ethylene glycol) methyl ether methacrylate) (MI-POEGMEMA) (Mn=26,000 g/mol) was synthesized to generate a polymer-albumin conjugate, which was able to condense positive charged proteins, here lysozyme (Lyz) as a model. The PEGylated albumin polyion complex micelle with lysozyme led to nanoparticles between 15-25 nm in size 1

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depending on the BSA to Lyz ratio. The activity of the encapsulated protein was tested using Sprouty 1 (C-12) (Spry1) proteins, which can act as an endogenous angiogenesis inhibitor. Condensation of Spry1 with the PEGylated albumin could improve the anticancer efficacy of Spry1 against the breast cancer cells lowering the IC50 value of the protein. Furthermore, the high anticancer efficacy of the POEGMEMA-BSA/Spry1 complex micelle was verified by effectively inhibiting the growth of three-dimensional MCF-7 multicellular tumour spheroids. The PEGylated albumin complex micelle has great potential as a drug delivery vehicle for a new generation of cancer pharmaceuticals.

Keywords Polymer-protein conjugates, polyion complex micelle, albumin, protein delivery

Introduction Proteins play an irreplaceable role in life, taking part in all vital life activities at the molecular level. To date, protein drugs have become increasingly common in modern medical care.1 Although proteins show promising potential as tumor suppressors, their intrinsic properties may prevent their administration. The development of new drug carriers is therefore requisite to deliver proteins in their active forms to specific cells and organs. Major barriers in delivering proteins for therapeutic purposes include issues like their large size, their surface charge and in particular their hydrolytic instability.2 When administered, most proteins are prone to quick degradation. Also low cellular uptake and slow endosomal escape can present a major obstacle.1 To combat these shortcomings, a range of techniques have been developed, which include the fabrication of polymer or lipid nanoparticles.3 Mode of interaction between protein and polymer can be either by the formation of a covalent bond, electrostatic forces or even van der Waals forces among others. Among the current approaches, the formation of polyion complexes represents a rational way to protect fragile proteins in a layer of (bio)polymers.4 Electrostatic charges are formed at ambient temperature using benign conditions thus ensuring high stability of the complex. Typically, polyion complex micelles (PIC) are created from block copolymers, where one block is responsible for binding with the oppositely charged payload while the other block ensures good water-solubility and cargo protection.5, 6 The block copolymers are usually water-soluble, but once they condense an oppositely charged molecule they associate into nano2


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sized micelles. This approach is common for the delivery of oligonucleotides,7 polysaccharides such as heparin8 and proteins.4, 9, 10 Kataoka and co-workers pioneered this area by designing a range of charged block copolymers which undergo condensation with proteins to create PICs with high protein activity.4, 9, 10

Acknowledging the need to trigger the release of proteins on demand, stimuli-responsive PIC

were created. Elegant approaches include the degradation of functional groups in acidic environment resulting in charge-conversion, which lead to the repulsion of the protein and thus an accelerated release.11

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Combined with a light-responsive feature, PIC micelles allow the

targeted release of the active protein at the desired site only opening the doors to highly specific carriers.13 Next to pH-responsive features, the PIC micelle can be unloaded by changes in glucose concentration14 and oxidation state.15 In an alternative approach, non-bioactive proteins were used to transport a bioactive cargo.16 In particular albumin has been identified as a safe drug delivery vehicle that is now widely used to deliver various drugs.7, 17 Besides its capability to deliver hydrophobic drugs,18 albumin also displays a high negative surface charge that allows polyion complex formation with biomolecules such as enzymes.19 The resulting particles however are usually well above 200 nm since the condensation process is sufficiently stabilized to allows the formation of small nanoparticles. In this manuscript, the approach to deliver therapeutic proteins using polyion complex micelles will be combined with the albumin technology to deliver drugs. Improvement to existing strategies will be the replacement of the usually charged synthetic polymer block in polyion complex micelles by a biocompatible protein, viz albumin. In addition, existing albumin technologies are improved by the addition of a stabilizing PEG-based polymer. Albumin will therefore take on the role of the charged block copolymer to condense with the opposite charged bioactive protein. The advantage of albumin over synthetic polymer is the easy digestion of the carrier in the endosomes by proteases, which will enable the liberation of the drug.20 Multi-step synthesis procedure to create a stimuli-responsive polymer is therefore not required. MIPOEGMEMA has been conjugated to albumin to stabilize the nanoparticle during nanoparticle formation leading to a hydrophilic corona and a protein core. (Scheme1). The drug delivery system was tested using the positive charged protein Sprouty1 (C -12) (Spry1), which is currently under investigation for its role in cancer.21 The hybrid polyion complex micelles were 3


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tested in their ability to deliver Spry1 using the established 2D breast cancer cell (MDA-MB-231 and MCF-7) model, followed by a 3D MCF-7 multicellular tumor spheroid model.

Scheme1 Preparation of POEGMEMA-BSA/Spry1 (or Lyz) complex micelles, including RAFT polymerization of OEGMEMA, end-group deprotection via Retro-Diels-Alder reaction, conjugation of MI-POEGMEMA and albumin and formation of complex micelles. 4


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Experimental Section Materials Unless otherwise specified, all chemicals were of reagent grade and used as received. Anhydrous methanol (Sigma-Aldrich, 99.8%), benzyl chloride (Aldrich, 99%), bovine serum albumin (BSA, Sigma, >96%), chloroform-D (CIL, 99.8%), Sprouty 1(C-12) (Santa Cruz), lysozyme from chicken egg white (Sigma-Aldrich, >90%), diethyl dimethyl sulfoxide (DMSO, Ajax, 98%), elemental sulphur (Ajax, 98%), ethanolamine (Ajax, 97%), furan (Aldrich, >99%), maleic anhydride (Fluka, >99%), N,N-dimethylformamide (DMF, Ajax, 99.8%), sodium phosphate dibasic (Sigma-Aldrich, 98%), sodium phosphate monobasic (Sigma-Aldrich, >99%), sodium hydroxide (Aldrich, 98%), sodium chloride (Univar, reagent), 2,2',2'',2'''-(ethane-1,2diyldinitrilo), ethylene diamine tetraacetic acid (EDTA, Sigma-Aldrich, reagent), Oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA, Mn = 300 g/mol, Aldrich, reagent), silica gel (SigmaAldrich, 60 Å,70-230 mesh), tetrahydrofuran (THF, Fisher Scientific, HPLC Grade, >99.9%), and

toluene (Ajax, 99%) were used as received. The synthesis of 4-cyanopentanoic acid dithiobenzoate (CPADB) and the subsequent furan protected maleimide-terminated product (MCPADB) are described elsewhere.22 Synthetic Procedures Syntheses Synthesis of POEGMEMA via RAFT polymerization. OEGMEMA (3 g, 0.01 mol), RAFT agent MCPADB (0.049 g, 1 × 10-4 mol), and initiator AIBN (0.0025 g, 1.5 × 10-5 mol) were mixed to achieve a ratio of [Monomer]:[RAFT]:[Initiator]=100:1:0.15. The reaction mixture was purged with nitrogen for 40 min at 0 °C. The polymerization was carried out in bulk at 70 °C for 3.5 h. The reaction was terminated by placing the vial into ice bath for 5 min and introducing air. Finally, the polymer was precipitated in anhydrous diethyl ether and dried under reduced pressure. The conversion and the theoretical molecular weight (Mn) of the polymer were determined by 1H NMR (CDCl3). The polydispersity index (Ð) was measured by gel permeation chromatography (GPC) using DMAc as the mobile phase.

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Retro-Diels-Alder reaction. 1.5 g polymer was dissolved in 25 mL toluene and the solution was refluxed at 110 °C for 7 h to remove the furan protecting group. The solvent was evaporated and the product was further dried under vacuum to give the MI-POEGMEMA. The formation of maleimide group was confirmed by 1H NMR with the presence of new proton peak at 6.75 ppm and the disappearance of the signals at 6.5, 5.3 and 2.8 ppm. Conjugation of albumin and MI-POEGMEMA. Stock solutions of both MI-POEGMEMA (Mn = 26,000 g/mol) and BSA were prepared where each substance was dissolved in PBS (pH7.4, with 20 mM EDTA) buffer with a concentration of 5 mM. The two solutions were then mixed at different ratios at room temperature. The molar ratios of BSA and MI-POEGMEMA were 5:1, 2:1, 1:1, 1:2 and1:5, respectively. In each sample, the amount of BSA was kept constant and its final concentration was 0.5 mM in 1 mL of mixture. All the mixtures were stirred for 18 h to facilitate the thiol-ene click reaction between the thiol group on the BSA and the maleimide group on POEGMEMA polymer chain. The product was purified by ultrafiltration (MWCO=30,000 Da), aiming to remove the EDTA and the unreacted polymer in the raw product. The purified POEGMEMA-BSA conjugation solutions were brought to the same albumin concentration of 50 µM in PBS buffer (pH7.4). The conjugation products were characterized with SDS-PAGE, Ellman’s assay. Preparation of BSA/lysozyme or POEGMEMA-BSA/lysozyme polyion complex micelles. 500 µL of lysozyme solution in water at different concentrations was added drop wise to 500 µL of BSA (3.3 mg/mL) or POEGMEMA-BSA (the concentration of BSA=3.3 mg/mL) solution. The mixture was kept stirring for 4 h to yield polyion complex micelle of different ratios between the two proteins. DLS and TEM were used to characterize the size, distribution, zeta potential and shape of the particles. Preparation of POEGMEMA-BSA/Spry1 polyion complex micelles. 500 µL of sprouty1 solution (1.75µg/mL) in water was added drop wise to 500 µL of POEGMEMA-BSA (the concentration of BSA is 3.3 µg/mL for the 1 to 1 sample and 1.65 µg/mL for the 2 to 1 sample) solution. The mixture was kept stirring for 4 h to yield polyion complex micelle. DLS was used to characterize the size, distribution and zetapotential of the particles. Synthesis of FITC labeled lysozyme. A 2mg/mL lysozyme solution was made using 14 mg lysozyme in 7 mL MilliQ water. A 1mg/mL fluorescein isothiocyanate (FITC) solution was 6


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made using 2 mg of FITC in 2 mL DMSO. The FITC solution was dropped into the lysozyme solution. After this the conjugation reaction was conducted with stirring at 4 oC overnight. The mixture was dialyzed at 4 oC for 3 days to obtain a bright yellow solution. After lyophilization, the pale yellow powder was obtained. The molar ratio of lysozyme to FITC was determined using UV-Vis. The FITC-lysozyme was then used to condense the POEGMEMA-BSA according to aforementioned procedure. Analysis Proton nuclear magnetic resonance spectroscopy, 1H NMR. All NMR measurements were performed on a Bruker DPX-300 with a 1H/ X inverse broadband z gradient BBI probe at 300 MHz frequency and 16 scans as default. Samples were dissolved and analysed in deuterated chloroform (CDCl3). DOSY NMR was carried out on Bruker AVANCE III HD 600 MHz in PBS buffer (Ph7.4) which was made from deuterated water (D2O). DMAc gel permeation chromatography, DMAc GPC. GPC measurements were performed using a Shimadzu modular system containing a DGU-12A degasser, an LC-10AT pump, a SIL-10AD automatic injector, a CTO-10A column oven and a RID-10A refractive index detector. A Phenomenex 50×7.8 mm guard column and four 300×7.8 mm columns (500, 103, 104, 105 Å pore size, 5 µm particle size) were used for analyses. DMAc (HPLC grade, 0.05% w/v BHT, 0.03% w/v LiBr) with a flow rate of 1 mL/min was used as the mobile phase. The injection volume was 50 µL. The samples were prepared by dissolving the samples in DMAc at a concentration of 2-3 mg/mL, followed by filtration through a 0.45 µm filter. The unit was calibrated using commercially available linear polystyrene standards (0.5-1000 kDa, Polymer Laboratories). Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was performed using a premixed electrophoresis buffer which contains 25 mM Tris, 192 mM glycine and 0.1% SDS (Tris/Glycine/SDS buffer) to determine the conjugation efficiency between the HSA and the PDMAEMA. A mixture of 8 proteins (6.5K-200K) (Bio-Rad) was used as molecular weight standards. Samples and protein molecular weight marker were diluted by a pre-made Laemmli sample buffer with reducing agent and then heated at 95 °C for 5 min to denature the protein. Commercially available 4-20% precast polyacrylamide gel, 8.6 × 6.8 cm 7


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(W × L) was then loaded with the protein samples and ran at a constant 120 V for 70 min. The samples were stained using Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad) for 2 h and washed with premixed eluent (ethanol: water: acetic acid = 5:4:1 (v:v:v)). Gel images were recorded using a Bio-Rad GS-800 calibrated densitometer. Native BSA was also used as a control. Dynamic light scattering, DLS. The particle size, size distribution and zeta potential of polyion micelles were measured using DLS. Concentration of albumin in relevant samples is 1.5 mg/mL. The data were obtained using Malvern Nano-ZS as particle size analyzer (laser, 4 mW, λ=632 nm; measurement angle 12.8° and 175°). Samples run for at least three times at 25 °C. Transmissionelectron microscopy, TEM. TEM analysis was performed to investigate the morphology of the nanoparticles with a Philips CM200 facility operating at 200 kV. Samples were prepared by placing a drop of solution on carbon-coated copper grids and draining the excess sample with filter paper. Samples were stained with uranyl acetate (2% aqueous solution) and then air-dried for 2 h. Cellular uptake observed using laser scanning confocal microscopy. MCF-7 breast cancer cells were seeded in 35mm Fluorodish (World Precision Instruments) at a density of 60,000 cells per dish and cultured for 3 days with DMEM medium supplemented with 10% fetal bovine serum. FITC-labeled micelle solutions were loaded to MCF-7 cells at a working concentration of 100 µg/mL and incubated at 37oC for 2 h. After incubation, the cells were washed thrice with PBS (pH 7.4). The cells were then stained with Hoechst 33342 for 10 min followed by staining with 100 nM LysoTracker Red DND-99 (Invitrogen) for 1 min. The dye solution was quickly removed and the cells were gently washed with PBS. Finally, the cells were mounted in PBS and observed under a laser scanning confocal microscope system (Zeiss LSM 780). The system was equipped with a Diode 405-30 laser, an argon laser and a DPSS 561-10 laser (excitation and absorbance wavelengths: 405 nm, 488 nm and 561 nm, respectively) connected to a Zeiss Axio Observer. The ZEN2011 imaging software (Zeiss) was used for image acquisition and processing. Flow Cytometry. MDA-MB-231 and MCF-7 cells were seeded in 6-well plates at a density of 5×105 cell/well and incubated at 37 °C with 5 % CO2 for 1 day prior to micelle 8


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treatment. During treatment, the medium was replaced with DMEM cell culture medium containing FITC-labeled micelles (40 µg/mL working concentration, excitation wavelength is 494 nm and the emission wavelength is 512 nm) at 37 °C for 2 h. The cell monolayer was washed 3 times with cold PBS and treated with trypsin/EDTA to detach the cells. The cells were collected, centrifuged and resuspended in cold serum free culture medium. The cells suspensions were used for flow cytometry analysis on BD FACSCanto™ II Analyser (BD Biosciences, San Jose, USA), collecting results from at least 50,000 events. Live cell imaging and data analysis. MCF-7 cells were seeded in 6-well glass bottom plates at a density of 3×105 cell/well and incubated at 37 °C with 5 % CO2 for 1 day prior to micelle treatment. The old medium in the cell culture plate was discarded and replaced with 1 mL of fresh twice-concentrated DMEM medium and 1 mL of sterile samples (MilliQ water as a control, Spry1 and POEGMEMA-BSA/Spry1 polyplex). Subsequently, the plate was subjected to the Nikon Eclipse TiE life cell imaging microscope to monitor the cell growth for 48 h. Automated time-lapse image acquisition was used for periods of every two hours under the control of NIS-Elements AR software (Nikon). NIS-Elements AR software was used for imaging analysis. This chamber was kept at 37 oC and supplemented with 5% CO2 throughout the entire experiment. Results shown are representatives of three independent experiments. Cytotoxicity testing. The cell suspensions of MDA-MB-231 and MCF-7 cell lines were seeded into 96-well cell culture plates at a cell density of 40,000 cells/mL, 100µL/well and incubated for 1 day at 37°C and 5% CO2. The medium in the cell culture plate was then discarded and replaced with 100 µL of fresh twice-concentrated DMEM medium and the cells were incubated with the addition of 100 µL of micelle solutions for 72 h. Before loading into the cell culture plates, the micelle solutions were prepared by sterilization with UV irradiation for 20 min and then serial diluted (in MilliQ water) where each micelle solution was half the concentration of the solution before it. The cell viability was then determined with sulforhodamine B (SRB) assay. The culture medium was discarded and 100 µL of 10% TCA was added to each well, followed by incubation of the plates for 30 min at 4 °C. The supernatant was discarded and the plates washed 5 times with DI water. 100 µL of SRB solution 0.4% (w/v) in 1% acetic acid was added to each well, and the plates were incubated for 15 minutes at room temperature. After staining, any unbound dye was removed by washing 5 times with 1% acetic 9


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acid and the plates were air-dried. Bound stains were solubilised with 200 µL of 10 mM Tris buffer and absorbance was measured on a Bio-Rad BenchMark microplate reader (λ = 490 nm), with data analysed and plotted using GraphPad Prism 6.0. Breast multicellular tumour spheroid (MCTS) preparation and drug treatment. The cell density of MCF-7 suspension was adjusted to 1.5×105 cells/mL. 10 µL of cell suspension was gently dropped onto the lid of a 100 mm cell culture dish. The lid was then slowly turned over and placed onto the dish filled with 10 mL sterile PBS to maintain the humidity of inner dish chamber. The cells were incubated and kept undisturbed at 37ºC at 5 % CO2. After culturing for 7 days, the MCTS were transferred to a 96-well suspension culture plate (Corning) and cultured for 1 day before further experiments. Sterile MilliQ water (as a control), Spry1 and POEGMEMA-BSA/Spry1 complex micelles were added to the spheroids which had been grown for 1 day in the 96-well suspension culture plates. The concentration of the Spry1 in each well was 571 nM. Control and complex micelles were loaded again after 3 days. The spheroids were incubated at 37 ºC for 6 days. The morphology of the MCTS at 0 day and 6 days was recorded using a Leica DM IL inverted microscope equipped with a ProgRes® Scan camera (Warner Instruments, LLC) and the sizes were analyzed using the software ProgRes® CapturePro.

Results and discussion The covalent conjugation between polymer and albumin is a well-known procedure 23-25 and an array of techniques is already available.26, 27 The conjugation to Cys-34 of albumin includes the reaction with maleimides,7, 17, 28 activated disulfides (pyridyl disulfide group),29 and vinyl sulfones30. The polymers that were attached to albumin range from the hydrophobic PMMA22 to the hydrophilic PHPMA,17 PNIPAAm28 and PEO.31 MI-POEGMEMA was prepared with the help of reversible addition fragmentation chain transfer polymerization (RAFT)32 and a furan protected maleimide chain transfer agent.27 The subsequent deprotection yielded an α-functional polymer (Scheme 1).33 The conversion of OGMEMA monomer was determined by 1H NMR (CDCl3) and the molecular weight and the polydispersity were evaluated by DMAc GPC. After 3.5 h of reaction time, POEGMEMA86 homopolymer with a monomer conversion of 87% and a theoretical molecular weight of 26,000 10


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g/mol (Mn,gpc=31,600 g mol-1 and Ð= 1.2, ESI, Figure S1) was obtained. Successively, the deprotection of the furan-protected polymer was conducted via a retro Diels-Alder reaction by refluxing the protected polymers in toluene under nitrogen atmosphere (Scheme 1). The success of the Retro-Diels-Alder reaction was confirmed using 1H-NMR where the signals at 6.5, 5.3 and 2.8 ppm disappeared and were replaced by a signal at 6.75 ppm belonging to the newly formed double bond (ESI, Figure S2). The reactive polymer then underwent conjugation with the only available thiol functional group on Cys-34 residual of albumin to create the POEGMEMA-BSA hybrid macromolecules.34 The conjugation efficiency of MI-POEGMEMA to albumin targeted the reactive thiol Cys-34 on albumin. The amount of active Cys-34 of the commercial BSA could be measured using Ellman’s reagent35 and it was determined that the albumin used in this work contained 60% reactive Cys-34. MI-POEGMEMA was reacted with albumin at various molar ratios (5:1, 2:1, 1:1 0.5:1 and 0.2:1) at pH 7.4 in PBS buffer at a constant BSA concentration. The conjugation of BSA and MI-POEGMEMA was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by the presence of new band at molecular weight of around 92 kDa (66 kDa for BSA and 26 kDa for MI-POEGMEMA). As the MIPOEGMEMA content decreases the band at around 92 kDa lost its intensity which indicates that less conjugate was formed. Ellman’s assay (Figure S3), which is used to determine the amount of active Cys-34 in the BSA, was employed to analyze the conjugation efficiency quantitatively. As shown in Table 1, the relative amount of BSA reacted in each mixture was calculated to be between 63% (5 to 1) and 16% (0.2:1) depending on the amount of MI-POEGMEMA. An excess of MI-POEGMEMA therefore leads to the complete reaction of active BSA fraction.

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Figure 1 SDS-PAGE image of MI-POEGMEMA and BSA conjugation with different POEGMEMA and BSA ratios. [A] protein standard, molar ratios of POEGMEMA to albumin 5:1 [B], 1:1 [C] and 0.2:1 [D]; [E] BSA Table 1 Conjugation efficiency of MI-POEGMEMA and BSA with different molar ratios according to Ellman’s assay. MI-POEGMEMA to BSA molar ratio 5:1 2:1 1:1 0.5:1 0.2:1

BSA conjugation efficiency/ % (% of BSA with available SH functionality consumed) 63 (~100%) 56 (93%) 46 (76%) 31 (51%) 16 (26%)

MI-POEGMEMA conjugation efficiency/ % 12.6 28 46 62 80

Further evidence of the occurring conjugation between MI-POEGMEMA and BSA was obtained by measuring the diffusion coefficients in D2O PBS buffer. The diffusion parameters were measured using DOSY NMR and the diffusion coefficients were calculated. Results are summarized in Table 2. The comparison of the diffusion coefficients of POEGMEMA-BSA conjugates (D= 8.31*10-11 m2s), BSA (D= 2.29*10-10 m2s) and MI-POEGMEMA (D= 1.28*10-10 m2s), which is indirectly related to the hydrodynamic radius confirms the successful conjugation. In addition, DOSY NMR of POEGMEMA-BSA was found to be a suitable technique to visualize the different species in solution. In agreement with the SDS-PAGE, free BSA and POEGMEMA-BSA conjugates were identified (Table 2). Table 2 Diffusion coefficient of MI-POEGMEMA, POEGMEMA-BSA conjugates and BSA.

2

Log (m s) Diffusion coefficient (m2s)

MI-POEGMEMA -9.89 1.28*10-10

POEGMEMA-BSA -10.08 8.31*10-11

BSA -9.64 2.29*10-10

The cytotoxicity of the various polymer-protein conjugates was tested against breast carcinoma cell lines (MDA-MB-231 and MCF-7), which revealed that the 2to1 POEGMEMABSA conjugates showed slight toxicity, probably due to excess reactive maleimide. In contrast, the 1to1 POEGMEMA-BSA conjugates were entirely non-toxic (Figure S4). For further investigations, conjugates based on equimolar molar ratio of MI-POEGMEMA and BSA were 12


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used since this pathway minimized the amount of unreacted BSA while keeping the amount of added MI-POEGMEMA at a minimum due to the undesired toxicity caused by the MIPOEGMEMA according to Figure S4. The resulting conjugate was isolated using a MWCO=30,000 ultrafiltration tube, which removed large amounts of unreacted polymer. However, traces of MI-POEGMEMA were still present. The final mixture was used as is. Prior to testing of the encapsulation of therapeutic proteins, lysozyme (Lyz) (Isoelectric point IEP= 11.3) was used as the cationic protein model to form the polyplex with the POEGMEMA-BSA macromolecule at different molar ratio (POEGMEMA-BSA/Lyz polyplex). To be able to evaluate the effect of the conjugated POEGMEMA chain, BSA without conjugated polymer was condensed with Lyz and used as control. Dynamic light scattering (DLS) analysis was employed to determine the hydrodynamic diameter and the zeta potential of the two types of albumin polyplex (Figure 2a and b, Table S2 and Figure S5). The concentration of BSA in both samples was kept constant. The measured hydrodynamic diameter of the BSA/Lyz complex was small and the result fluctuated noticeably, which indicated the absence of well-defined particles. The hydrodynamic diameter did not change significantly with increasing Lyz concentration although the zeta potential indicated the neutralizing effect of the added Lyz. Left standing, the particles grew in size and settled to the bottom of the vial (Figure 2e). TEM analysis, recorded after the fresh solution was prepared, did not reveal any well-defined particles (Figure 2c). In contrast, the POEGMEMA-BSA/Lyz polyplex micelle was observed to form nanoparticals immediately with sizes ranging between 15 to 25 nm depending on the Lyz/BSA ratio. Furthermore, the zeta potential of the POEGMEMA-BSA conjugates and polyplex formed from these conjugates was close to zero indicating the shielding effect of POEGMEMA polymer. It was notable that with the increase of the Lyz concentration, the nanoparticles grew in size while simultaneously increasing in stability, in particular after reaching a molar ratio of Lyz to POEGMEMA-BSA of 2.5. TEM analysis further confirmed the quality of the resulting POEGMEMA-BSA/Lyz polyplex (Figure 2d) at the molar ratio of Lyz to POEGMEMA-BSA of 2.5. In contrast to BSA/Lyz, the POEGMEMA-BSA/Lyz was stable over an extended period of time as evidenced by a clear solution without precipitation (Figure 2e). The POEGMEMA polymer chain, conjugated to BSA, plays a crucial role in stabilizing the complex micelle and influencing particle size and shape.

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Figure 2 Hydrodynamic diameter and zeta potential variation of of (a) BSA/Lyz and (b) POEGMEMA-BSA/Lyz polyplex with the increase of lysozyme. TEM images of (c) BSA/Lyz and (d) POEGMEMA-BSA/Lyz polyplex (molar ratio lysozyme to BSA is 2.5). The scale bar was 100 nm. The concentration of BSA was 1.65 mg/mL in each sample. (e) The appearance of the BSA/Lyz polyplex solution, and (f) the appearance of the POEGMEMA-BSA/Lyz polyplex solution.

The formation of POEGMEMA-BSA/Lyz polyplex was also confirmed by DOSY NMR. According to the Stokes-Einstein equation, the diffusion coefficient is indirectly proportional to the hydrodynamic radius. Therefore, a decline in the measured diffusion coefficient can confirm the occuring aggregation. Although large micelles are not directly visible due the broadening of the proton resonance, the low measured diffusion coefficient displayed in Table 3 confirms the interaction between Lyz and POEGMEMA-BSA.

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Table 3 Diffusion coefficient of the POEGMEMA-BSA/Lyz mixture.

Lyz Log (m2s)

-9.1 2

Diffusion coefficient (m s)

7.9*10

-10

POEGMEMABSA

POEGMEMABSA/Lyz aggregate

-10.02

-10.32

9.5*10

-11

4.78*10-11

The cellular uptake of FITC labeled POEGMEMA-BSA/Lyz complex micelle by the breast cancer cells was monitored with confocal microscopy (Figure 3). The molar ratio of the FITCLyz and POEGMEMA-BSA conjugates was 2.5 and the properties of the FITC labeled micelle was close to the non-labeled one. (Table S3) The cell nuclei were stained blue with Hoechst 33342, lysosomes were stained red with LysoTracker DND-99 and Lyz was labeled with FITC. After incubation with MDA-MB-231 and MCF-7 cells for 2 h, the micelles (FITC-Lyz in green) were engulfed by the cells and could be located in the lysosomes (red) in both cell lines. It was evident that the Lyz wrapped in the albumin complex micelles were safely transported into the cells by endocytosis. In contrast FITC labelled Lyz did not show any cellular uptake as evidenced by the absence of any fluorescent activity inside the cell (ESI, Figure S6) Complementing the micrographs, the internalization of the POEGMEMA-BSA/Lyz into the MDA-MB-231 and MCF-7 cells was analyzed quantitatively by flow cytometry (Figure 4). The increase of fluorescence intensity indicates that the complex micelles were successfully internalized by the two cell lines, which is in agreement with Figure 3. Furthermore, the uptake efficiency of the POEGMEMA-BSA/Lyz polyplex was determined to be approximate 60% and 80%, respectively (Figure 4).

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Figure 3 Confocal microphotographs of MDA-MB-231 cells (top) and MCF-7 (bottom) after incubation with micelles at 37 oC for 2 hours. Lyz (Green) was labeled with FITC. Cell nuclei (blue) were stained with Hoechst 33342. Lysosomes (Red) were stained with LysoTracker Red DND-99. Scale bar is 20 µm.

Figure 4 Flow cytometry analysis of the FITC labelled lysozyme delivered using POEGMEMA-BSA polyion complex micelles internalized into the MDA-MB-231 (top) and MCF-7 (bottom) carcinoma cells after 2 h incubation.

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The investigations using Lyz provide crucial evidence that stable polyion complex micelles can be formed and the resulting nanoparticles are efficiently taken up by cancer cells. However, no evidence was provided so far on the stability of the delivered protein inside the carrier and its ability to free itself from the matrix. We therefore tested this technology platform using a bioactive protein. Protein of choice is Sprouty 1 (Spry1), which with an IEP of 8.65 is similar to Lyz positively charged at physiological conditions. With a molecular weight of 35 kDa it is bigger than Lyz, which has a molecular weight of 14 kDa. Since discovery of Sprouty (Spry) in 1998, numerous investigations have supported its promising role in regulating various physiological processes such as vasculogenesis, bone morphogenesis, and renal uretic branching, etc.36-38 The Sprouty family is composed of four members in mammals (Spry1–4), orthologous to a single Drosophila melanogaster (dSpry).39 Sprouty proteins are established down-regulators of receptor tyrosine kinase (RTK) signaling.38 Among many signaling pathways, receptor tyrosine kinases (RTKs) can activate the mitogen-activated protein kinase (MAPK) signaling pathway that subsequently leads to a variety of cellular changes, including proliferation, differentiation and motility.40 As a consequence, they restrain proliferation of many cell types and are conceivably expected to be deregulated in malignant conditions. Accordingly, Sprouty deregulation has been reported in different cancer types and shown to impact cancer development, progression, and metastasis.37 The expression of Spry1 and Spry2 was also found to be down-regulated in prostate, breast and melanoma cancer.41, 42 The role of Spry1 in human ovarian cancer has been subject to further investigation revealing that Spry1 was differentially expressed in normal ovarian cells versus ovarian cancer cell-lines.

43, 44

While the cancer-related characteristics of cancer cells are

multifactorial, it can be seen that ovarian cancer cells with a high expression of Spry1 are generally regarded as being non-aggressive, non-metastatic and even not easily taken up as orthotropic tumors in nude mice. However, cells lacking Spry1 are recognized as aggressive, chemo-resistant and metastatic cell-lines. Based on this point, it has been hypothesized that cellular Spry1 over-expression inhibits tumor growth and metastasis. Moreover, early results from gene transfection and silencing confirm the significant effect of Spry4 influence on cell proliferation, migration, adhesion and invasion. 45

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Table 4 Hydrodynamic diameter, size distribution and zetapotential of the POEGMEMABSA/Spry1 polyplex. The concentration of BSA is 3.3 µg/mL for the 1 to 1 sample and 1.65µg/mL for the 2 to 1 sample. Sample Name

Number Mean (d.nm)

PdI

Zetapotential (mV)

Spry1 to POEGMEMA-BSA= 1 to 1

24.7±1.14

0.41

-5.96±0.55

Spry1 to POEGMEMA-BSA= 2 to 1

17.22±0.71

0.39

-6.78±0.49

Figure 5 Spry1 and PEG-BSA/Spry1 complex micelle treatment against MCF-7 cell line for 48 h. Water was used as a control. The concentration of Spry1 in the two samples is the same. The ratio of Spry1 and POEGMEMA-BSA was 1 to 1. The scale bar is 50 µm.

Spry1 was mixed with POEGMEMA-BSA resulting in nanoparticles of similar properties to the Lyz particles (Table 4). TEM analysis was not possible due to the extremely low 18


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concentration of these solutions. The cytotoxicity towards MDA-MB-231 and MCF-7 cell lines of the POEGMEMA-BSA/Spry1 complex micelle was subsequently investigated using two different molar ratios. Live cell imaging (Figure 5) revealed already the toxicity expressed by both the Spry1 and Spry1 polyplex compared to the control. The IC50 values obtained from the cell proliferation curves were plotted against the concentration of Spry1 (Figure 6). The IC50 values of all the POEGMEMA-BSA/Spry complex micelles were observed to be lower than free Spry1, an indication that the protein drug was not destroyed during the formulation process. Moreover, the enhanced activity of the protein drug may be assigned to better cellular uptake and good release in the cytosol.

Figure 6 Cytotoxicity assays of free Sprouty and POEGMEMA-BSA/Spry1 complex against breast carcinoma cell lines (MDA-MB-231 and MCF-7) for 48 h, and data are expressed as mean ± standard deviation, n=8. High efficacy of the POEGMEMA-BSA/Spry1 complex micelles was demonstrated when investigating its inhibiting behavior in three dimensional MCF-7 multicellular tumor spheroids. Compared to the monolayer cell testes, multicellular spheroids are considered to be closer to real tissues in terms of cell metabolism and gene profiles. As a consequence, they have attracted increasing interests in the drug delivery field,46 as they can provide additional information on the movement of drug carriers.47, 48 The MCF-7 spheroids were incubated in water (as a control), Spry1 and POEGMEMA-BSA/Spry1 complex micelles for 6 days at 37 ºC. The inhibiting effects of the Spry1 and the Spry1 polyplex micelles on the MCF-7 spheroids were demonstrated by the size variation of the spheroids. The morphology of spheroids is shown in Figure 7. The size of spheroids was about 270 µm at day 0. After being 19


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incubated for 6 days, the spheroids in the Spry1 polyplex showed the most obvious inhibition to the cell proliferation and the size kept decreasing during the culturing period. It could be observed that the Spry1 also inhibited the cell proliferation in the spheroids, while the sizes of the MCF-7 spheroids in the control group swelled to more than 400 µm.

(a)

(b)

Figure 7 (a) Breast carcinoma MCF-7 spheroids were treated with Spry1 and POEGMEMA-BSA/Spry1 complex micelle for 6 days. MilliQ water was used as a control. The ratio of Spry1 and POEGMEMA-BSA was 1 to 1. The scale bar is 100 µm. (b) Size variation of spheroids in each group with time. Data are expressed as mean ± standard deviation, n=3. a: **, statistical difference, p˂ 0.01. b: ***, statistical difference, p

PEGylated Albumin-Based Polyion Complex ... - ACS Publications

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