Biodegradable “Smart” Polyphosphazenes with ... - ACS Publications

May 19, 2017 - Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States. •S Supporting Information...
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Biodegradable “Smart” Polyphosphazenes with Intrinsic Multi-Functionality as Intracellular Protein Delivery Vehicles Andre P. Martinez, Bareera Qamar, Thomas R. Fuerst, Silvia Muro, and Alexander K. Andrianov Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 22, 2017

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Biodegradable “Smart” Polyphosphazenes with Intrinsic Multi-Functionality as Intracellular Protein Delivery Vehicles Andre P. Martinez,†,‡ Bareera Qamar,§ Thomas R. Fuerst,†,# Silvia Muro, †,¶,* and Alexander K. Andrianov†,‡,* †

Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky

Dr., Rockville, MD 20850, United States; §

Neurobiology and Physiology Program of the Department of Biology, 1210 Biology-

Psychology Building, University of Maryland, College Park, MD 20742, United States; #

Department of Cell Biology and Molecular Genetics, 1109 Microbiology Building, University

of Maryland, College Park, MD 20742, United States; ¶

Fischell Department of Bioengineering, 2330 Jeong Kim Building, University of Maryland,

College Park, MD 20742, United States.

ABSTRACT:

A series of biodegradable drug delivery polymers with intrinsic multifunctionality

have been designed and synthesized utilizing polyphosphazene macromolecular engineering approach. Novel water-soluble polymers, which contain carboxylic acid and pyrrolidone moieties attached to inorganic phosphorus-nitrogen backbone, were characterized by a suite of

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physico-chemical methods to confirm their structure, composition, and molecular sizes. All synthesized polyphosphazenes displayed composition dependent hydrolytic degradability in aqueous solutions at neutral pH. Their formulations were stable at lower temperatures, potentially indicating adequate shelf life, but were characterized by accelerated degradation kinetics at elevated temperatures, including 37°C. It was found that synthesized polyphosphazenes are capable of environmentally triggered self-assembly to produce nanoparticles with narrow polydispersity in the size range between 150 and 700 nm. Protein loading capacity of copolymers has been validated via their ability to non-covalently bind avidin without altering its biological functionality. Acid induced membrane disruptive activity of polyphosphazenes has been established with an onset corresponding to endosomal pH range and being dependent on polymer composition. The synthesized polyphosphazenes facilitated cellsurface interaction followed by time-dependent, vesicular mediated, and saturable internalization of a model protein cargo into cancer cells, demonstrating potential for intracellular delivery.

INTRODUCTION The emergence of proteins and peptides as therapeutic agents has inspired a search for advanced delivery technologies, which can improve biodistribution, stability, and reduce undesirable immunogenicity of these macromolecular drugs.1-7 Diverse drug delivery systems have been successfully introduced to combat the multiple challenges therapeutic proteins face in the recipient organism. Such systems can minimize clearance of proteins by the reticuloendothelial system through providing ‘stealth’ characteristics,5,

8, 9

enable ‘passive’

targeting to the disease site through the enhanced permeability and retention effect (EPR),10-12 actively target specific cells, and modulate cellular uptake as well as intracellular trafficking.7, 13,

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14

Most advanced drug carriers require multifunctionality, which introduces new levels of

sophistication in material engineering, resulting from the need to integrate multiple and frequently poorly compatible functionalities on the molecular or nanoscale levels.14-16 Along with complying with some common requirements, such as biocompatibility, biodegradability, and protein loading capability, novel carriers are expected to incorporate a complex set of elements, which can provide for biological sensing, recognition, protection, and adequate biological response.2 Intracellular transport of macromolecular drugs remains one of the key problems in drug delivery.17 Many therapeutic proteins have their targets inside the cell and low permeability of cell membranes to macromolecules represents a serious obstacle for the development of proteinbased drug formulations. Although various promising strategies have been introduced to address the problem, such as cell-penetrating peptides18,

19

or pH-sensitive, long-circulating

immunoliposomes,20 they still face substantial challenges, which include specificity and stability, high manufacturing costs and scale-up problems.17 Synthetic polymers present an attractive alternative solution due to their well-established chemistry, lack of immunogenicity, general biocompatibility, potential for EPR effect, and long blood circulating times.15 In particular, the concept of the “smart polymeric carrier” was introduced as a promising approach for intracellular delivery of macromolecular drugs.13,

21-24

‘Smart polymers” can provide elements

for conjugation or complexation of drugs, incorporate an optional cell-targeting component, and enable endosomal escape by ‘sensing’ changes in environmental pH. Despite the potential and already established proof, the concept has not yet been successfully adopted to biodegradable polymers, which is a prerequisite for their use in the biomedical field, especially when injectable formulations are considered.13, 25

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Polyphosphazenes offer a unique platform for developing advanced materials for biological applications as they combine an intrinsic biodegradability with a versatile synthetic route, which allows for unprecedented structural diversity.26, 27 These synthetic macromolecules consisting of a phosphorus and nitrogen backbone and organic side groups integrate a number of distinct features that can uniquely position them for drug delivery applications. Synthetic ‘toolkit’ methods for creating new structures via macromolecular substitution of the polymer precursor, tunable degradation, flexibility of the backbone, high density of functional groups, and established manufacturing processes,26,

27

can provide unconventional approaches to solving

challenges in the drug delivery field. The present paper describes the synthesis and characterization of biodegradable ‘smart’ polyphosphazenes for the delivery of macromolecular drugs. The simple binary copolymer design of these water-soluble polyphosphazenes, which includes phenoxypropionic acid and propylpyrrolidone side groups, is shown to provide intrinsic biological multi-functionality: tunable biodegradability, environmentally triggered self-assembly in nanoparticulate carriers, protein binding characteristics, and endosomolytic properties. The potential of these polyphosphazenes to facilitate cellular uptake of proteins is demonstrated in cell culture using cancer cells. MATERIALS AND METHODS Materials.

1-Methyl-2-pyrrolidinone,

NMP,

heptane,

sodium

hydride,

citric

acid

monohydrate, sodium phosphate monobasic dihydrate, egg white avidin and avidin conjugated to fluorescein isothiocyanate, FITC (Sigma-Aldrich, Saint Louis, MO), bis(2-methoxyethyl) ether, diglyme, N-(3'-aminopropyl)-2-pyrrolidinone, APP (Acros Organics, Morris Plains, NJ), ethanol

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(Warner-Graham, Cockeysville, MD), hydrochloric acid, potassium hydroxide (Alfa Aesar, Haverhill, MA), HyPure™WFI Quality Water (GE Life Sciences, Pittsburgh, PA), methyl 3(4hydroxyphenyl)propionate, MHP (TCI, Portland, OR), acetonitrile (EM Science, Darmstadt, Germany), phosphate buffered saline pH 7.4, PBS (Life Technologies, Carlsbad, CA), poly(acrylic acid) standards (American Polymer Standards, Mentor, OH), porcine red blood cells (Innovative Research, Novi, MI), sodium chloride (Fisher Scientific, Waltham, MA), sodium phosphate dibasic heptahydrate (VWR, Radnor, PA), biotinylated mouse IgG (BD Biosciences PharminGen, San Jose, CA), Texas Red goat anti-mouse IgG (Life Technologies, Carlsbad, CA), Dulbecco’s Modified Eagle’s Medium with 4.5g/L glucose, L-glutamine and sodium pyruvate (Corning Life Sciences, Tewksbury, MA) were used as received. Phosphonitrilic chloride trimer, hexachlorocyclotriphosphazene was generously donated by Fushimi Pharmaceutical Co. Ltd. (Kagawa, Japan). Polydichlorophosphazene (PDCP) was synthesized by a ring-opening polymerization reaction in a pressure reactor.28 The degree of polymerization (2,200) was determined on the basis of reaction mixture viscosity and converting PDCP into poly[di(carboxylatophenoxy)phosphazene)] as described previously.28 Characterization. Gel permeation chromatography, GPC was performed using a Hitachi HPLC system with L-2450 diode array detector, L-2130 pump, and L-2200 autosampler (Hitachi LaChrom Elite system, Hitachi, San Jose, CA) and Ultrahydrogel Linear size exclusion column (Waters Corporation, Milford, MA). PBS, pH 7.4 with 10% of acetonitrile was employed as a mobile phase with a flow rate of 0.5 mL/min. Samples were prepared at a concentration of 0.5 mg/mL in PBS, pH 7.4 and were filtered using Millex 0.22 µm filters (EMD Millipore, Billerica, MA) prior to the analysis. GPC traces of synthesized polymers are shown in Supplementary Figure S1. Molecular weights were calculated using EZ-Chrome Elite software (Agilent

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Technologies, Santa Clara, CA). Calibration curve was obtained using narrow poly(acrylic acid) standards (American Polymer Standards Corporation, Mentor, OH). Dynamic light scattering, DLS was carried out using a Malvern Zetasizer Nano series, ZEN3600 and analyzed using Malvern Zetasizer 7.10 software (Malvern Instruments Ltd., Worcestershire, UK). Samples were prepared in PBS, pH 7.4 and filtered using Millex 0.22 µm filters prior to the analysis. UV-Vis readings for hemolysis assays were performed using a Thermo Scientific Multiscan Spectrum spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Data was analyzed using SkanIt 2.4.4 software (Thermo Fisher Scientific, Waltham, MA). Circular dichroism (CD) measurements were carried out using a Chiroscan qCD spectrometer (Applied Photophysics, Leatherhead Surrey, United Kingdom) in a 1 mm cell at ambient temperature. Asymmetric Flow Field Flow Fractionation, AF4 was performed using a Postnova AF2000 MT series (Postnova Analytics GmbH, Landsberg, Germany). The system was equipped with two PN1130 isocratic pumps, PN7520 solvent degasser, PN5120 injection bracket and UV-Vis detector (SPD-20A/20AV, Shimadzu Scientific Instruments, Columbia, MD). A regenerated cellulose membrane with molecular weight cutoff of 10 kDa (Postnova Analytics GmbH, Landsberg, Germany) and a 350 µm spacer were used in a separation micro-channel employing both laminar and cross flows of an eluent - PBS (pH 7.4). The collected data was processed using AF2000 software (Postnova Analytics GmbH). Multi-angle static light scattering, MALS analysis was performed using a Wyatt miniDAWN TREOS MALS detector (Wyatt Technology Corporation, Santa Barbara, CA) assembled in line

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with Optilab T- rEX refractometer (Wyatt Technology Corporation, Santa Barbara, CA), 1200 series isocratic pump, 1200 series variable wavelength detector (Agilent Technologies, Santa Clara, CA) and Ultrahydrogel column (Waters Corporation, Milford, MA). PBS, pH 7.4 was used as a mobile phase. Samples were prepared at a concentration of 0.5 mg/mL in PBS, pH 7.4 and filtered using Millex 0.22 µm filters prior to the analysis. Molecular weights were determined using ASTRA V 5.3.4.14 software (Wyatt Technology Corporation, Santa Barbara, CA). Fluorescent microscopy examination was conducted using ECLIPSE 80I fluorescent microscope (Nikon Instruments, Melville, NY). Synthesis

of

Mixed

Substituent

Polyphosphazenes.

Synthesis

of

poly{[carboxylatoethylphenoxy][3-(2-oxo-1-pyrrolidinyl)propylamino]phosphazenes}, PPA was carried out via subsequent addition of nucleophiles, MHP and APP, to PDCP followed with hydrolysis of ester bearing copolymer to yield polyphosphazene polyacid. Copolymers with the targeted content of phenoxypropionic groups of 20, 40 and 70 % (mol.) were synthesized (20PPA, 40PPA, and 70PPA). The synthesis of 70PPA is described below as an example. 5 g of methyl 3(4-hydroxyphenyl)propionate, MHP was suspended in deionized water, treated with 6 M sodium hydroxide solution (0.7 molar equivalents) and lyophilized to produce an off white powder of the MHP, sodium salt. 0.30 g (1.2 mmol) of MHP, sodium salt was suspended in 10 mL of diglyme and added dropwise under anhydrous conditions to the flask containing solution of 0.093 g (1.6 mmol) of PDCP in 10 mL of diglyme. The contents were heated to 120°C with stirring under nitrogen flow, kept at this temperature for 1.5 hours and then allowed to cool to ambient temperature. 0.465 mL (3.2 mmol) of N-(3'-aminopropyl)-2-pyrrolidinone,

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APP was dissolved in 60 ml of 1-methyl-2-pyrrolidinone, NMP and added dropwise to the reaction mixture while stirring. The reaction was kept at ambient temperature overnight and then heated to 95°C for deprotection of ester groups. 14 mL of 6 M sodium hydroxide was added dropwise with stirring and the suspension was allowed to cool. The supernatant was decanted, precipitated polymer was recovered by dissolving in deionized water, purified by two precipitations with ethanol, and dried under vacuum. 40PPA and 20PPA were synthesized similarly, however the amounts of MHP were adjusted to reflect the targeted polymer compositions. In addition, MHP sodium salt was prepared under anhydrous conditions by reacting MHP with sodium hydride at a molar ratio of 1.1 : 1.0. The volumes of NMP were also scaled up relative to the content of APP in the polymers in order to maintain solubility throughout the synthesis. 1H and 31P NMR spectra for 70PPA are displayed in Supplementary Figure S2 and shifts for all copolymers are shown below. 70PPA.

1

H-NMR (400 MHz, D2O): δ [ppm] = 6.8 (br, 4H, −CH=); 2.6 (br, 2H, Ar−CH2−);

2.2 (br, 2H, −CH2−COO); 2.0 (br, 2H, −CH2−CO−NR2−); 1.5 (br, 2H, −CH2−); 1.0 (br, 2H, −CH2−). 31P-NMR (162 MHz, D2O): δ [ppm] = -4.0 (br, 2P, −N=P(NH−)2, −N=P(NH−)(O−Ar)); -18.0 (br, 1P, −N=P(O−Ar)2). 40PPA. 1H-NMR (400 MHz, D2O): δ [ppm] = 7.0 (br, 4H, −CH=); 3.2-2.8 (br, 6H, −NH−CH2−, −CH2−, −NR-CH2−); 2.7 (br, 2H, Ar−CH2−); 2.2 (br, 2H, −CH2−COO); 2.1 (br, 2H, −CH2−CO−NR2−); 1.7 (br, 2H, −CH2−); 1.2 (br, 2H, −CH2−). 31P-NMR (162 MHz, D2O): δ [ppm] = 0.0 (br, 1P, −N=P(NH−)2); -3.2 (br, 2P, −N=P(NH−)2, −N=P(NH−)(O−Ar)), -17.1 (br, 1P, −N=P(O−Ar)2).

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20PPA. 1H-NMR (400 MHz, D2O): δ [ppm] = 7.1 (br, 4H, −CH=); 3.4-2.8 (br, 6H, −NH−CH2−, −CH2−, −NR-CH2−); 2.7 (br, 2H, Ar−CH2−); 2.3 (br, 2H, −CH2−COO); 1.8 (br, 2H, −CH2−CO−NR2−); 1.7-1.2 (br, 4H, −CH2−, −CH2−). 1P-NMR (162 MHz, D2O): δ [ppm] = 2.4 (br, 1P, −N=P(NH−)2); 1.2 (br, 1H, −N=P(NH−)(O−Ar)). Hydrolytic Degradation of Polyphosphazene Copolymers. Polymers were dissolved to a concentration of 0.50 mg/mL in phosphate buffered saline (pH 7.4). Solutions were stored at 4°C, ambient temperature, 37°C, and 65°C. 0.50 mL samples were taken for DLS and GPC analysis at various time intervals. Representative GPC traces for 40PPA as a function of degradation time are shown in Supplementary Figure S3. Non-Covalent Polymer-Protein Complexes - Protein Loading and Functional Activity. Non-covalent binding of model protein, avidin, with polyphosphazene copolymers was evaluated using AF4. The AF4 analysis was performed at 0.015 mg/mL of polyphosphazene and 0.10 mg/mL of avidin in PBS (pH 7.4), which was also used as an eluent. AF4 profiles were recorded at 210 nm. The results for polymer – protein formulation were compared with elution profiles of individual components. The percentage of avidin in the complex was determined on the basis of unbound protein detected in the polyphosphazene – avidin formulation. Functional activity of avidin in non-covalent complex with polyphosphazene was evaluated by examining its affinity to fluorescently labeled substrate, FITC-biotin. 40PPA and avidin solutions in PBS (pH 7.4) were mixed to form a complex with a concentration of 0.25 mg/mL of both polymer and protein. Next, FITC-biotin was added to the solution to achieve resulting concentration of 0.01 mg/mL. A control sample was prepared by premixing solutions of avidin and FITC-biotin followed by addition of 40PPA, generating final concentrations of 0.25 mg/mL,

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0.25 mg/mL and 0.01 mg/mL of polymer, avidin, and FITC biotin respectively. Concentrations were chosen to achieve a roughly 1:1 ratio between FITC biotin and avidin binding sites. Samples were then analyzed by AF4 as described above. Water-insoluble complexes of avidin and polyphosphazene for the analysis by fluorescent microscopy were prepared as follows. 100 µL of 0.2 mg/mL avidin and 100 µL of 1mg/mL 70PPA in PBS (pH 7.4) were mixed and vortexed for one minute. After incubating the resulting heterogeneous mixture for 5 minutes, 20 µL of 0.2 mg/mL FITC-biotin in 1xPBS was added and the solution was vortexed again for one minute. The sample was centrifuged and the supernatant was decanted. The pellet was resuspended in 200 µL of PBS with vortexing and analyzed by fluorescent microscopy. Environmentally Triggered Self-Assembly of Polyphosphazenes. Self-assembly in polyphosphazene solutions was studied by DLS. Acid induced transitions: 1 mL of 0.1 mg/mL polyphosphazene solutions in PBS were titrated to pH 5 by adding 10 µL aliquots of 0.1 M hydrochloric acid upon vortexing. Spermine induced nanogel formation: four 20 µL aliquots of 50 mg/mL spermine tetrahydrochloride were added to 1 mL of 0.1 mg/mL 70PPA solution in PBS to achieve 3.7 mg/mL concentration of the cross-linker. The sample was vortexed after each addition. Evaluation of Hemolytic Activity. The membrane disruptive activity of multifunctional carriers was tested as described previously.25,

29, 30

50 µL of fresh Porcine Red Blood Cells

(RBC) as a 10% suspension in phosphate buffered saline (PBS) (Innovative Technology Inc., Novi, MI) was re-suspended in 200 µL of PBS. 50 µL of re-suspended RBC was added to 925 µL of 50 mM of phosphate or citric acid/disodium phosphate buffer at the appropriate pH, vortexed, and to this mixture 25 µL of 2.0 mg/mL polymer in PBS was added followed by

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vortexing. Samples were incubated at 37 °C for one hour on a shaker table. Cells were then centrifuged at 14,000 rpm for 5 minutes, and the absorbance of the supernatant was then measured at 541 nm using Multiskan Spectrum microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA). To determine 100% hemolysis, RBCs were suspended in distilled water and lysed by ultrasound (Branson Sonifier, Model 450). All hemolysis experiments were conducted in triplicate. Binding and Uptake in Cell Culture. Oral adenosquamous carcinoma Cal27 cells (American Type Culture Collection) were seeded on 12-mm2 glass coverslips and grown to confluence at 37◦C, 5% CO2 and 95% relative humidity in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin (herein referred to as complete medium). For binding and uptake experiments, cells were incubated with 40PPA/FITC-avidin or 70PPA/FITC-avidin (both at 0.3 mg/ml polymer carrier and 0.5 mg/ml avidin cargo), versus 0.5 mg/ml FITC-avidin alone as a control, at 4°C versus 37°C where indicated. To best mimic physiological conditions, incubations were carried out in the presence of complete medium for different time periods, i.e., 30 minutes, 2 hours or 5 hours. Cell were then washed to remove unbound materials, fixed with 2% paraformaldehyde, and nuclei stained with 4',6-diamidino-2phenylindole (DAPI). Samples were imaged using an Olympus IX81 microscope (Olympus, Inc., Center Valley, PA), 60× oil immersion objective (UPlanApo, Olympus, Inc., Center Valley, PA), ORCA-ER camera (Hamamatsu Corporation, Bridgewater, NJ), and SlideBook™ 4.2 software (Intelligent Imaging Innovations, Denver, CO). Fluorescence images were taken under the green and blue channels to monitor FITC and DAPI, respectively, and bright field images provided visualization of full cells and cell-cell borders. To monitor the amount of FITC-avidin associated to cells, Image-Pro 6.3 (Media Cybernetics, Bethesda, MD) was used to quantify FITC mean

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intensity and sum intensity per cell, normalized to that of the background in surrounding areas (as indicated in each figure). For uptake experiments, cells were incubated 40PPA/FITC-avidin, 70PPA/FITC-avidin or control FITC-avidin as described for binding experiments, but surface-bound versus internalized materials were differentiated. For this purpose, cells were washed after incubation to remove unbound materials, fixed and incubated with biotin-conjugated mouse IgG, which can only access surface-bound FITC-avidin but not internalized FITC-avidin. Surface-bound counterparts were finally stained using a Texas Red-labeled secondary antibody. Using this protocol, FITCavidin located on the cell-surface appears yellow (Texas Red + green FITC stains), while internalized FITC-avidin appears green alone. Fluorescence microcopy analysis can then be used to quantify the percentage of uptake with respect to the total amount of FITC-avidin associated with cells, as well as the total cell area occupied by internalized counterparts, as described before.31, 32 Importantly, because the fluorescence intensity of FITC is affected at acidic pH, all experiments based on microscopy imaging involved cell fixation. After fixation the vacuolar H+ATPase (as all other cell proteins) is inactive and acidic pH cannot be maintained in endosomal and lysosomal compartments, hence enabling visualization of FITC-avidin regardless of location. Two independent experiments were analyzed cell-by-cell, for a total sample size of n ≥100 cells. Data were calculated as mean ± standard error of the mean (SEM). Statistical significance for two-way comparisons was determined using Student’s t-test for both p