Biodegradable “Smart” Polyphosphazenes with Intrinsic Multi-Functionality as Intracellular Protein Delivery Vehicles

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 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 cell-surface 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, 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 protein-based drug formulations. Although various promising strategies have been introduced to address the problem, such as cell-penetrating peptides^{18, 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}

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 (Warner-Graham, Cockeysville, MD), hydrochloric acid, potassium hydroxide (Alfa Aesar, Haverhill, MA), HyPure™WFI Quality Water (GE Life Sciences, Pittsburgh, PA), methyl 3(4-hydroxyphenyl)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 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. Molecular weights were calculated using EZ-Chrome Elite software (Agilent 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).

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 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, 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. NMR data for the synthesized copolymers are shown below.

70PPA. ¹H-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).

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.

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, 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 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. 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-2-phenylindole (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 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, FITC-avidin 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<0.05 and p<0.5.

RESULTS

Synthesis and Physico-Chemical Characterization of Polyphosphazene Copolymers.

Mixed substituent polyphosphazene copolymers containing carboxylic acid and pyrrolidone side groups were synthesized via chemical substitution of macromolecular precursor – polydichlorophosphazene (Scheme 1). Diglyme was used as a prime solvent due to its PDCP stabilizing properties.²⁸ However, due to poor solubility of pyrrolidone containing polyphosphazenes in diglyme, it was necessary to use NMP as a co-solvent. The ratios of diglyme and NMP were determined empirically to ensure that phase separation would not occur and complete substitution of chlorine atoms could be achieved. In order to control copolymer composition, MHP, the nucleophilic reagent containing the carboxylic acid group, was added to PDCP first in the amounts corresponding to the targeted content of these groups in the copolymer. After allowing it to react for 1.5–2 hours, APP, the pyrrolidone-containing nucleophile was added in the excess to complete the substitution process. The ester moiety of substituted polyphosphazenes was then deprotected using aqueous base to yield carboxylic acid groups. Three copolymers were synthesized with the targeted content of ionic groups between 20 and 70 % mol.

Scheme 1.

Synthesis of polyphosphazene copolymers

^a The inset shows content of carboxylic acid groups (-COOH, %, mol.) in polyphosphazene copolymers versus concentration of MPP in the reaction mixture relative to concentration of chlorine atoms of PDCP (%, mol.).

Table 1 summarizes compositions, molecular weights, and molecular sizes of each polyphosphazene. All synthesized copolymers were fully soluble in water and PBS (pH 7.4). The compositions of copolymers was determined by NMR and correlated well with the content of carboxylic acid ester component employed in the reaction mixture (inset in Scheme 1). Hydrodynamic radii and molecular weights of polyphosphazene copolymers with a high content of carboxylic acid groups (40 and 70 %) were characterized by similar hydrodynamic radii and molecular weights in excess of 100,000 g/mol. Interestingly, the copolymer with 20% of acidic groups displayed somewhat smaller dimensions and mass, which may be due both to more compact conformation and greater hydrolytic instability of polymer with a high content of pyrrolidone groups.

Table 1.

Physico-Chemical Characterization of polyphosphazene copolymers

Polymer	Content of -COOH, % mol.		Dz,a nm	Mw,b g/molx10−3
	1H NMR	31P NMR		MALS	GPCc
20PPA	22	17	11	34	18
40PPA	43	40	15	150	82
70PPA	73	70	15	110	62

^az average diameter by DLS

^bweight average molecular weight

^cgel permeation chromatography based on poly(acrylic acid) molecular weight standards.

Hydrolytic Degradation of Polyphosphazene Copolymers.

Synthesized copolymers were first evaluated for their ability to undergo hydrolytic degradation at near physiological and potential storage conditions. Solutions of polyphosphazenes in PBS (pH 7.4) were incubated at various temperatures (4°C, ambient temperature, 37°C, and accelerated conditions, 65°C) and analyzed at various time intervals for molecular mass and size losses by DLS and GPC (Figure 1, A–D). The results confirm degradability of all synthesized polymers with a direct correlation between the degradation rate and content of pyrrolidone groups in polyphosphazene with the highest rate observed for the polymer with the maximum content of pyrrolidone groups – 20PPA (Figure 1, curves 3). These results generally agree with previous findings on hydrolytic instability of polyphosphazenes containing pyrrolidone groups.³³ Degradation of all polyphosphazenes in solution was highly dependent on the temperature. While polymers showed accelerated hydrolytic degradation at physiological temperature (37°C) and especially at 65°C, polyphosphazenes showed minimal degradation at ambient temperature and were stable at potential storage conditions for at least a two month period. No sign of degradation was observed for material stored in a dry state for at least several months.

Figure 1.

Polymer molecular weight loss (% of initial) versus time for 70PPA (1), 40 PPA (2) and 20PPA (3) at 4°C (A), ambient temperature (B), 37°C (C), and 65°C (D) (PBS, pH 7.4; 0.5 mg/mL polyphosphazene; molecular weights were analyzed by GPC).

Biocompatibility and Acid-Induced Membrane Destabilizing Activity.

In vitro evaluation of drug carrier biocompatibility with blood components is a necessary part of early preclinical screening.³⁴ Synthesized polyphosphazenes were tested for potential membrane destabilizing behavior in the hemolysis studies. Each of the copolymers did not show any hemolytic activity at neutral pH, but exhibited membrane disruptive properties at acidic conditions, which correspond to the range of endosomal pH (pH 5.0–6.5)³⁵ (Figure 2). The pH onset of hemolytic activity was observed to be dependent on polymer composition with an increase of carboxylic acid side groups in the polymer leading to a progressive rise of the pH at which membrane disruption occurs. This generally correlates with the dependence of threshold of polymer aggregation upon acidification of solution experimentally observed by DLS. Polyphosphazenes with greater content of carboxylic acid groups show earlier onset of aggregation and thus are more likely to undergo conformation changes leading to membrane destabilization at higher pH values.

Figure 2.

Hemolysis of Red Blood Cells as a function of pH for 70PPA (1), 40PPA (2), and 20PPA (3) (polymer concentration - 0.05 mg/mL, 50 mM phosphate or citric acid/disodium phosphate buffer, 0.9 % of sodium chloride).

Environmentally Induced Self-Assembly of Polyphosphazenes into Nanoparticulate Carriers.

It was found that aqueous solutions of synthesized polyphosphazenes undergo spontaneous self-assembly into nanoparticulate structures in acidic environment. Figure 3 (charts A-C) displays DLS profiles for polyphosphazenes in neutral (pH 7.4) and acidified (pH 5.0) phosphate buffer solutions. As seen from the Figure, dramatic shifts towards larger sizes are observed for all polymers upon decrease in pH, with resulting nanoparticle size varying between 160 and 710 nm (volume average diameters). The nano-assemblies appear to be stable at acidic conditions, but the phenomenon is fully reversible once the solutions are brought back to neutral pH. Although the observed pH-induced self-assembly of polyphosphazenes is reversible, the nano-assemblies formed at low pH can be further ionically cross-linked using calcium chloride, spermine, and their combinations^36–39 to achieve the appropriate stability and protein release profiles under the physiological conditions at neutral pH.⁴⁰

Figure 3.

Self-assembly of polyphosphazenes in acidic media as demonstrated by DLS profiles for 20PPA (A), 40PPA (B), and 70 PPA (C) at pH 7.4 and pH 5.0 (volume average diameters are shown, 0.1 mg/mL of polyphosphazene, phosphate buffer) and 70PPA in the presence of spermine (D) (volume average diameter is shown, 0.1 mg/mL of 70PPA, 3.7 mg/mL of spermine, phosphate buffer, pH 7.4).

It was also found that synthesized copolymers could form nano-sized supramolecular assemblies upon direct addition of multivalent cross-linkers without prior acidification. Figure 3D shows formation of nanoparticulates with volume average diameter of 360 nm upon addition of spermine to 70PPA in a phosphate buffer at neutral pH.

Protein Loading and Functional Activity - Non-Covalent Protein-Polyphosphazene Complexes.

Polyphosphazene copolymers were evaluated for their ability to serve as proteins carriers. Their molecular design was based on the assumption that carboxylic acid groups of the polymer can serve as potential sites for covalent and non-covalent attachment of the biological cargo. Avidin, a glycoprotein characterized by high isoelectric point, was selected as a convenient model and AF4 method was used to investigate these interactions. Figure 4A shows that all polyphosphazenes underwent spontaneous assembly with protein with the formation of non-covalent complexes. As expected, the percent of bound protein increased as the content of acidic groups on the polymer rose. Both 40PPA and 70PPA were able to bind more than ten avidin molecules per polymer chain. A somewhat higher protein binding capacity of the 40PPA, as calculated per chain, may suggest an optimum balance between ionic and hydrogen bond interactions in this system. As was previously found, hydrogen bonds may play a significant role in intermolecular interactions between polyelectrolytes and glycosylated proteins.⁴¹

Figure 4.

Non-covalent loading of protein on polyphosphazene carriers. (A) Avidin bound to polyphosphazenes as a percent of total protein in formulation and number of protein molecules per polymer chain (AF4 analysis, 210 nm, 0.015 mg/mL of polyphosphazene, 0.10 mg/mL of avidin, PBS, pH 7.4); (B) AF4 profiles of avidin-polyphosphazene-FITC-biotin complexes resulting from (1) avidin-polyphosphazene complex reacting with FITC-biotin and (2) FITC-biotin-avidin complex reacting with polyphosphazene (0.25 mg/mL of 40PPA, 0.25 mg/mL of avidin, and 0.010 mg/mL of FITC-biotin, PBS); (C) Fluorescent microphotograph of water-insoluble avidin-polyphosphazene-FITC-biotin complexes (0.5 mg/mL of 70PPA, 0.1 mg/mL of avidin, and 0.02 mg/mL of FITC, PBS, pH 7.4).

Another desirable feature of the delivery carrier is its non-interference with biological functions of the loaded protein. This was evaluated by reacting avidin-polyphosphazene complex with fluorescently labeled avidin substrate – FITC-biotin. Avidin – FITC-biotin complex, which was prepared first and then mixed with polyphosphazene, was used as a control. Figure 4B shows that AF4 profiles for both formulations were practically identical. This experiment demonstrates that avidin in complex with polyphosphazene fully maintains its capacity to bind its substrate, which suggests that non-covalent protein-polyphosphazene interactions are gentle enough not to interfere with the biological function of the drug cargo.

To independently demonstrate both protein binding capacity of synthesized polymers and maintenance of protein functionality, insoluble complexes of polymers with avidin were prepared and examined by fluorescent microscopy. For 70PPA the formation of precipitate was achieved at 0.5 mg/mL of polymer and 0.1 mg/mL of protein in PBS (pH 7.4). The resulting insoluble avidin-polyphosphazene complex was then treated with fluorescently labeled biotin, centrifuged and re-suspended in PBS for the analysis by fluorescent microscopy. Figure 4C demonstrates fluorescence of polymer-protein complex, which qualitatively confirms both strong affinity of avidin to polyphosphazene and reactivity of immobilized protein towards the substrate.

Interactions of Protein-“Loaded” Polyphosphazenes with Cells.

The potential of polyphosphazenes to facilitate interaction of protein cargo with cells was examined using oral adenosquamous carcinoma Cal27 cells. Given the proven interaction of the polymers with avidin, FITC-labeled avidin was used as a model cargo to enable visualization and analysis by fluorescence microscopy. Cells were incubated in serum-containing media with polymer/avidin complexes (40PPA versus 70PPA) and compared to control, unbound FITC-avidin (Figure 5A–B). After only 30 minutes of incubation, significant amount of avidin associated with cells when presented as a polymer complex. For instance, measured as fluorescence mean intensity (which normalizes the cellular fluorescence to the cell area, allowing us to compare cells of different size), it was observed that 40PPA and 70PPA enhanced avidin association with cells by 21- and 20-fold, respectively. Measured as fluorescence sum intensity (which expresses the total cell fluorescence regardless of cell size), it was seen that 40PPA and 70PPA also enhanced avidin association with cells, i.e., by 50- and 41-fold, respectively.

Figure 5.

PPA copolymers enhance cell association and impact cellular distribution of a model protein cargo. (A) Fluorescence microscopy visualization, (B) quantification of the background-corrected mean fluorescence intensity (black bars) and sum fluorescence intensity (white bars) of Cal27 cells after 30 min incubation with FITC-avidin alone versus FITC-avidin complexed to 40PPA or 70 PPA. (C) Quantification of the number of 100–200 vesicular objects and (D) background-corrected mean fluorescence intensity after 30 minute, 2 hour, or 5 hour incubation with FITC-avidin complexed to 40PPA or 70 PPA. Green = FITC-avidin; blue = cell nuclei marked with DAPI; scale bar = 10 μm. Data are mean ± standard error of the mean (SEM). *Compares copolymer complexes against FITC-avidin control; #compares 70PPA against 40PPA; &compares each time point to the previous one, all by Student’s t-test (p<0.05 is represented with two symbols and p<0.5 is represented with one symbol).

Interestingly, when examining complex-cell interactions over time, an increase in the number of 100–200 nm diameter vesicles positive for FITC-avidin fluorescence was observed, i.e., up to 60 vesicular objects for 70PPA and 38 for 40PPA by 5 hours (Supplementary Figure S1 and Figure 5C). A possible interpretation is that cells may internalize polyphosphazene-complexed FITC-avidin into endocytic vesicles. Figure 5D shows that the fluorescence mean intensity decreased over time for both copolymers, which can be explained by changes in the fluorescence distribution, e.g., accumulation in endosomes vs. spread cell-surface accumulation, as discussed above. This result could also be due to decreased fluorescence intensity of FITC in acidic intracellular vesicles, such as endosomes and lysosomes, as reporteded.⁴² However, because samples had been fixed prior to imaging (see Methods), it is expected that all cell compartments are neutral and decay in FITC fluorescence mean intensity should be due rather to differences in subcellular localization. Although indirectly, this result indicates that polyphosphazene copolymers may facilitate cellular uptake of protein cargo.

Internalization of Protein-“Loaded” Polyphosphazene by Cells.

To confirm the previous hypothesis, Cal27 cells were similarly incubated with unbound FITC-avidin control or FITC-avidin complexed to 40PPA or 70PPA, yet in this experiment cell-surface versus internalized counterparts were differently stained using a well established method:³² after 30 minute incubation, unbound materials were washed off, cells were fixed (not permeabilized), and cell-surface bound counterparts were labeled using biotin-IgG followed by Texas Red-labeled secondary antibody. As shown in Figure 6A, this allows visualization of cell-surface bound materials in yellow (green + red), while internalized counterparts appear green alone. Quantification of the percentage of internalization of the total FITC-avidin label associated to cells represents the uptake rate (Figure 6B). This parameter was enhanced in the presence of polyphosphazene copolymers (1.4- and 4.1-fold increase for 40PPA and 70PPA, respectively, over unbound FITC-avidin) with 65% uptake for 70PPA after only 30-minute incubation with cells. The total area occupied by FITC-avidin internalized in the presence of copolymers surpassed that of the unbound control by 4.5-fold for 40PPA and 21-fold for 70PPA (Figure 6B). Therefore, this confirms internalization of a model protein cargo by polyphosphazene copolymer, suspected from the previous assay.

Figure 6.

PPA facilitates cell uptake of protein cargo. (A) Fluorescence microscopy visualization and (B) quantification of the percentage of internalized cargo, and the area occupied by internalized cargo (see Methods) in Cal27 cells, after 30 min incubation with FITC-avidin complexed to 40PPA or 70 PPA versus control unbound counterpart. (C) Quantification of the percentage of internalized cargo and (D) the area occupied by internalized cargo (see Methods) in Cal27 cells, after 30 minute, 2 hour, and 5 hour incubation with FITC-avidin complexed to 40PPA or 70 PPA. Yellow = FITC-avidin counterstained at the cell surface with biotin-IgG + Texas Red-secondary antibody; green = internalized FITC-avidin; blue = cell nuclei (DAPI); scale bar = 10 μm. Data are mean ± standard error of the mean (SEM). *Compares copolymer complexes against FITC-avidin control; #compares 70PPA against 40PPA; &compares each time point to the previous one, all by Student’s t-test (p<0.05 is represented with two symbols and p<0.5 is represented with one symbol).

Furthermore, the uptake of FITC-avidin carried by polyphosphazenes was time dependent and saturable (Supplementary Figure S2 and Figure 6C). The extent of uptake for 70PPA/FITC-avidin complex increased from 2.5% at 30 minutes to 65% at 2 hours, yet it practically did not change thereafter: 67% at 5 hours (Figure 6C). Comparatively, the percentage of uptake for protein cargo complexed to 40PPA was generally slower and did not saturate within this time frame: 8% at 30 minutes, 23% at 2 hours, and 41% at 5 hours. Similarly, the cell area occupied by internalized FITC-avidin was greater when this cargo was complexed to 70PPA versus 40PPA, although this difference was reduced with time: 5.5-fold and 2.1-fold greater area for 70PPA by 2 hours and 5 hours, respectively (Figure 6D).

DISCUSSION

Development of multifunctional polymeric carriers for intracellular delivery of proteins is one of the most promising approaches to improving efficacy of macromolecular therapeutics. This includes engineering of sophisticated nanoparticulates,^{3, 16} synthesis of amphiphilic block copolymers for the preparation of micellar or vesicular drug carriers,^{43, 44} dendrimers,⁴⁵ and covalent water-soluble protein – polymer conjugates.^{8, 15} Stimuli responsive ‘smart’ polymers, which mimic features of fusogenic peptides typically found in pathogenic organisms, represent an especially attractive approach.^{13, 35} The concept has been successfully developed for non-cytotoxic polyanions, which become membrane active under acidic conditions, hydrophobically modified copolymers of acrylic and methacrylic acids, N-isopropylacrylamide, and poly(styrene-alt-maleic anhydride) alkylamide.^{13, 46} The non-biodegradability of such carriers still presents a substantial limitation.^{13, 25} Biodegradable poly(maleic acid) was chemically modified with hydrophobic octyl groups in an attempt to overcome this obstacle. This, however resulted in carriers with membrane destabilizing activity below pH 4, which limits the development of such molecules for drug delivery applications.²⁵

Therefore, one of major remaining challenges in the development of smart polymeric carriers for intracellular protein delivery lies in a need of integrating molecular biodegradability with multiple biological functionalities. This has to be also achieved in a generally simple molecular design, which can be acceptable for future production and clinical development of the material. To this end, we designed polyphosphazenes containing just two moieties - phenoxypropionic and propylpyrrolidone groups. The relatively hydrophobic carboxylic acid group was selected to provide protein attachment sites and environmental responsiveness, which can be potentially translated into self-assembly and endosomolytic behavior. The pyrrolidone side group was chosen to sustain water-solubility, modulate biodegradability and conformational behavior of macromolecules. Such side chain functional synthetic design is also attractive because the polymer can be easily further modified with labeling groups or targeting ligands.⁴⁷

Polyphosphazenes with variable compositions were synthesized, applying macromolecular substitution approach with the use of mixed solvents to maintain adequate solubility of the resulting macromolecules. This methodology provided for adequate synthetic control over the ratio of side groups in polyphosphazenes, which is an important factor in further development of such polymer carriers.

Synthesized polymers were then screened to empirically validate main biologically relevant design elements.

Firstly, evaluation of their hydrolytic degradability revealed temperature sensitive breakdown in aqueous solutions, which can be modulated through the content of pyrrolidone groups. This suggests that synthesized polyphosphazenes are degradable under physiological conditions, however they may provide for an adequate shelf life when stored in solutions under refrigeration or in dry state.

Secondly, biocompatibility with blood components was assessed in the hemolysis assay,³⁴ in which all synthesized polyphosphazenes demonstrated lack of cellular toxicity at neutral pH. However, acidification of solution below pH 6 triggered membrane disruptive activity of polymers, a behavior that is typically characteristic to hydrophobically modified polyacids.^{13, 25} Generally, acidic conditions cause these polymers to undergo coil to globule conformational changes, which in turn causes adsorption to the outer leaflet of the bilayer, membrane expansion and disruption.²⁵ Since the threshold of polymer membranolytic activity generally corresponds to endosomal pH (pH 5.0–6.5)³⁵ and correlation between hemolytic efficiency and endosomal disruption had been previously established,⁴⁸ synthesized polyphosphazenes may present an interest as endosomolytic carriers facilitating delivery into the cytoplasm.^{7, 13, 25, 49} While interesting, potential for cytosolic delivery is beyond the scope of this initial investigation and represents the focus of a future, independent study. It must be noted however, that it is necessary to optimize such membranolytic properties for a protein-carrier formulation as covalent or non-covalent conjugation with another macromolecule can affect the onset of the activity.⁵⁰

Thirdly, synthesized polyphosphazenes demonstrated ability to form nanoparticles through self-assembly induced either by acidification or addition of physiologically benign multivalent cross-linker, spermine. The practical importance of the above results for delivery of protein drugs is in the potential ability to optimize the size of nanoparticulates for tumor targeting through EPR effects.^{3, 51} It has been already shown that the size of ionically cross-linked polyphosphazenes can be further modulated not only through the composition and molecular weights of the copolymers, but also via varying pH, ionic strength, use of surfactants and hydrogen bond forming excipients.^{36, 38, 39, 50, 52}

It should be noted that the formation of stable polyphosphazene nanoparticulates in acidic solutions was somewhat unexpected. Although polyphosphazene homopolymers with acidic functionalities are known to be non-soluble under these conditions,^{53, 54} this may be the first report of polyphosphazene polyacids forming stable nanoparticles with relatively narrow size distribution at low pH. The formation of these nano-assemblies is probably due to the stabilizing effect of hydrophilic pyrrolidone groups of the polymer, however such behavior should be more anticipated for amphiphilic block copolymers.⁵⁵ Although synthesized polyphosphazenes were not designed as block copolymers, it is possible that some blocky structures can be formed during the synthesis, which can lead to nano-scale domain segregation.⁵⁶ Also, the supreme flexibility of polyphosphazene backbone²⁶ can potentially contribute to a more organized arrangements of such blocky domains.

Finally, a model protein cargo, avidin, was successfully loaded on polyphosphazene copolymers through spontaneous non-covalent protein-polymer complexation in aqueous solutions. The binding capacity of polymers, which, as expected, was dependent on copolymer composition, was evaluated and it was demonstrated that polymers with higher content of carboxylic acid groups were capable of carrying multiple protein molecules per chain. Importantly, functional activity of complexed avidin was evaluated and was shown to be practically identical to that of unbound protein.

After experimental validation of design features of new polyphosphazenes, we were enticed to conduct preliminary evaluation of their protein delivery capability in carcinoma cell assays. These experiments were conducted for copolymers that demonstrated high avidity to avidin – 40PPA and 70PPA. It was found that both copolymers drastically enhanced association of protein with cells (21–50 fold increase compared to experiments with unbound protein), although copolymer with higher content of acidic groups exhibited better performance. Furthermore, it appears that polyphosphazene copolymers are well suited to facilitate uptake of protein cargo by cells, which was demonstrated by additional staining of surface accumulated protein molecules, which allowed differentiation between cell-internalized and cell-adsorbed protein. The cellular uptake findings were also supported by an increase with time in the number of vesicular-size objects, which may indicate protein accumulation in endocytic vehicles.

It is tempting to speculate that the mechanism by which polymers facilitate cellular association relates to polyphosphazene ability to establish non-covalent interactions with proteins and polysaccharides in solution as it was shown above for avidin. The same interactions would be expected to occur with proteins and polysaccharides on the cell surface. Although such interactions are reported to play an important role in the uptake of particulates,^{15, 57} it should be noted that polyphosphazene formulations retained their solubility and were not nanoparticulates with a defined surface. Regardless, it can be speculated that primary interaction on the cell surface would facilitate uptake by either inducing endocytic processes (if interactions do occur through receptors of these pathways) or via an adsorptive mechanism as the cell internalizes other extracellular components or recycles the plasmalemma.⁷ Modification of polyphosphazenes with active targeting moieties, such as antibodies, peptides, aptamers, etc., may provide means to further modulate cellular uptake as well as attain specific delivery to target cells.⁷ Optimization of pH sensitive membrane disruptive properties, which are typically correlated to endosomolytic activity, through macromolecular composition of the complex⁵⁰ can be an additional pathway to improving the delivery, especially to cytoplasm, which we will examine next.

Overall, the ability of polyphosphazene copolymers to dramatically increase association and internalization of cargo protein with cancer cells holds potential for drug delivery applications. The fact that these results were observed after a relatively short incubation time and in cell medium supplemented with serum is encouraging, as those represent conditions more physiologically relevant to the in vivo environment.

CONCLUSIONS

The minimalistic molecular design of polyphosphazenes in the present study affords biodegradability and surprisingly impressive multi-functionality while maintaining a reasonably direct pathway to further development and potential manufacturing. Temperature and composition dependent degradation profiles observed in this study can provide ample opportunities for modulating pharmacokinetics and solving shelf life challenges. Perhaps one of the most important and yet to be further explored features of polyphosphazene copolymers is their demonstrated ability to self-assemble in aqueous solutions upon variations in environmental conditions. While nanogel formation upon cross-linking with multivalent spermine was not completely unexpected, the pH induced aggregation leading to narrow polydispersity nano-assemblies can introduce new dimensions in nano-scale engineering of delivery vehicles with required hydrophobicity, size, hydration sphere, and electrostatic potential. Synthesized biodegradable polyphosphazenes have already demonstrated their potential for intracellular delivery by facilitating cell-surface interactions and uptake of cargo protein by cancer cells.

ABBREVIATIONS

PDCP

polydichlorophosphazene

PPA

poly{[carboxylatoethylphenoxy][3-(2-oxo-1-pyrrolidinyl) propylamino]phosphazene}

DAPI

4′,6-diamidino-2-phenylindole

DLS

dynamic light scattering

FITC

fluorescein isothiocyanate

MALS

multi-angle laser light scattering

NMR

nuclear magnetic resonance

AF4

Asymmetric Flow Field Flow Fractionation

D_z

z-average diameter

M_w

weight average molecular weight

GPC

gel permeation chromatography

NMP

1-methyl-2-pyrrolidinone

MHP

methyl 3(4-hydroxyphenyl)propionate

APP

N-(3′-aminopropyl)-2-pyrrolidinone

PBS

phosphate buffered saline