Hydrolytically Degradable PEGylated Polyelectrolyte

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Hydrolytically Degradable PEGylated Polyelectrolyte Nanocomplexes for Protein Delivery Alexander K. Andrianov, Alexander Marin, Andre Paul Martinez, Jacob L. Weidman, and Thomas R. Fuerst Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00785 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Hydrolytically Degradable PEGylated Polyelectrolyte Nanocomplexes for Protein Delivery

Alexander K. Andrianov,†,* Alexander Marin, † Andre P. Martinez,†, ¶ Jacob L. Weidman, † Thomas R. Fuerst,†,# †

Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Dr., Rockville, MD 20850, United States

#

Department of Cell Biology and Molecular Genetics, 1109 Microbiology Building, University of Maryland, College Park, MD 20742, United States ¶

Present address: DSM Biomedical 735 Pennsylvania Drive, Exton PA 19341, United States

ABSTRACT: Novel oppositely charged polyphosphazene polyelectrolytes containing grafted poly(ethylene glycol) (PEG) chains were synthesized as modular components for the assembly of biodegradable PEGylated protein delivery vehicles. These macromolecular counterparts, which contained either carboxylic acid or tertiary amino groups, were then formulated at near physiological conditions into supramolecular assemblies of nanoscale level - below 100 nm. Nanocomplexes with electroneutral surface charge, as assessed by zeta potential measurements, were stable in aqueous solutions, which suggests their compact polyelectrolyte complex “core” – hydrophilic PEG “shell” structure. Investigation of PEGylated polyphosphazene nanocomplexes

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as agents for non-covalent PEGylation of the therapeutic protein – L-Asparaginase (L-ASP) in vitro demonstrated their ability to dramatically reduce protein antigenicity, as measured by antibody binding using enzyme linked immunosorbent assay (ELISA). Encapsulation in nanocomplexes did not affect enzymatic activity of L-ASP, but improved its thermal stability and proteolytic resistance. Gel permeation chromatography (GPC) experiments revealed that all synthesized polyphosphazenes exhibited composition controlled hydrolytic degradability in aqueous solutions at neutral pH and showed greater stability at lower temperatures. Overall, novel hydrolytically degradable polyphosphazene polyelectrolytes capable of spontaneous selfassembly into PEGylated nanoparticulates in aqueous solutions can potentially enable a simple and effective approach to modifying therapeutic proteins without the need for their covalent modification. INTRODUCTION Clinical applications of protein and peptide therapeutics – an advanced generation of drugs characterized by high biological activity and specificity - are rapidly expanding.1-5 Despite their unique advantages, the development of new macromolecular drugs is often impeded by their suboptimal pharmacokinetic profiles, in particular short in vivo half-life and undesirable intrinsic immunogenicity.3, 4, 6 PEGylation – a technology, which is based on the covalent modification of protein surface with a synthetic polymer, polyethylene glycol (PEG) is currently one of the most successful techniques addressing these shortcomings.7-11 Hydrophilic PEG chains form a steric barrier around the protein, which shields immunogenic sites on its surface from the body’s immune system12,

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and increases the hydrodynamic volume of the protein, resulting in a

reduced renal clearance. 12, 14, 15 Although PEGylation has been proven efficient in prolonging in vivo half-life and achieved a significant commercial success, the technology still suffers from


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significant limitations. In particular, covalently bound PEG chains can block active sites of the protein resulting in the reduction of therapeutic efficacy.16 Also, covalently PEGylated proteins may contain unreacted materials and reaction by-products, which requires expensive multistep purifications.16-18 A non-covalent PEGylation has been recently suggested as an alternative technology, which can potentially address these shortcomings.19 Functionalized PEGs were complexed with proteins through ionic or hydrophobic bonds,17,

20-25

coordination bonds,16 and host-guest

interactions,26 and formation of PEGylated micelles.27 However, the single point non-covalent attachment of PEG failed to demonstrate sufficient stability in vivo.16 Multivalent attachment through ionic interactions of PEGylated semi-synthetic pentosan polysulfate and dextran sulfate,28 as well as poly(N,N-dimethylaminoethylmethacrylate)29, 30 were also investigated and resulted in an improved thermal stability and protease resistance. An alternative approach has been suggested, which is based on the encapsulation of proteins into polyelectrolyte complex micelles, which form through the spontaneous self-assembly of two oppositely charged ionic block copolymers containing PEG chains.31-36 This pathway also includes the formation of non-covalent complexes of proteins with the polyion, but the complex is further augmented by reacting with the oppositely charged macromolecule. The hydration sphere created by PEG chains provides a stabilizing steric effect and results in nanoparticles of a narrow polydispersity. However, the use of non-biodegradable polyelectrolytes may limit further development of such systems for clinical applications. Although some of the suggested block copolymers are based on polypeptides, which can undergo enzymatic and microbial degradation, such degradation can be potentially challenging to control and can lead to irreproducible in vivo


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performance.37 Therefore, it is important to develop hydrolytically degradable polyion complex micelles. Polyphosphazenes - synthetic macromolecules, which are based on a biodegradable phosphorus-nitrogen backbone and organic side groups - offer an appealing approach for developing PEGylated polymers for protein drug delivery. Water-soluble polyphosphazenes are capable of hydrolytic degradation, which can be modulated via careful selection of side groups. Two pendant groups of the monomer unit can potentially enable high charge density, which is required for multivalent ionic interactions with proteins and oppositely charged counterparts, while still allowing for extensive PEGylation. Finally, the macromolecular substitution pathway, which relies on the use of a reactive macromolecular precursor, polydichlorophosphazene (PDCP), and organic nucleophiles, enables versatile derivatization chemistry. A number of polyphosphazenes containing PEG side groups of various chain lengths have been synthesized.38-45 These were typically either amphiphilic or hydrophilic neutral macromolecules designed as delivery vehicles for small molecule drugs. In particular, polyphosphazene copolymers were synthesized as water-soluble carriers for platinum based anticancer drugs38, 39 or for assembling into micelles, which can be loaded with a poorly soluble chemotherapy agent - doxorubicin.40 Polyphosphazene with poly(ethylene oxide-co-propylene oxide) (PEO-co-PO) grafts were also prepared and conjugated with platinum (IV)42 or organoruthenium and organorhodium complexes43 to yield anticancer prodrugs, as well as with imidazoquinoline derivatives for use as immunotherapies.44 PEGylated polyphosphazenes were also employed for assembling hydrogels based on the inclusion complex between the PEG side groups and α -cyclodextrin.45


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Polyphosphazene polyelectrolytes have also been also synthesized and investigated as ionically cross-linked hydrogels and immunoadjuvants (polyanions)46-56 and gene delivery carriers (polycations)57-67 however their PEGylated derivatives are yet to be explored. The present paper describes the synthesis and characterization of polyphosphazene polyelectrolytes containing grafted PEG chains. Novel polyphosphazenes, which comprise carboxylic acid or tertiary amino pendant groups, demonstrated the ability to spontaneously selfassemble into stable PEGylated polyelectrolyte complexes, and improve the stability and reduce the antigenicity of the therapeutic protein, L-Asparaginase (L-ASP) in vitro. They also showed temperature and composition dependent hydrolytic degradability.

MATERIALS AND METHODS Materials. Heptane, sodium hydride, citric acid monohydrate, sodium phosphate monobasic dihydrate, methoxypolyethylene glycol amine (5000 g/mol), PEG-NH2, bovine serum albumin, BSA, bis(2-methoxyethyl) ether, diglyme (Acros Organics, Morris Plains, NJ), ethanol (WarnerGraham, Cockeysville, MD), hydrochloric acid, potassium hydroxide (Alfa Aesar, Haverhill, MA), HyPure™WFI Quality Water (GE Life Sciences, Pittsburgh, PA), sodium carbonate (Amresco, Solon, OH), methyl 3(4-hydroxyphenyl)propionate, MHP (TCI, Portland, OR), polysorbate 20, Tween-20 (Spectrum Chemical, Gardena, CA), 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), native E. coli Lasparaginase protein (Abcam, Cambridge, MA), anti-L-asparaginase (rabbit) antibody, anti-Lasparaginase (rabbit) antibody peroxidase conjugated (Rockland Immunochemicals Inc., Pottstown, PA), asparaginase activity colorimetric/fluorometric assay kit (BioVision Inc.,


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Milpitas, CA), TMB peroxidase EIA substrate kit (Bio-Rad Laboratories, Hercules, CA), 3dimethylamino-1-propanol, trypsin from bovine pancreas (Sigma-Aldrich, Milwaukee, WI), porcine red blood cells (Innovative Research, Novi, MI), sodium chloride (Fisher Scientific, Waltham, MA), sodium phosphate dibasic heptahydrate, sodium bicarbonate (VWR, Radnor, PA), biotinylated mouse IgG (BD Biosciences PharminGen, San Jose, CA), Texas Red goat antimouse IgG (Life Technologies, Carlsbad, CA), and Dulbecco’s Modified Eagle’s Medium, DMEM 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.68 The structure was confirmed by 31P NMR (singlet at -19 ppm; mixture diglyme/deuterated chloroform - 1:3 (v/v)) and its concentration was determined gravimetrically by precipitating with heptane. 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 Information (Figure S2 and S3). Molecular weights were calculated using EZ-Chrome Elite software (Agilent Technologies, Santa Clara, CA). A calibration curve was obtained using narrow polyethylene oxide standards (American Polymer Standards Corporation, Mentor, OH).


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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 a phosphate buffer or 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). Synthesis of Anionic Polyphosphazenes (AP-PEGs). Synthesis of anionic graft copolymers poly[di(carboxylatoethylphenoxy)phosphazene]-graft-poly(ethylene glycol), AP-PEGs, were carried out via subsequent addition of nucleophiles PEG-NH2 and MHP to PDCP followed by hydrolysis of a resulting ester bearing copolymer to yield polyphosphazene polyacid. Copolymers with the content of PEG-NH2 groups of 1, 5, and 16% (mol) were synthesized (APPEG1, AP-PEG5, AP-PEG16). The synthesis of AP-PEG16 is described below as an example.


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0.092 g (0.79 mmol) of PDCP in 15 mL of diglyme was warmed to 60°C while stirring. 1.2 g (0.24 mmol) PEG-NH2 was dissolved in 15 mL diglyme, heated to 60°C, and stirred as 37 µL (0.26 mmol) triethylamine were added. The PEG-NH2 solution was added to PDCP solution. This solution was stirred for 5 hours at 60°C, then stirred overnight at ambient temperature. 0.58 g (3.22 mmol) MHP was dissolved in 10 mL diglyme and heated under nitrogen to 120°C for 30 minutes. Heating was turned off, and a suspension of 0.074 g (3.10 mmol) sodium hydride in 6 mL diglyme was added slowly once the reaction mixture was cooled. The MHP/sodium hydride solution was stirred at ambient temperature for one hour and was then added to the PDCP/PEGNH2 solution, while stirring under nitrogen. The combined solution was heated to 120°C and stirring was continued for 2.5 hours. Heating was turned off and 20 mL 13 N KOH was added once temperature fell below 100°C. The contents were stored at 4°C overnight and suspended precipitate formed. Polymer was collected by filtration then dissolved into deionized water. Polymer was twice precipitated with diglyme and redissolved with deionized water. Next, polymer was precipitated with acetone, redissolved in deionized water, and precipitated again with acetone before drying under vacuum. Polymer was further purified by dissolving in 10 mM ammonium bicarbonate, fractionating on a P-50 Sephadex column, and lyophilizing. AP-PEG1 and AP-PEG5 were synthesized similarly, however the amounts of reagents were adjusted as follows: AP-PEG1: 0.183 g (1.58 mmol) PDCP, 0.4 g (0.08 mmol) PEG-NH2, 11 µL (0.08 mmol) triethylamine, 1.15 g (6.39 mmol) MHP, 0.150 g (6.25 mmol) NaH. AP-PEG5: 0.092 g (0.79 mmol) PDCP, 0.4 g (0.08 mmol) PEG-NH2, 11 µL (0.08 mmol) triethylamine, 0.58 g (3.22 mmol) MHP, 0.074 g (3.10 mmol) NaH.


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AP-PEG1. 1H-NMR (400 MHz, D2O): δ [ppm] = 6.6 (br, 4H, −CH=); 3.2 (br, 4H, −CH2−CH2−O); 2.6 (br, 2H, Ar−CH2−); 2.2 (br, 2H, −CH2−COO); 31P-NMR (162 MHz, D2O): δ [ppm] = -18.2 (br, 1P, −N=P(O−Ar)2). AP-PEG5. 1H-NMR (400 MHz, D2O): δ [ppm] = 6.6 (br, 4H, −CH=); 3.6 (br, 4H, −CH2−CH2−O); 2.6 (br, 2H, Ar−CH2−); 2.3 (br, 2H, −CH2−COO); 31P-NMR (162 MHz, D2O): δ [ppm] = -4.4 (br, 1P, −N=P(NH-CH2-)2); -17.1 (br, 1P, −N=P(O−Ar)(NH-CH2-)); -18.2 (br, 1P, −N=P(O−Ar)2). AP-PEG16. 1H-NMR (400 MHz, D2O): δ [ppm] = 6.7 (br, 4H, −CH=); 3.6 (br, 4H, −CH2−CH2−O); 2.6 (br, 2H, Ar−CH2−); 2.4 (br, 2H, −CH2−COO); 31P-NMR (162 MHz, D2O): δ [ppm] = -4.4 (br, 1P, −N=P(NH-CH2-)2); -17.3 (br, 1P, −N=P(O−Ar)(NH-CH2-)); -19.8 (br, 1P, −N=P(O−Ar)2). Synthesis of Cationic Polyphosphazene (CP-PEG). Synthesis of cationic graft copolymer poly[di(dimethylaminopropyloxy)phosphazene]-graft-poly(ethylene

glycol),

CP-PEG,

was

performed using subsequent addition of amine-functionalized poly(ethylene glycol) (PEG-NH2) and 3-dimethylamino-1-propanol (DMAP). 0.80 g (0.16 mmol) of PEG-NH2 was dissolved in 20 mL of diglyme under anhydrous conditions. 25 µL (0.18 mmol) of triethylamine was added to the solution, which was then heated at 60°C and stirred to complete dissolution. 0.184 g (1.58 mmol) of PDCP solution in 20 mL of diglyme was heated to 60°C to allow both solutions to reach the same temperature. The polymer solution was added dropwise into the PEG-NH2 solution under stirring, and allowed to react for 6.5 hours. Then 0.757 mL (6.41 mmol) of DMAP and 0.982 mL (7.05 mmol) of triethylamine were added to the reaction mixture, and it was left at 60°C overnight. The heating was turned off; the reaction mixture was allowed to cool, and then kept in the freezer (-32 °C) overnight. Solid


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precipitate was separated by centrifuging at 4°C and then stored in the freezer. For further purification, the precipitate was dissolved to 10 mg/mL in a 10 mM ammonium bicarbonate solution and purified through fractionation using a P-50 Sephadex column. The aliquots containing polymer were collected and lyophilized twice, then stored dry in a freezer at -32°C. 1

H-NMR (400 MHz, D2O): δ [ppm] = 3.6 (br, 4H, (−CH2−CH2−O)); 3.3 (br, 3H, O−CH3); 2.8

(br, 6H, −N−(CH3)2); 3.5 (br, 2H, −O−CH2); 2.0 (br, 2H, −O−CH2-CH2); 3.1 (br, 2H, −O−CH2CH2-CH2); 1.2 (br, 1H, −NH). 31

P-NMR (162 MHz, D2O): δ [ppm] = -2.4 (br, 1P, −N=P(-O-CH2-)2); 11.4 (br, 1P, −N=P(-

O−CH2-)(-NH-CH2-)). Antigenicity of Protein in NP-PEG Complexes as Evaluated by Antibody Binding. The amount of L-ASP available for interaction with antibody was measured using an enzyme-linked immunosorbent assay (ELISA). 10 µL of Anti-L-Asparaginase (rabbit) antibody was mixed with 10 mL 0.05 M carbonate-bicarbonate buffer (pH 9.6). 100 µL aliquots of this solution were added to a 96-well plate and incubated overnight at 4°C. Next, the solution was removed and the plate was washed with PBS. To prevent non-specific interaction, 300 mL of blocking buffer (1% BSA in PBS) was added to each well and incubated for 1 h at room temperature. The plate then was rinsed with washing buffer (0.05% Tween-20 in PBS). Formulations containing 0.01 mg/mL of L-ASP with various concentrations of NP-PEG (complex of AP-PEG5 and CP-PEG) in PBS were diluted to a final concentration of 25 ng/mL L-ASP. 100 µL of these solutions were added to each well and incubated for 1 hour at room temperature. The plate was then washed with washing buffer, 100 µL of anti-L-asparaginase (rabbit) antibody peroxidase conjugated (0.5 µg/mL in PBS containing 0.5% BSA and 0.05% Tween) was added to each well and incubated for 30 minutes at room temperature, then rinsed with washing buffer. 100 µL of TMB peroxidase


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EIA substrate kit solution was added into each well and incubated for 20 minutes. The reaction was stopped by adding 100 µL 1 M sulfuric acid. Optical density at 450 nm was measured by Multiscan Spectrum microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA). The data were presented as a residual antigenicity (RA), which was calculated using the following equation: RA = ODPoly / OD0 x 100, where OD0 and ODPoly are the optical densities of the solution without polymer and in the presence of polymer. Proteolytic Stability. Various solution formulations of L-ASP were incubated at 37 °C in the presence of 0.005 mg/mL trypsin for predetermined time periods. L-ASP activity was measured by its ability to hydrolyze asparagine to aspartic acid, which was then detected fluorescently at Ex/Em = 535/590 nm using a coupled enzymatic reaction (BioVision, Inc., Milpitas, CA). Samples were first diluted 10-fold and then 10 µl of diluted solution was mixed with 50 µl of assay buffer in a well of a 96-well plate. To this mixture, 50 µl of assay reagent solution was added and fluorescence intensity was recorded in 3-minute intervals for 30 minutes. L-ASP activity rate was calculated using the linear part of the curve. Proteolytic resistance was evaluated based on the residual activity of L-ASP - the ratio between activity rates before and after incubation with trypsin, expressed as a percent. Thermal Stability. Various solution formulations of L-ASP were incubated at 60 °C for predetermined time periods. Activity of L-ASP was measured as described above. Hydrolytic Degradation of Polyphosphazenes. Polymers were dissolved to a concentration of 0.50 mg/mL in 1xPBS then filtered through a 0.22 µm membrane. Solutions were stored at 4°C, ambient temperature, 37°C, and 65°C. Samples were taken for GPC analysis at various time intervals.


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RESULTS AND DISCUSSION Synthesis and Characterization of PEGylated Polyphosphazene Polyelectrolytes. Macromolecular modules for the construction of “core-shell” structured nano-assemblies were designed to include three main features – ionic moieties for enabling electrostatic interactions in the core, grafted PEG chains for forming the hydrophilic shell, and hydrolytically labile bonds to facilitate polymer degradation. PEG with molecular weight of 5,000 g/mol, which is frequently employed for covalent PEGylation of proteins, and L-ASP in particular,69 was selected for grafting to polyphosphazene backbone. It has been also demonstrated that modification of LASP with PEG of the above molecular weight effectively reduced antigenicity of the protein and improved its proteolytic resistance, which was not achievable with smaller PEG chains.70 To enable electrostatic interactions between component macromolecules in aqueous solutions, phenylpropionic acid and dimethylaminopropyl pendant groups were introduced into anionic (AP-PEG) and cationic polyphosphazenes (CP-PEG), respectively. All ionic functionalities were linked to the phosphazene backbone through oxygen atoms, whereas PEG chains were grafted using their terminal aminogroups creating links that can potentially amplify hydrolytic degradation of the copolymer.41,

71

PEGylated ionic polyphosphazenes were synthesized using

macromolecular substitution approach as shown in Figure 1. The macromolecular precursor PDCP was first reacted with a targeted amount of monofunctional PEG containing a primary amino end group to create a graft copolymer structure. This step was followed by the replacement of chlorine atoms of the polyphosphazene main chain with pendant groups containing anionic (in AP-PEG) and cationic (in CP-PEG) functionalities. In the case of APPEG, an excess of the ester containing nucleophile, MHP, was then added to complete the substitution reaction followed by hydrolyzing the ester functionality to reveal carboxylic acid


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groups. The substitution of CP-PEG was completed by adding excess of DMAP in the presence of triethylamine. Three AP-PEGs with varying content of PEG and one CP-PEG were synthesized for further investigation of their complexation. The structure and composition of synthesized polymers were analyzed by 1H NMR and

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P NMR (for representative spectra see

Supporting Information, Figure S1) and their molecular weights were determined by GPC. Table 1. Physico-Chemical Characterization of Polyphosphazene Polyions. Polymer

PEG*

Mw**

Đ***

% (mol)

% (w/w)

(kg/mol)

AP-PEG1

1

25

450

1.67

AP-PEG5

5

59

150

1.72

AP-PEG16

16

81

150

1.81

CP-PEG

13

89

340

1.81

* Calculated based on 1H NMR data; ** As measured by GPC (PBS, pH 7.4 containing 10% of acetonitrile was used as a mobile phase, polyethylene oxide were used as standards); ***Đ – molecular weight dispersity as measured by GPC. Table 1 summarizes compositions and molecular weights of synthesized macromolecules as determined by NMR and GPC. All graft copolymers were fully soluble in water and PBS (pH 7.4) and showed unimodal molecular weight distribution (Supporting Information, S2 and S3). It was also found that the utilized sequential substitution approach provided adequate control of polymer composition. The content of PEG in each polyphosphazene correlated well with its concentration in the reaction mixture expressed as a molar part of chlorine atoms of PDCP (Supporting Information, S4, data shown for AP-PEG). The somewhat lower observed molecular weights of AP-PEG5 and AP-PEG16 (Table 1) may potentially indicate although minimal, but still detectable degradation of these polymers during the synthesis. Though this requires further investigation, it is possible that higher content of bulky PEG groups in these polymers may


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create steric hindrance for the following substitution with MHP producing minute quantities of residual chlorine atoms, which in turn can cause chain breakdown in aqueous environment. It needs to be mentioned that although the molar content of PEG grafts in polyphosphazenes was relatively low - 1 – 16 %, the percent of PEG by weight was in the range between 25 and 89 %. PEGylated

Polyphosphazene

Polyelectrolyte

Complexes.

Anionic

and

cationic

polyphosphazenes were then evaluated for their ability to spontaneously assemble into polyelectrolyte complexes in aqueous solutions. It was observed that adding CP-PEG to any of AP-PEG solutions at neutral pH resulted in a gradual increase of sample turbidity (Figure 2A). As seen from the Figure, the faster onset and steeper slope of turbidimetric titration curves was observed for AP-PEG1, which has the highest content of carboxylic acid groups (Figure 2A, curve 1). Polyphosphazene with the highest density of PEG grafts (AP-PEG16 - curve 3) showed lowest levels of turbidity and required more cationic polymer to achieve them. An increase in turbidity was also detected when CP-PEG and AP-PEG were mixed at near physiological conditions. Figure 2B shows the results of turbidimetric titration for the AP-PEG5 – CP-PEG system in PBS, pH 7.4 (curve 1). However, the presence of salt in this solution resulted in a slower development of sample turbidity upon addition of cationic polymer when compared to same polymers mixed in phosphate buffer, free from sodium chloride (Figure 2B, curve 2). These results indicated the ability of oppositely charged polyphosphazenes to form polyelectrolyte complexes in aqueous solutions and provided compelling reasons for further investigation of the system using asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) methods. AF4 traces of CP-PEG and AP-PEG5, as well as their mixtures, are shown in Figure 2C. Similarly to size-exclusion HPLC, this elution-based method allows for the separation of


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macromolecules and nanoparticles by size and detection by UV absorbance; however, as opposed to chromatographic methods, the upper size limit for the analyte can reach as high as 100 µm.72 The separation is carried out in a single liquid phase and an external flow of the mobile phase is applied perpendicularly to the direction of sample flow through a channel equipped with semi-permeable membrane.72 Figure 2C demonstrates that the addition of CPPEG to AP-PEG5 resulted in a substantial decrease in UV peak area, which was proportional to the amount of cationic polymer added (traces 1-3), as compared to AP-PEG5 alone (trace 4). However, minimal changes in the elution time of the sample, which is generally related to the size of analyte, were observed. CP-PEG alone showed only negligible UV absorbance at the employed detection wavelength (Figure 2, trace 5). The observed changes in AF4 profiles upon addition of cationic polymer appear to be consistent with polyelectrolyte complex formation. The decrease in the UV absorbance at 210 nm may be related to the experimentally detected turbidity of polyelectrolyte complexes discussed above and hydrophobic nature of the complex core, which can potentially increase non-specific adsorption to the analytical membrane. The dimensions of complexes were further investigated by DLS. A representative size distribution profile by intensity for the complex formed by AP-PEG5 and CP-PEG at 1:1 (w/w) ratio shows unimodal distribution (Figure 2D) with z-average hydrodynamic diameter of 42 nm and a relatively narrow dispersity - polydispersity index of 0.27. The dependence of the normalized hydrodynamic diameter of the complex (D/DCP-PEG) on the composition of formulation is shown in Figure 3A. As seen from the Figure, formation of the complex was characterized by a significant increase in size compared to its macromolecular components. The polydispersity parameter of the complexes, as determined by DLS, varied between 0.5 and 0.25, with minimum achieved at about 70% of CP-PEG content (Figure 3B). The observed count rate,


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which is representative of light scattering intensity (Figure 3C), peaked at the component ratios corresponding to maximum size values – 60-80% of CP-PEG (%w/w). Z-potential of APPEG5/CP-PEG formulations rose steadily as the content of polycation increased (Figure 3D). Electroneutrality point, which suggests the formation of stoichiometric polyelectrolyte complexes, was reached at the 1:1 (w/w) ratio of CP-PEG to AP-PEG5 in the formulation. This corresponds to the ratio of amine/carboxylic acid groups of approximately 0.75, which is in agreement with previous findings that polyphosphazene polyacids may not be completely ionized in neutral solutions.50,

52

Typically, unless stabilized in the form of micelles or

coacervates,73, 74 the formation of stoichiometric polyelectrolyte complexes between oppositely charged polyelectrolytes results in their subsequent aggregation and precipitation75, 76 However, electrostatically neutral formulation of AP-PEG5 and CP-PEG (1:1 (w/w) ratio) showed no sign of aggregation under these conditions and remained stable for at least several days. The observed increase in macromolecular dimensions, low polydispersity, and increase in solution turbidity (scattering intensity) of AP-PEG/CP-PEG formulations as compared to their macromolecular components, along with the stability of electroneutral formulation provides compelling support for the formation of nano-assemblies having a compact polyelectrolyte complex core and stabilizing hydrophilic PEG shell. PEGylated Polyphosphazene Complexes Reduce Antigenicity (Antibody Binding) and Stabilize L-Asparaginase (L-ASP) in Vitro. L-ASP, the enzyme that converts asparagine into aspartate and ammonia is an effective antineoplastic agent, used in acute lymphoblastic leukemia chemotherapy.77 Despite its well-proven clinical efficacy, the use of unmodified L-ASP has been limited by the development of hypersensitivity reactions and neutralizing antibodies, as well as the need for frequent administration.78 L-ASP enzyme was covalently linked to PEG, forming


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the PEGylated L-ASP (Pegaspargase - Oncaspar®), which eliminated most of these limitations.69,

78,

79

It was tempting to investigate whether non-covalent PEGylated

polyphosphazene complexes were also able to reduce antigenicity and improve stability of LASP. For the evaluation of their biologically relevant properties, PEGylated neutral polyelectrolyte complexes - NP-PEGs (CP-PEG+AP-PEG5) were prepared by first mixing aqueous solutions of the negatively charged enzyme (isoelectric point is reported to be between 4.6 and 5.5)77 with CP-PEG at neutral pH followed by the addition of AP-PEG5 to form a complex with 1:1 polymer mass ratio (Figure 1). L-ASP activity was evaluated by its ability to hydrolyze asparagine to aspartic acid, which was then measured fluorescently. No loss of activity was detected for NP-PEG formulations as compared to the activity of the enzyme in the absence of polyphosphazenes, and AF4 analysis did not reveal presence of unbound L-ASP in polymer formulations (Supporting information, Figure S5). Encapsulation of L-ASP did not affect the size and z-potential of the formulation. Initial reports on covalent PEGylation of L-ASP cited the need to overcome the immunogenicity of this enzyme.69,

70

Protein immunogenicity is a significant concern for

therapeutic drugs as it can affect both safety and efficacy of the drug.80 The consequences of protein immunogenicity vary from no evidence of clinical effect to severe, life-threatening responses, and its reduction can be positively reflected in the pharmacokinetic profile of the protein.12 The ability of PEGylated polyphosphazenes to shield antigenic sites of L-ASP was investigated by an enzyme-linked immunosorbent assay (ELISA). Figure 4A shows the residual antigenicity (the ability to bind antibody) of the enzyme as a function of added CP-PEG with (curve 1) or without (curve 2) subsequent addition of AP-PEG5. As seen from the Figure, the


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reduction in antigenicity of the protein was proportional to the amount of cationic polymer added. Moreover, formation of NP-PEG was important for further shielding of antigenic sites and resulted in a dramatic (over 10 fold) reduction in antigenicity. Thermal stability of L-ASP modified with cationic polyphosphazene and a polyelectrolyte complex was explored in aqueous solution at 60 °C. Figure 4B demonstrates that although the addition of CP-PEG to L-ASP resulted in the improved stability of the enzyme (curve 2 versus curve 1), a NP-PEG complex (curve 3) once again afforded best results leading to an almost 2.5 fold extension of half life compared to native enzyme. PEGylated polyphosphazenes were evaluated for their ability to protect L-ASP against proteolytic digestion by trypsin. The residual activity of L-ASP and its NP-PEG formulations versus time of incubation with trypsin are shown in Figure 4C (curves 1, 2, and 3 correspondingly). Similarly to studies on thermal stability, polyphosphazene formulations increased the proteolytic resistance of L-ASP with polyelectrolyte complex showing the best stability. The half-life for NP-PEG formulation exceeded that of the native enzyme over 8.5 fold (Figure 4D). It has to be noted that covalent PEGylation of L-ASP usually increases its stability approximately 7-10 fold.77 Overall, non-covalent modification of L-ASP with polyphosphazene polyelectrolyte complexes resulted in a 10-fold reduction in protein antigenicity, as well as 2.5 and 8 fold enhancements in thermal stability and proteolytic resistance of this enzyme. Hydrolytic

Degradation

of

Polyphosphazene

Copolymers.

Finally,

PEGylated

polyphosphazenes were evaluated for their ability to undergo hydrolytic degradation at near physiological and potential storage conditions. Solutions of AP-PEGs and CP-PEG in PBS (pH 7.4) were incubated at various (4°C, 37°C, 65°C and ambient) temperatures and their residual


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molecular weight was analyzed at various time intervals by GPC.

Representative

chromatograms (AP-PEG16, 65 °C) show consistent decrease in polymer molecular weight (shift towards longer retention times) over time (Figure 5A). This was also accompanied with a gradual rise in the peak representing small molecules (retention time longer than 23 minutes), indicating the release of products corresponding to polyphosphazene side groups.52 Figures 5A5E summarize molecular weight changes for AP-PEGs at various temperatures. All PEGylated polyacids underwent rapid degradation at 65°C and somewhat slower breakdown at 37°C, with the rate of hydrolysis increasing as the content of PEG groups in polymer rose. A relatively slow degradation rate was observed at 4°C - about 10% molecular weight loss over a period of 100 days. Degradation profiles of CP-PEG generally followed the trends observed for AP-PEGs with rapid and complete degradation under accelerated conditions and slower hydrolysis at lower temperatures (Figure 5F). These results validate hydrolytic degradability of all synthesized polymers under near physiological conditions, as well as suggest short-term solution stability of PEGylated polyphosphazenes – less than 20% molecular weight decrease over one month period.

CONCLUSIONS Spontaneous supramolecular assembly of biodegradable polyelectrolytes into stable PEGylated nanocomplexes in aqueous solutions presents an appealing approach for encapsulation and delivery of pharmaceutical agents. In particular, this methodology can potentially eliminate complexity and reduce expenses of chemical conjugation reactions and purification processes, which

are

routinely

associated

with

traditional

covalent

PEGylation

of

proteins.

Polyphosphazenes appear to offer some critical advantages for realizing this objective. Novel, oppositely charged polyelectrolytes with variable content of grafted PEG chains were synthesized as potential modular components of non-covalently associated nano-assemblies.


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Investigation of their interactions in aqueous solutions revealed experimental support for the formation of stable polyelectrolyte complexes with overall hydrodynamic diameters under 100 nm. The observed increase in size of interacting macromolecules, low polydispersity of some formulations, and stability of electrostatically uncharged nano-assemblies strongly suggest their polyelectrolyte complex “core” - stabilizing hydrophilic PEG “shell” structure. The potential of polyphosphazene polyelectrolyte complexes as PEGylated delivery vehicles was validated in vitro using L-ASP as a therapeutic protein. It was demonstrated that non-covalent modification of L-ASP with PEGylated polyphosphazene complexes resulted in a dramatic reduction in protein antigenicity, as well as substantial improvement in thermal stability and proteolytic resistance of this enzyme. Finally, PEGylated polyphosphazene polyelectrolytes demonstrated hydrolytic degradability in aqueous solutions, which suggests clinical suitability and potential for modulating pharmacokinetic profiles. Notably, their degradation rates were considerably slowed at lower temperatures indicating short-term stability in solutions. It is tempting to speculate that hydrolytically degradable PEGylated polyelectrolyte complexes provide an alternative approach to protein stabilization and delivery that can potentially simplify production processes, result in contaminant free formulations, and even broaden the scope of protein drugs to which PEGylation technology can be applied.

Figure Captions Figure 1. Synthesis of CP-PEG and AP-PEG and schematic presentation of their spontaneous complexation in aqueous solution.


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Figure 2. (A) Turbidimetric titration of AP-PEG1 (1), AP-PEG5 (2), and AP-PEG16 (3) with CP-PEG (4 mg/mL AP-PEG and 4 mg/L CP-PEG solutions; 10 mM phosphate buffer, pH 7.4, turbidity data plotted versus the ratio of amino and carboxylic acid groups in solution, measurements were performed in triplicates, error bars represent standard deviation); (B) Turbidimetric titration of AP-PEG1 with CP-PEG in PBS (1) and 10 mM phosphate buffer (2) (4 mg/mL AP-PEG5 and 4 mg/L CP-PEG solutions; pH 7.4, measurements were performed in triplicates, error bars represent standard deviation); (C) AF4 profiles of AP-PEG5 formulations with 0.05 (1), 0.1 (2), 0.25 mg/mL CP-PEG (3) as compared with controls: AP-PEG5 (4) and 0.25 mg/mL CP-PEG (5) (0.25 mg/mL AP-PEG5, detection wavelength - 210 nm); (D) Representative DLS profile of AP-PEG - CP-PEG formulation (0.5 mg/mL AP-PEG, 0.5 mg/mL of CP-PEG, Dz – z-average hydrodynamic diameter, pdi – polydispersity index; PBS, pH 7.4). Figure 3. (A) Normalized hydrodynamic diameter, (B) polydispersity index, (C) count rate, and (D) z-potential of CP-PEG and AP-PEG5 formulation as a function of CP-PEG content (DCP-PEG and D – volume average peak hydrodynamic diameters of CP-PEG and formulation, correspondingly; 1 mg/mL total polymer concentration, 10 mM phosphate buffer, pH 7.4). All measurements were performed in triplicates, error bars represent standard deviation. Figure 4. Effect of PEGylated complexes on stability and antigenicity of L-ASP. (A) Residual antigenicity of L-ASP in (1) NP-PEG-ASP and (2) CP-PEG-ASP versus concentration of CPPEG (1 mg/mL AP-PEG; 0.01 mg/mL L-ASP; ELISA; PBS, pH 7.4); (B) thermal stability of (1) L-ASP, (2) CP-PEG-ASP and (3) NP-PEG-ASP (2 mg/mL NP-PEG, 0.5 mg/mL CP-PEG; 0.05 mg/mL L-ASP; 60°C, pH 7.4); (C) Proteolytic resistance of (1) L-ASP, (2) CP-PEG-ASP and (3) NP-PEG-ASP against trypsin as shown by time dependence of residual enzymatic activity and (D) their half-life (1.0 mg/mL NP-PEG (CP-PEG-AP-PEG5), 0. 5 mg/mL CP-PEG; 0.01


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mg/mL L-ASP; 0.005 mg/mL Trypsin, 37°C, pH 7.4). All measurements were performed in triplicates, error bars represent standard deviation. Figure 5. Hydrolytic degradation of AP-PEGs and CP-PEG. (A) HPLC profiles of AP-PEG16 at various timepoints after incubation at 65 °C; (B-E) Residual MW versus degradation time for AP-PEG16 (1, circles), AP-PEG5 (2, triangles) and AP-PEG1 (3, diamonds) at (B) 65 °C; (C) 37 °C; (D) ambient temperature; and (E) 4 °C (0.5 mg/mL polymer, PBS, pH 7.4); (F) Residual MW of CP-PEG versus degradation time at (1) 65 °C; (2) 37 °C; (3) ambient temperature; and (4) 4 °C (0.5 mg/mL polymer concentration, PBS, pH 7.4). Error bars represent standard deviation of GPC measurements performed in triplicates. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge. Representative NMR spectra, GPC profiles, AF4 profiles, reaction mixture – polymer composition graph. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; fax 240-314-6225; phone 1 240-314-6456. ORCID: 0000-00016186-6156 Author Contributions The manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript. Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported in part by MPower Maryland, Maryland Innovation Initiative (MII) award (A.K.A, A.M.) and intramural IBBR funding (A.M., A.P.M, J.L.W.). Authors are thankful to Fushimi Pharmaceutical Co. for kindly providing hexachlorocyclotriphosphazene and to Dr. Raquel Godoy-Ruiz (Protein Production and Biophysics (PPB) Center for Biomolecular Therapeutics (CBT) of the University of Maryland School of Medicine) for her assistance and the use of separation equipment in polyphosphazene purification. ABBREVIATIONS PDCP, polydichlorophosphazene; AP-PEG, poly[di(carboxylatoethylphenoxy)phosphazene]graft-poly(ethylene glycol), CP-PEG, poly[di(dimethylaminopropyloxy)phosphazene]-graftpoly(ethylene

glycol);

MHP,

methyl

3(4-hydroxyphenyl)propionate;

DMAP,

dimethylaminopropanol; PBS, phosphate buffered saline; DLS, dynamic light scattering; MALS, multi-angle laser light scattering; NMR, nuclear magnetic resonance; GPC, gel permeation chromatography; AF4, asymmetric flow field flow fractionation; CD spectroscopy, circular dichroism spectroscopy; Dz, z-average hydrodynamic diameter; Mw, weight average molecular weight; PDI, polydispersity index; GPC, gel permeation chromatography; ELISA, enzyme-linked immunosorbent assay; pI, isoelectric point. REFERENCES 1.

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54. Andrianov, A. K.; DeCollibus, D. P.; Gillis, H. A.; Kha, H. H.; Marin, A.; Prausnitz, M. R.; Babiuk, L. A.; Townsend, H.; Mutwiri, G., Poly[di(carboxylatophenoxy)phosphazene] is a potent adjuvant for intradermal immunization. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, (45), 18936-18941. 55. Andrianov, A. K.; Chen, J.; Payne, L. G., Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazene solutions. Biomaterials 1998, 19, (1-3), 109-115. 56. Payne, L. G.; Jenkins, S. A.; Woods, A. L.; Grund, E. M.; Geribo, W. E.; Loebelenz, J. R.; Andrianov, A. K.; Roberts, B. E., Poly[di(carboxylatophenoxy)phosphazene] (PCPP) is a potent immunoadjuvant for an influenza vaccine. Vaccine 1998, 16, (1), 92-98. 57. Kwon, S.-K., Synthesis of Water-Soluble Aminoaryloxy-Methylamino Cosubstituted Polyphosphazenes as Carrier Species for Biologically Active Agents. Bull. Korean Chem. Soc. 2001, 22, (11), 1243-1247. 58. de Wolf, H.; de Raad, M.; Snel, C.; van Steenbergen, M.; Fens, M.; Storm, G.; Hennink, W., Biodegradable Poly(2-Dimethylamino Ethylamino)Phosphazene for In Vivo Gene Delivery to Tumor Cells. Effect of Polymer Molecular Weight. Pharm. Res. 2007, 24, (8), 1572-1580. 59. de Wolf, H. K.; Luten, J.; Snel, C. J.; Oussoren, C.; Hennink, W. E.; Storm, G., In vivo tumor transfection mediated by polyplexes based on biodegradable poly(DMAEA)-phosphazene. J. Controlled Release 2005, 109, (1-3), 275-287. 60. Yang, Y.; Xu, Z.; Chen, S.; Gao, Y.; Gu, W.; Chen, L.; Pei, Y.; Li, Y., Histidylated cationic polyorganophosphazene/DNA self-assembled nanoparticles for gene delivery. Int. J. Pharm. 2008, 353, (1-2), 277-282.


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61. Yang, Y.; Xu, Z.; Jiang, J.; Gao, Y.; Gu, W.; Chen, L.; Tang, X.; Li, Y., Poly(imidazole/DMAEA)phosphazene/DNA self-assembled nanoparticles for gene delivery: Synthesis and in vitro transfection. J. Controlled Release 2008, 127, (3), 273-279. 62. Yang, Y.; Zhang, Z.; Chen, L.; Gu, W.; Li, Y., Galactosylated Poly(2-(2aminoethyoxy)ethoxy)phosphazene/DNA Complex Nanoparticles: In Vitro and In Vivo Evaluation for Gene Delivery. Biomacromolecules 2010, 11, (4), 927-933. 63. Yang, Y.; Zhang, Z.; Chen, L.; Gu, W.; Li, Y., Urocanic Acid Improves Transfection Efficiency of Polyphosphazene with Primary Amino Groups for Gene Delivery. Bioconjugate Chem. 2010, 21, (3), 419-426. 64. Luten, J.; van Nostrum, C. F.; De Smedt, S. C.; Hennink, W. E., Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J. Controlled Release 2008, 126, (2), 97-110. 65. Luten, J.; van Steenbergen, M. J.; Lok, M. C.; de Graaff, A. M.; van Nostrum, C. F.; Talsma, H.; Hennink, W. E., Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazenebased polyplexes exhibit receptor-specific gene expression. Eur. J. Pharm. Sci. 2008, 33, (3), 241-251. 66. Luten, J.; Van Steenis, J. H.; Van Someren, R.; Kemmink, J.; SchuurmansNieuwenbroek, N. M. E.; Koning, G. A.; Crommelin, D. J. A.; Van Nostrum, C. F.; Hennink, W. E., Water-soluble biodegradable cationic polyphosphazenes for gene delivery. J. Controlled Release 2003, 89, (3), 483-497.


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Figure 1. Synthesis of CP-PEG and AP-PEG and schematic presentation of their spontaneous complexation in aqueous solution. 595x446mm (72 x 72 DPI)



Figure 2. (A) Turbidimetric titration of AP-PEG1 (1), AP-PEG5 (2), and AP-PEG16 (3) with CP-PEG (4 mg/mL AP-PEG and 4 mg/L CP-PEG solutions; 10 mM phosphate buffer, pH 7.4, turbidity data plotted versus the ratio of amino and carboxylic acid groups in solution, measurements were performed in triplicates, error bars represent standard deviation); (B) Turbidimetric titration of AP-PEG1 with CP-PEG in PBS (1) and 10 mM phosphate buffer (2) (4 mg/mL AP-PEG5 and 4 mg/L CP-PEG solutions; pH 7.4, measurements were performed in triplicates, error bars represent standard deviation); (C) AF4 profiles of AP-PEG5 formulations with 0.05 (1), 0.1 (2), 0.25 mg/mL CP-PEG (3) as compared with controls: AP-PEG5 (4) and 0.25 mg/mL CP-PEG (5) (0.25 mg/mL AP-PEG5, detection wavelength - 210 nm); (D) Representative DLS profile of APPEG - CP-PEG formulation (0.5 mg/mL AP-PEG, 0.5 mg/mL of CP-PEG, Dz – z-average hydrodynamic diameter, pdi – polydispersity index; PBS, pH 7.4). 595x793mm (72 x 72 DPI)


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Figure 3. (A) Normalized hydrodynamic diameter, (B) polydispersity index, (C) count rate, and (D) zpotential of CP-PEG and AP-PEG5 formulation as a function of CP-PEG content (DCP-PEG and D – volume average peak hydrodynamic diameters of CP-PEG and formulation, correspondingly; 1 mg/mL total polymer concentration, 10 mM phosphate buffer, pH 7.4). All measurements were performed in triplicates, error bars represent standard deviation. 595x793mm (72 x 72 DPI)


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Figure 4. Effect of PEGylated complexes on stability and antigenicity of L-ASP. (A) Residual antigenicity of LASP in (1) NP-PEG-ASP and (2) CP-PEG-ASP versus concentration of CP-PEG (1 mg/mL AP-PEG; 0.01 mg/mL L-ASP; ELISA; PBS, pH 7.4); (B) thermal stability of (1) L-ASP, (2) CP-PEG-ASP and (3) NP-PEG-ASP (2 mg/mL NP-PEG, 0.5 mg/mL CP-PEG; 0.05 mg/mL L-ASP; 60°C, pH 7.4); (C) Proteolytic resistance of (1) L-ASP, (2) CP-PEG-ASP and (3) NP-PEG-ASP against trypsin as shown by time dependence of residual enzymatic activity and (D) their half-life (1.0 mg/mL NP-PEG (CP-PEG-AP-PEG5), 0. 5 mg/mL CP-PEG; 0.01 mg/mL L-ASP; 0.005 mg/mL Trypsin, 37°C, pH 7.4). All measurements were performed in triplicates, error bars represent standard deviation. 595x793mm (72 x 72 DPI)



Figure 5. Hydrolytic degradation of AP-PEGs and CP-PEG. (A) HPLC profiles of AP-PEG16 at various timepoints after incubation at 65 °C; (B-E) Residual MW versus degradation time for AP-PEG16 (1, circles), AP-PEG5 (2, triangles) and AP-PEG1 (3, diamonds) at (B) 65 °C; (C) 37 °C; (D) ambient temperature; and (E) 4 °C (0.5 mg/mL polymer, PBS, pH 7.4); (F) Residual MW of CP-PEG versus degradation time at (1) 65 °C; (2) 37 °C; (3) ambient temperature; and (4) 4 °C (0.5 mg/mL polymer concentration, PBS, pH 7.4). Error bars represent standard deviation of GPC measurements performed in triplicates. 595x793mm (72 x 72 DPI)


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For TOC only 595x446mm (72 x 72 DPI)


Hydrolytically Degradable PEGylated Polyelectrolyte

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