Bioinspired pH-Responsive Polymers for the Intracellular Delivery of

4.26 pH-Responsive Polymers for the Intracellular Delivery of Biomolecular Drugs. D.S.W. Benoit , W. Gray , N. Murthy , H. Li , C.L. Duvall. 2017,472-...
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Bioconjugate Chem. 2003, 14, 412−419

Bioinspired pH-Responsive Polymers for the Intracellular Delivery of Biomolecular Drugs Niren Murthy,† Jean Campbell,‡ Nelson Fausto,‡ Allan S. Hoffman,*,† and Patrick S. Stayton*,† Department of Bioengineering and Department of Pathology, University of Washington, Seattle, Washington 98195. Received July 12, 2002; Revised Manuscript Received October 14, 2002

The biotechnology and pharmaceutical industries have developed a wide variety of potential therapeutics based on the molecules of biology: DNA, RNA, and proteins. While these therapeutics have tremendous potential, effectively formulating and delivering them have also been a widely recognized challenge. A variety of viruses and toxins have evolved multi-functional biomolecules to solve this problem by directing cellular uptake and enhancing biomolecular transport to the cytoplasm from the low pH endosomal compartment. In the study reported here, we have designed and synthesized bio-inspired, pH-responsive polymeric carriers, which we call “encrypted polymers”, that mimic the multi-functional design of biology. These encrypted polymers target and direct cellular uptake, as well as enhance cytosolic delivery by disrupting endosomal membranes in a pH-dependent fashion. We show that the encrypted polymeric carriers significantly enhance the delivery of oligonucleotides and peptides to the cytoplasm of cultured macrophages, demonstrating the potential of this approach for delivery of biotherapeutics and vaccines.

INTRODUCTION

The exciting potential of biomolecular therapeutics is well established, but numerous biological barriers to these relatively fragile drugs have proven to represent significant delivery challenges. The bioavailability of proteins and nucleic acids are typically low compared to traditional small molecule therapeutics, due to factors such as poor stability and susceptibility to enzymes. A widespread barrier for the activity of vaccines and biomolecular drugs that function intracellularly is cytoplasmic delivery. Proteins, peptides, and nucleic acids usually enter cells through the process of fluid-phase or receptor-mediated endocytosis and are initially localized in the endosomal compartment. A high percentage of these biomolecules are subsequently trafficked to lysosomes, where they are degraded. For example, the percentage of pinocytosed protein molecules released to the cytoplasm of cultured mouse fibroblast cells has been estimated to be less than 5% (1) and that of oligonucleotides to be less than 20% (2). Thus, there is a significant need to design and synthesize carriers that can enhance the intracellular delivery of biotherapeutics, in particular to overcome the important barrier of lysosomal trafficking (3-8). The * Corresponding authors. Address: Department of Bioengineering, Box 352255, University of Washington Seattle, WA 98195. A.S.H.: telephone 206-543-9423, fax 206-543-6124, email: [email protected]. P.S.S.: telephone 206-6858148, fax: 206-685-8256, e-mail: [email protected]. † Department of Bioengineering, University of Washington. ‡ Department of Pathology, University of Washington. 1 Abbreviations: DMAEMA; dimethylaminoethyl methacrylate; BMA, butyl methacrylate; BA, butyl acrylate; SBA, styrene benzaldehyde monomer; SA, styrene acetal monomer; AIBN, azobisisobutyronitrile; ODN, oligodeoxynucleotide; AS-ODN, antisense oligodeoxynucleotide; DMEM, Dulbecco’s modified eagle’s medium; iNOS, inducable nitric oxide synthetase; NO, nitric oxide; LPS, lipopolysaccharide; PTSOH, p-toluene sulfonic acid.

issue of cytoplasmic delivery is particularly important for vaccine development, where antigenic proteins/peptides must reach the cytoplasm of antigen-presenting cells to enter the MHC1 pathway for subsequent stimulation of CD8+ lymphocytes. Other biomolecular drugs or vaccine components such as plasmid DNA, antisense oligonucleotides, ribozymes, and immunotoxins must also be transported to the cytoplasm to reach their eventual intracellular molecular targets and compartments. The use of endosomal releasing proteins and peptides in gene and protein delivery systems has been widely investigated (9, 10), but potential limitations of cost, stability, and immunogenicity make alternative synthetic carrier systems desirable. In this report we describe a new strategy for the design and synthesis of polymeric drug carriers that enhance the cytoplasmic delivery of biomolecules into macrophages by disrupting the endosomal membrane at the acidic pHs of the endosome. We have termed these polymers “encrypted” by analogy to encrypted domains in biological proteins that become exposed and activated by proteolytic processing at controlled timepoints (11). The challenge of intracellular delivery varies with cell type, and the macrophage is a particularly difficult model because of the high degradative activities of the endosomal/phagosomal compartment. Macrophages are an important medical target because they play a key role in many inflammatory diseases as well as in the foreign body response to implants. Macrophages are also efficient antigen-presenting cells, where they play a role in the development of memory T and B cells, as well as the activation of naı¨ve CD8+ T-cells. There is considerable interest in developing delivery vehicles that can enhance cytoplasmic entry in macrophages of biotherapeutics such as antisense oligonucleotides and antigenic proteins and peptides. EXPERIMENTAL METHODS

Materials. The peptide FITC-(His)6-(Gly)4-Cys was purchased from the SynPep Corporation. The phospho-

10.1021/bc020056d CCC: $25.00 © 2003 American Chemical Society Published on Web 01/15/2003

Bioinspired pH-Responsive Polymers

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Scheme 1. Synthesis of the Styrene Acetal Monomer (III)

rothioate oligonucleotides (ODNs), antisense for inducable nitric oxide synthase (iNOS) (CCA-GGG-GCAAGC-CAT-GTC-TG) (12), and scrambled iNOS (GACGTG-CG-AGT-CAG-CAC-TGC) (12) used in this study were purchased from Integrated DNA Technologies. Chemicals and solvents used for the synthesis of monomers and polymers were obtained from Aldrich, unless otherwise indicated. Mannopyranosylphenyl-isothiocyanate was purchased from Sigma. THF was distilled from sodium/benzophenone under a nitrogen atmosphere immediately before use. H NMRs of monomers were taken on a Bruker 200MHZ machine, and H NMRs of copolymers were taken on a AM-400MHZ machine. UV spectroscopy was performed on a Shimadzu 480 machine. Gel permeation chromatography was performed on a Waters GPC system using Styragel columns. The cell line Raw 264.7 was obtained from the ATCC. Styrene Aminobenzaldehyde (I). A solution containing methyl amino benzaldehyde (2 mmol), chloromethyl styrene (5 mmol) and triethylamine (2 mmol) was prepared in a 10 mL round-bottom flask, fitted with a stir bar. The reaction was heated to 68 °C, with stirring for 10 h. The reaction was then purified by column chromatography on a column containing 30 g of silica gel. The column was eluted with 50 mL of 20/80 ethyl acetate/ hexane, 75 mL of 30/70 ethyl acetate/hexane, and then 75 mL of 35/65 ethyl acetate/hexane. The yield was 50%. H NMR in CDCl3 gave the following: 3.2 singlet 3H C-N-CH3, 4.7 singlet 2H CH2-N-C, 5.25 doublet 1H Ar-CdC-H, 5.8 doublet 1 H Ar-CdC-H, 6.7-6.9 multiplet 3H one hydrogen from Ar-CHdC and two aromatic hydrogens ortho to amino group, 7.2-7.5 two doublets 4H aromatic hydrogens on styrene ring, 7.8 doublet 2H aromatic hydrogens meta to amino group and ortho to aldehyde, 9.9 singlet 1H Ar-CHdO. UV analysis of the product gave a 346 of 4.1 × 104 M-1 cm-1 in DMF.

Hydroxypropyl-mercaptopyridine (II). A solution containing 2,2 dithiopyridine (0.109 mol), 1.6 mL of glacial acetic acid, and 125 mL of methanol was prepared in a 250 mL round-bottom flask with stir bar. Mercaptopropanol (0.054 mol) was dissolved in 20 mL of methanol and added to the dithiopyridine solution from a dropping funnel (Scheme 1). After 3 h of reacting at room temperature, the reaction was concentrated under vacum, giving a green oil. This oil was then purified by flash chromatography over 200 g of silica gel. The column was eluted with 600 mL of 40/60 ethyl acetate/hexane and then 600 mL of 50/50 ethyl acetate/hexane. After one column purification, the product still contained impurities from unreacted 2,2-dithiopyridine and was again purified over 150 g of silica gel, eluting this time with 1 L of 1/2 ethyl acetate/hexane and then one liter of 1/1 ethyl acetate/hexane. The yield was 60%. H NMR in CDCl3 gave the following: 1.9 pentet 2H C-CH2-CO, 2.9 broad singlet 1H O-H, 3.0 triplet 2H S-S-CH2, 3.8 triplet 2H C-CH2-O, 7.1 multiplet 1H aromatic hydrogen meta to nitrogen, 7.7 multiplet 2H aromatic hydrogens para to nitrogen and ortho to thiol derivatized carbon, 8.5 quartet 1H aromatic hydrogen ortho to nitrogen. UV analysis of the product gave a 372 of 5.8 × 103 M-1 cm-1 in DMF, after reduction with DTT. Styrene Acetal Monomer (III). A 100 mL roundbottom flask was fitted with stir bar and hydroxypropylmercaptopyridine (10 mmol), styrene aminobenzaldehyde (2 mmol), and para-toluenesulfonic acid (PTSOH) (1 millimole), and 5 Å molecular sieves (5 g) were placed inside the flask. Approximately 20 mL of THF was distilled from sodium benzophenone directly into this 100 mL flask. The reaction was then placed in an ice bath, under a nitrogen atmosphere, for 12 h. The reaction was quenched with 3 mL of triethylamine and filtered using methylene chloride as the rinsing solvent. The filtrate

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Scheme 2. PEGylation of Polymer Backbone E1

was then concentrated under vacum and loaded onto 30 g of basic alumina (Brockman’s activity I). The column was eluted with 250 mL of 20/80 ethyl acetate/hexane stabilized with 2 mL of TEA. The yield was 50%. H NMR in deuterated benzene gave the following: 2.0 pentet 4H O-C-CH2-C-S-S, 2.75 singlet 3H C-N-CH3, 2.9 triplet 4H C-CH2-S-S, 3.6 multiplet 4H O-CH2-C, 4.4 singlet 2H CH2-N-C, 5.2 doublet 1H Ar-CdC-H, 5.6 singlet 1H acetal hydrogen, 5.7 doublet 1H Ar-Cd C-H, 6.6-6.9 multiplet 3H one hydrogen from Ar-CHd C and two aromatic hydrogens ortho to amino group, 7.1 multiplet 2H aromatic hydrogens meta to nitrogen on pyridal ring, 7.2-7.4 multiplet 4H aromatic hydrogens from styrene ring, 7.5-7.7 multiplet 6H; two aromatic hydrogens are meta to the methoxy group and four aromatic hydrogens from the pyridal ring, 8.4 doublet 2H aromatic protons ortho to nitrogen on pyridal ring. The presence of the acetal and thiopyridal groups were further confirmed by UV analysis. The acetal had a 346 of 4.4 × 103 M-1 cm-1 in DMF, which increased to 4.1 × 104 M-1 cm-1 upon hydrolysis to the benzaldehyde form with 1 N HCl. UV analysis of the product after reduction with DTT in DMF gave a 372 of 1.2 × 104 M-1 cm-1, indicating the presence of the thiopyridone. Synthesis and Characterization of Encrypted Polymer E1. A 150 mg (0.23 mmol) sample of the styrene-acetal monomer (III), 250 mg (1.5 mmol) of dimethylaminoethyl methacrylate (DMAEMA), 300 mg (2.1 mmol) of butyl methacrylate (BMA), and 20 mg (0.12mmol) of 2,2-azobisisobutyronitrile (AIBN) were placed in a 5 mL round-bottom flask, fitted with a stopcock and stir bar. The reaction was subjected to three freeze-thaw-vacuum cycles and polymerized at 60 °C for 10 h. The polymer was isolated by dissolving the reaction in 3 mL of THF and precipitating it into 40 mL of ice cold hexane; the resulting solid was filtered and vacuum-dried. The molecular weight of the polymer was determined by GPC in THF (Styragel gel columns) with poly(methyl methacrylate) (PMMA) standards; the Mw of the polymer was 100 kD and the Mn was 44 kD. The composition of this terpolymer was determined by H NMR, and UV spectroscopy to be DMAEMA/BMA/SA ) 47/48/5. The mole ratio of the monomers DMAEMA/BMA was determined by H NMR to be 4.7/4.8 (H NMR in deuterated chloroform AM-400). The mole percent of III was determined by quantifying the thiopyridals groups in the polymer, after reduction with dithiothreitol (DTT) (372 nm in DMF). The polymer backbone E1 was PEGylated (see Scheme 2) by reacting, in an Eppendorf tube, 25 mg (15µmol of thiopyridal groups) with 130 mg (26

µmol) of PEG-thiol (5 kD) in 1 mL of DMF, containing 10 µL of triethylamine (TEA). The PEGylation reaction was monitored by UV spectroscopy at 372 nm, for the released thiopyridone; after 12 h the reaction had gone to completion. The reaction mixture was precipitated in 20 mL of ice cold ether, filtered, and vacuum-dried; the yield was 80% (see Figure 2a for chemical structure). Synthesis of Thiol-PEG-Lys6. A 2.10 g (0.61 mmoles) sample of Dithiol PEG (MW ) 3400) was slowly added to a solution of 128 mg of maleimido-caproic acid (0.61 mmoles), 60 µL of TEA, in 10 mL of DMF and stirred for 2 h. Ellmans analysis of the product indicated that 50% of the thiol groups of the dithiol-PEG had reacted with maleimido-caproic acid. The reaction was then added to 0.53 g of 2,2-dithiopyridine (2.2 mmoles) in 2 mL of DMF, stirred for 30 min, precipitated into cold ether, filtered, and vacuum-dried. A 2.10 g sample of thiopyridal-PEGcaproic acid, 190 mg of N-hydroxysuccinimde (NHS), and 190 mg of dicyclohexylcarbodimide (DCC) were dissolved in 4 mL of dry methylene chloride, stirred for 4 h, filtered, and rotavapped down to a solid and then crystallized twice from ethyl acetate. The product was characterized by UV spectroscopy, and reduction of the thiopyridal group with DTT and hydrolysis of the NHS group indicated a 1:1 ratio of NHS groups to thiopyridal groups; the yield was 20%. An 8.2 mg sample of hexalysine, 8 mg of thiopyridalPEG-NHS, and 5 µL of triethylamine were dissolved in 50 µL of dry DMF and reacted overnight. The reaction was then diluted in 2.5 mL of deionized water and run through a PD-10 column to remove unreacted hexalysine. The high-MW fractions of the PD-10 column were lyophilized for 2 days. The recovered white solid was dissolved in 1 mL of 10 mM pH 7.4 phosphate buffer and loaded onto a high trap ion exchange column, equilibrated with 10 mM pH 7.4 phosphate buffer. The column was washed with 5 mL of 10 mM pH 7.4 phosphate buffer, and the product was eluted with 2.5 mL of 3 M NaCl. The product was run through a PD-10 column, and deionized water was used as the eleunt. The high-MW fractions of the PD-10 column were lyophilized; the yield was 25%. The product had a thiopyridal/ amine ratio of 1:5, the thiopyridal content was determined by reducing the product with DTT and UV analysis at 372 nm (DMF), and the amine content was determined by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay. Synthesis of (Man)3-(Lys)3-PEG-SH. A 50 mg sample of thiopyridal-PEG-(Lys)6 (0.01 mmol) was mixed with 10 mg of mannopyrannosylphenyl-isothiocyanate (0.03 mmol) in 0.5 mL of DMSO with 3µl of TEA. The reaction

Bioinspired pH-Responsive Polymers

Figure 1. Schematic diagram of the encrypted polymer design.The polymers are designed to be PEGylated and serum stable at pH 7.4, but to be disruptive to the endosomal membrane at the acidic pHs within the endosome. The polymers have the following components: a membrane-disruptive backbone (red line), acid-degradable linkers (purple circle), PEG grafts (aqua ellipsoid), conjugated or ionically complexed drug molecules (blue circle), hexalysine peptide (green line), and targeting ligands (black arrow). At pH 7.4 the polymers are PEGylated (“masked”); however, after endocytosis the aciddegradable linker hydrolyzes and the polymer backbone becomes de-PEGylated (“umasked”) and membrane-disruptive, causing endosomal disruption. The PEGs may be conjugated to the backbone via both acid-degradable linkages and disulfide bonds. The latter are reduced in the cytoplasm to release the free drug. In the study reported here, two examples are presented. In the first case, a model peptide drug with a terminal cysteine is conjugated to the backbone via -S-S- bonds, and in the second case an antisense oligonucleotide (AS-ODN) is ionically complexed to cationic lysine groups that have been linked to the terminal ends of the PEG molecules. In the latter case, mannose groups have been linked along with the lysine groups to the ends of the PEGs for targeting the ODN to the RAW cells.

was allowed to proceed overnight and was then dialyzed against water in a Pierce Slidealyzer. The thiol content was quantified by reducing the thiopyridal group, and the mannose content was determined by performing the resorcinol/sulfuric acid assay. The mannose to thiol content was 3:1, leading to a lysine/mannose ratio of 1/1. The product was reduced with DTT, purified with a PD10 column, and lyophilized. Synthesis of Encrypted Polymer E2. A 1.6 mg sample of (Man)3-(Lys)3-PEG-SH (0.26 µmol) was mixed with 353 µg of the polymer backbone E1 (0.21 µmol of thiopyridal groups) in 40 µL of DMF, and allowed to react overnight. UV analysis of the released thiopyridone (372 nm DMF) indicated that 84% of the thiopyridal groups on the polymer backbone E1 had reacted with (Man)3(Lys)3-PEG-SH. Synthesis of Encrypted Polymer E3. A 200 mg (1.4 mmol) sample of BMA, 250 mg (1.5 mmol) of DMAEMA, 400 mg (3.0 mmol) of butyl acrylate (BA), 100 mg (0.153 mmol) of III, and 35 mg (0.21 mmol) of AIBN were placed in a 5 mL round-bottom flask fitted with a stir bar and stopcock. The reaction flask was subjected to three freeze-thaw-vacuum cycles and heated to 60 °C for 1.5 h. The reaction was then dissolved in 3 mL of THF with 1% TEA and precipitated in 20 mL of ice-cold hexane, decanted, and vacuum-dried. A white solid was recovered, which was precipitated again from THF into hexane, decanted, and vacuum-dried. GPC of the polymer (Styragel gel columns, with PMMA standards in THF) gave a MW of 58 kD and an Mn of 38 kD. The monomer composition of the E3 backbone was determined by H

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Figure 2. Chemical structures of the “encrypted” polymeric carriers. (a) The membrane-disruptive backbone for polymer E1 is a terpolymer of three monomers: BMA, DMAEMA, and SBA. The acid-degradable linker is an acetal, specifically, paraaminobenzaldehyde-acetal. The PEG grafts have a molecular weight of 5 kD and in this study have been terminated with various functional groups. Polymer E1 has its PEG grafts terminated with methoxy groups. Polymer E2 has its PEG grafts terminated with Lysine3-Mannose3. (b) The membrane-disruptive backbone for polymer E3 is a copolymer of four monomers: BMA, BA, DMAEMA, and SBA. Polymer E3 has its PEG grafts terminated with methoxy groups.

NMR and UV spectroscopy, giving DMAEMA/BMA/BA/ SA ) 25.3/20.5/51.5/2.7. The molar ratios of the three main monomers of the E3 backbone, DMAEMA/BMA/BA, were determined by H NMR to be 2.5/2.0/5.0 (H NMR deuterated chloroform AM-400). The molar content of III was determined by reducing the polymer backbone with DTT, in DMF, and measuring the UV absorption at 372 nm. The polymer backbone E3 was PEGylated by reacting 15 mg of the polymer backbone (4.6 µmol of thiopyridal groups) with 160 mg (16µmol) of PEG-thiol (5kD) in 1.5 mL of a 2/3 mixture of THF/DMF. The reaction was allowed to proceed overnight, and analysis of the released thiopyridone indicated that 100% of the thiopyridal groups had reacted. The reaction was precipitated in 20 mL of cold ether, filtered, and vacuum-dried overnight (see Figure 2b for structure). Synthesis of Cys-(Gly)4-(His)6-FITC-Polymer E3 Conjugate. A 4.5 mg sample of the polymer backbone E3 (1.4 µmol of thiopyridal groups) was dissolved in 270 µL of 1/1 THF-DMF and added to 1 mg of PEG-thiol (5kD) (0.2 µmoles) dissolved in 170 µL DMF. The PEGylation reaction was allowed to proceed for 4 h, and UV analysis indicated quantitative reaction of the PEG-thiol. Following this, 12.5 mg of the peptide Cys-(Gly)4-(His)6-

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FITC (1.2 µmoles) was added to the reaction (Figure 2c) and allowed to proceed overnight. UV analysis of the reaction indicated quantitative reaction of the peptide with the polymer backbone. The Cys-(Gly)4-(His)6-FITCpolymer E3 conjugate was used directly for cell culture experiments. Fluorescence Microscopy of Macrophages. RAW cells were split onto #1 Gold coverslips in a 35 mm dish and allowed to grow for 16 h, reaching approximately 50% confluency. The cells were washed once with DMEM high-glucose media and incubated with either the peptide Cys-(Gly)4-(His)6-FITC, at a 40 µg/mL concentration, in DMEM high glucose media, or the Cys-(Gly)4-(His)6FITC-polymer E3 conjugate (40 µg/mL peptide concentration), in a 1.5 mL volume for 12 h. The cells were washed once with DMEM high-glucose media and allowed to grow for 4 h in DMEM high-glucose media with 10% fetal bovine serum. The cells were then washed 6 times with PBS and fixed for 5 min with 3% paraformaldehyde and observed with a Zeiss digital fluorescent microscope at a 40× magnification. iNOS Assay. RAW cells were split onto a 96 well plate, at 4 × 105 cells per well, and grown for 16 h in DMEM high glucose media with 10% fetal bovine serum (FBS). The RAW cell media was then removed and the appropriate polymer/ODN sample was added to the cells, in serum free media, the cells were incubated with polymer/ODN samples for 16 h. The media was then aspirated off, and the cells were stimulated with 10 units/ mL of γ interferon and 10 µg/mL of lipopolysaccharide (LPS) for 8 h in serum containing media (to stimulate (iNOS) production). The cell medium was isolated, and the concentration of nitric oxide (NO) was detemined with the Griesse reagent (1% sulfanilamide, 0.1% naphthylethylene-diamine hydrochloride, and 5% phosphoric acid), a 1:1 mixture of the cell medium with the Griesse reagent was made, incubated for 10 min at room temperature, and its absorbance at 540 nm was measured. RESULTS AND DISCUSSION

Polymer Design. The encrypted polymers are designed as multifunctional carriers that incorporate three primary functionalities of viruses and toxins: (a) a targeting agent that directs receptor-mediated endocytosis, (b) a pH-responsive element that selectively disrupts the endosomal membrane, and (c) the biomolecular therapeutic component which is delivered as a free and active agent into the cytoplasm. Our encrypted polymers contain a masked, membrane-disruptive backbone that is unmasked and activated in the low pH environment of the endosome. This design is shown schematically in Figure 1, and three specific compositions that we have synthesized are presented in Figure 2a,b (see Schemes 1 and 2 for synthesis). Copolymers of dimethylaminoethyl methacrylate (DMAEMA) with hydrophobic alkyl methacrylates (BMA) and alkylacrylates (BA) were chosen for the membrane-disruptive backbone, based on our previous work on endosomolytic polymers (13, 14). PEG was chosen as the solubilizing hydrophilic graft to “mask” the backbone because of its established ability to improve the stability, solubility, circulation lifetime, and biodistribution properties of a wide variety of drugs and delivery systems (15). The pH sensitivity of the encrypted polymer is provided through acid-degradable acetal bonds that link the PEGs and PEGylated drugs or targeting ligands to the polymer backbone. Hydrolysis Kinetics of PEG Grafts. The encrypted polymer carrier system is designed to remain PEGylated

Figure 3. The pH-dependent hydrolysis and hemolytic properties of the polymer carriers. (a) The hydrolysis of polymer E1 was measured at 37 °C, in phosphate buffer, at either pH 5.4 or 7.4, by observing the change in UV absorbance at 340 nm. The experiments were done in triplicate and the standard deviation was under 5% for all samples. (b) pH-dependent hemolysis by polymer E1. The ability of the polymer E1 to disrupt red blood cell membranes was measured at either pH 5.0 or 7.4. In each hemolysis experiment, 108 RBCs were suspended in 1 mL of phosphate buffer saline. The incubation time was 20 min at 37 °C. The experiments were done in triplicate. The protocol used to isolate and purify the red blood cells and to quantitate hemolysis is described in (13, 14).

during circulation at pH 7.4, but then to be rapidly dePEGylated after endocytosis, as the pH drops within the endosome to ca. 5.0. Endocytosed macromolecules are typically trafficked to lysosomes within 30-60 min after endocytosis, and therefore, we designed the linkage between the PEG and the membrane-disruptive backbone to hydrolyze within 30-60 min at pHs between 5 and 5.5. The hydrolysis kinetics of the PEG grafts from the polymer E1 are strongly pH-dependent, as expected, with a half-life of 15 min at pH 5.4 (Figure 3a). In contrast, at pH 7.4 less than 10% of the PEG grafts are hydrolyzed after 75 min, and only 38% are hydrolyzed after 12 h. The PEG grafts are released from the encrypted polymer backbone about 2 orders of magnitude faster at pH 5.4 than at pH 7.4. This 100-fold increase in hydrolysis kinetics agrees with the enhancement expected due to the 100-fold increase in the hydronium ion concentration

Bioinspired pH-Responsive Polymers

between pH 5.4 and 7.4. The hydrolysis rates of acetals can be manipulated over a wide range of time scales by changing the substituent group in the para position of the benzene ring to stabilize or destabilize the carbocation intermediate formed during hydrolysis (16). For example, by changing the para substituent from a nitrogen to an oxygen in polymer E1, the rate constant for hydrolysis is decreased by a factor of ca. 60 (data not shown). The ability to control the hydrolysis kinetics of the acid degradable linker is a critical design element of the encrypted polymer carrier system. Membrane Disruption by Polymer E1. The pHdependent membrane disruption properties of polymer E1 were characterized in a red blood cell (RBC) hemolysis assay at pH 5.0 and 7.4 (Figure 3b). After 20 min at pH 5.0, less than 5 µg/mL of the polymer E1 caused almost 60% hemolysis of 108 cells/mL, while in contrast there was no significant hemolysis at pH 7.4 under those same conditions. These kinetic properties match the time frame of vesicular evolution in macrophages from endosomes to lysosomes, which has been estimated to occur on the order of 30 min (18). The ability of the encrypted polymers to avoid degradative lysosomal trafficking was first tested with antisense oligonucleotides (AS-ODNs) in a cultured macrophage cell line. The antisense ODN was designed to inhibit secretion of NO by macrophages by blocking the expression of inducable nitric oxide synthetase (iNOS). Several phosphorothioate AS-ODNs have been developed which selectively inhibit iNOS; however, they require high extracellular concentrations, where toxicity can become significant (19-22). AS-ODN Delivery. The membrane-disruptive polymer backbone E1 was conjugated with heterobifunctional PEGs that were terminated with thiol groups at one end and with lysine3-mannose3 groups at the other end. To synthesize this derivatized PEG, a hexalysine peptide was first conjugated to a carboxyl derivatized PEG, and then three of the six lysines groups were statistically reacted with mannose-isothiocyanate, generating a heterobifunctional PEG with three residual lysine groups. This graft copolymer is called “encrypted polymer E2”. These lysine-grafted polymers ionically complexed ASODNs efficiently, as determined in a gel shift assay (see Figure 4). At low pHs, the acetal groups linking the PEG-lysine3-mannose3 graft to the backbone should be hydrolyzed, and since the PEG-lysine3-mannose3 has no membrane-disruptive activity by itself, polymers E2 and E1 should therefore have very similar membrane disruptive activities. Polymer E2 was complexed with ASODNs at various charge ratios and their ability to inhibit the iNOS catalyzed synthesis of NO in the RAW cells was measured. As a control, the encrypted polymers were complexed with a scrambled ODN and incubated under the same conditions. As shown in Figure 5, the polymer E2 is able to significantly enhance the delivery into macrophages of AS-ODNs at a 1/1 ( charge ratio. At this charge ratio, the AS-ODN complexed to polymer E2 caused approximately 80% inhibition of iNOS, whereas by itself the iNOS AS-ODN causes only 25% inhibition. In addition, in another control experiment, when the scrambled ODN was complexed to the encrypted polymer, there was no significant inhibition of iNOS activity. Peptide Delivery with Polymer E3. To test the ability of the encrypted polymers to enhance endosomal escape of polypeptides, the labeled peptide, FITC-(His)6(Gly)4-Cys, was conjugated to the encrypted polymer E1 backbone through its thiopyridal groups. A methoxyPEG-thiol was also grafted through the thiopyridal groups in order to enhance the solubility of the backbone

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Figure 4. Gel Shift assay with polymer E2. The polymer E2 was incubated at various charge ratios with 2 µg of the phosphorothioate oligonucleotide thiol-CCA-GGG-GCA-AGCCAT-GTC-TG in a 20 µL volume of 1X TAE buffer. The charge ratios indicated on the gel refer to the number of lysine residues from the grafts of polymer E2 vs the number of phosphate groups on the oligonucleotide backbone. The complexes were run on a 2% agarose gel at a 100 V for 45 min and then stained with ethidium bromide

Figure 5. AS-ODN delivery with polymer E2. Diagonal hatched lines: 104 µg/mL of polymer E2 mixed with 10 µg/mL of “scrambled” ODN. Dark filling: 104 µg/mL of polymer E2 mixed with 10 µg/mL of AS-ODN targeted against iNOS. Horizontal hatched lines: 10 µg/mL of AS-ODN targeted against iNOS. Vertical hatched lines: A control in which cells were incubated with 10 mg/mL of LPS and 10 units/mL of γ interferon. This sample was used as the 100% reference. After incubation with the appropriate ODN formulation, each of the samples was stimulated with 10 µg/mL of LPS and 10 units/ mL of γ interferon. The cell supernatants were harvested after 8 h of stimulation and assayed for NO content. (See Materials and Methods for details.)

polymer. Initial delivery to the macrophage RAW cells of the peptide conjugated to the polymer backbone E1 was inefficient and resulted in lysosomal localization (as suggested by the punctate localization seen in fluorescence micrographs). However, a key aspect of the encrypted polymer strategy is the flexibility in polymer

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Figure 6. Hemolysis by polymer E3 vs polymer E1. The membrane disruptive activities of polymer E3 and E1 were compared using a hemolysis assay at pH 5.0. In each hemolysis experiment, 108 RBCs were suspended in 1 mL of phosphate buffer saline. The incubation time was 20 min at 37 °C. The experiments were done in triplicate. The protocol used to isolate and purify the red blood cells and to quantitate hemolysis is described in (13, 14). The quantities of polymer indicated on the x axis refer to the mass of the polymer backbone generated after the PEG grafts are hydrolyzed from PEGylated polymers E1 and E3. The quantity of polymer backbone (x axis) was determined by estimating the weight percent of PEG in the polymers E3 and E1 and then subtracting it from the actual mass of polymer E1 and E3 used for the hemolysis assay.

design it allows, and therefore, we subsequently designed and synthesized a new backbone, E3, which contained an additional monomer, butyl acrylate (BA). This polymer backbone is significantly more hydrophobic than the E1 or E2 backbones (compare panels a and b of Figure 2). We chose to add BA to the polymer composition because it copolymerizes exceptionally well with the other methacrylate-based monomers. The copolymer E3 is strikingly more effective at RBC hemolysis than the encrypted polymer E1 (see Figure 6). For example, the copolymer backbone E3 is hemolytically active at nanogram per milliliter concentrations, making it approximately 2-3 orders of magnitude more efficient at membrane disrup-

Murthy et al.

tion on a molar basis than the best known peptides (9, 10) or the polycarboxylic polymers (e.g., poly[propylacrylic acid]) that we have previously studied (13, 14). Importantly, polymer E3 has no membrane disruptive activity at pH 7.4 at 25 µg/mL (highest concentration tested). The peptide FITC-(His)6-(Gly)4-Cys and methoxy-PEGSH (5kD) were conjugated via disulfide linkages to the polymer backbone E3. This conjugate was incubated with RAW cells, and the intracellular location of the FITClabeled peptide was determined by fluorescence microscopy. Figure 7b demonstrates that the peptide by itself was taken up by RAW cells but sequestered in punctate organelles, presumably lysosomes. The intracellular location of the peptide was dramatically altered after being conjugated to the PEGylated E3 backbone (Figure 7a) and was spread diffusely through the cytoplasm. This demonstrates that the conjugated E3 polymers were able to direct transport out of the macrophage endosomal compartments and release endocytosed peptides into the cytoplasm. CONCLUSIONS

In this report we describe a novel strategy for the design and synthesis of pH-responsive polymers as carriers that enhance the cytosolic delivery of fragile drugs, thus avoiding lysosomal degradation of the drugs. This strategy combines a relatively hydrophobic, membrane-disruptive polymer backbone that is “masked” at pH 7.4 by grafted PEG chains. The PEG grafts are linked to the backbones through two key linkers, first disulfide groups and then acid degradable acetal linkages. The acetal groups are designed to degrade in the acidic environment of the endosome, which then unmasks the membrane-disruptive backbone. This leads to disruption of the endosomal membrane and release into the cytosol of the grafted side chains, where the disulfide groups are reduced and release the drug. Peptide, protein, or antisense ODN therapeutics can be conjugated to the backbone of the polymer via the disulfide bonds. In the case of highly negative ODNs, they may also be electrostatically complexed to cationic groups that are either conjugated to the terminal ends of the grafted PEGs or included as a monomeric component of the polymer backbone. Targeting ligands may also be conjugated to the terminal ends of the PEGs.

Figure 7. Cytoplasmic delivery of peptides with polymer E3. (a) Fluorescence microscopy (40X magnification) of RAW cells treated overnight with the peptide Cys-(Gly)4-(His)6-FITC conjugated to polymer E3. (b) Lysosomal localization of the peptide Cys-(Gly)4(His)6-FITC. The peptide Cys-(Gly)4-(His)6-FITC was incubated with RAW cells overnight and visualized by fluorescence microscopy at 40× magnification.

Bioinspired pH-Responsive Polymers

This new type of pH-responsive polymer is at least 2-3 orders of magnitude more efficient at RBC membrane disruption than existing endosomal-disruptive polymers and peptides. We have shown that the encrypted polymers provide efficient endosomal escape in a model macrophage cell line, and are able to deliver functional antisense ODNs and peptides into the cytoplasm. This encrypted polymer strategy should therefore have broad applications in the design of peptide vaccine delivery systems for CD8+ T cells and in the development of antisense ODN delivery systems. These multifunctional polymeric carriers mimic the sophisticated protein complexes of viruses and toxins that direct uptake and enhance cytosolic transport of DNA or proteins. Like the biological systems, they combine targeting elements that direct cellular uptake, together with the sensing of pH changes within the endosome to activate membrane destabilization and cytosolic delivery. Their intrinsic modular design makes it possible to tailor the targeting and membrane destabilizing activities for a wide range of biotherapeutics and vaccine applications. ACKNOWLEDGMENT

The authors gratefully acknowledge the NIH (Grant No. GM-53771-02 to -04), University of Washington Office of Technology Transfer, Washington Research Foundation, and Center for Nanotechnology (fellowship to N.M.). We would also like to thank the University of Washington Engineered Biomaterials Research Center (UWEB) (NSF Grant No. EEC-9529161) for use of their cell culture facilities and the UWEB Optical Microscopy and Image Analysis Shared Resource (NSF Grant Nos. EEC9872882 and EEC 9529161). LITERATURE CITED (1) Berg, K., et al. (1999) Photochemical internalization: A novel technology for delivery of macromolecules into cytosol. Cancer Res. 59, 1180. (2) Bijsterboch, M. et al. (1997) In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells. Nucleic Acids Res. 25, 3290. (3) Haensler, J., and Szoka, F. C. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 4, 312. (4) Choi, Y. H., Lui, F., Choi, J. S., Kim, S. W., and Park, J. S. (1999) Characterization of a targeted gene carrier, lactosepolyethylene glycol-grafted poly-L-lysine and its complex with plasmid DNA. Hum. Gene Ther. 10, 2657. (5) Richardson, S., Ferruti, S., and Duncan, R. (1994) Poly(amidoamine)s as potential endosomolytic polymers: evaluation in vitro and body distribution in normal and tumour bearing animals. J. Drug Target. 6, 391. (6) Vinogradov, S. V., Bronich, T. K., and Kabanov, A. V. (1998) Self-assembly of polyamine-poly(ethylene glycol) copolymers with phosphorothioate oligonucleotides. Bioconjate Chem. 9, 805.

Bioconjugate Chem., Vol. 14, No. 2, 2003 419 (7) Wetering, P., et al. (1997) Structure-activity relationships of water soluble cationic methacrylate/methacrylamide polymers for nonviral gene therapy. Bioconjate Chem. 4, 687. (8) Boussif, O., Lezoualch, F., Zanta, F., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo. Proc. Natl. Acad. Sci. U.S.A. 92, 797. (9) Subbarao, N., Parente, R., Szoka, F., Nadasdi, L., and Pongracz, K. (1987) pH dependent bilayer destabilization by an amphiphatic peptide. Biochemistry 26, 2964. (10) Plank, C., Oberhauser, B., Mechtler, K., Koch, C., and Wagner, E. (1994) The influence of Endosome-disruptive peptides on gene transfer using synthetic virus such as gene transfer systems. J. Biol. Chem. 269, 12918. (11) Kaplan, F., and Shore, E. (1998) Encrypted morphogens of skeletogenesis: biological errors and pharmacologic potentials. Biochem. Pharmacol. 55, 373. (12) Bilecki, W., Okruszek, A., and Przewlocki, R. (1997) The Effect of Antisense Oligodeoxynucleotides on Nitric Oxide Secretion from Macrophages-Like Cells. Antisense Nucelic Acid Drug Dev. 7, 531-537. (13) Murthy, N., Robichaud, J. R., Tirrell, D. A., Stayton, P. S., and Hoffman, A. S. (1999) The design and synthesis of polymers for eukaryotic membrane disruption. J. Controlled Release 61, 137. (14) Murthy, N., Chang, I., Stayton, P. S., and Hoffman, A. S. (2001) pH-sensitive hemolysis by random copolymers of alkyl acrylates and acrylic acid. Macromol. Symp. 172, 149-155. (15) Woodle, M. C. In Poly(ethylene-glycol) Chemistry and Biological Applications (J. M. Harris and S. Zalipsky, Eds.) pp 60-77, American Chemical Society, Washington, DC. (16) Taft, R. W., and Kreevoy, M. M. (1955) The evaluation of inductive and resonance effects on reactivity. I hydrolysis rates of acetals and nonconjugated aldehydes and ketones. J. Am. Chem. Soc. 77, 5590. (17) Fife, T. H., and Jao, L. K. (1965) Substiteunt effects in acetal hydrolysis. J. Am. Chem. Soc. 30, 1492. (18) Mellman, I. (1996) Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575. (19) Arima, H., et al. (1997) Specific Inhibtion of Nitric Oxide Production in Macrophages by Phosphorothioate Antisense Oligonucleotides. J. Pharm. Sci. 86, 1079-1084. (20) Ding, M., et al. (1996) Antisense Blockade of Inducable Nitric Oxide Synthase in Glial Cells Derived From Adult SJL Mice. Neuroscie. Lett. 220, 89-92. (21) Wood, L., et al. (1999) Enhancement of Methylcholanthrene-induced Neoplastic Transformation in Murine C3H 10T1/2 Fibroblasts by Antisense Phosphorothioate Oligodeoxynucleotide Sequences. Cancer Lett. 147, 163-173. (22) Ding, M., et al. (1998) Antisense Knockdown of Inducable Nitric Oxide Synthase Inhibits Induction of Experimental Autoimmune Encephalomyelitis in SJL/J Mice. J. Immun. 160, 2560-2564.

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