The design and synthesis of polymers for eukaryotic membrane disruption

Journal of Controlled Release 61 (1999) 137–143

The design and synthesis of polymers for eukaryotic membrane disruption Niren Murthy a , John R. Robichaud a , David A. Tirrell b , Patrick S. Stayton a , Allan a, S. Hoffman * b

a Department of Bioengineering, University of Washington, Seattle, WA 98195, USA Department of Chemical Engineering, California Institute of Technology, California, CA, USA

Received 19 December 1998; received in revised form 3 May 1999; accepted 3 May 1999

Abstract The intracellular trafficking of drugs is critical to the efficacy of drugs that are susceptible to attack by lysosomal enzymes. It is therefore an important goal to design and synthesize molecules which can enhance the transport of endocytosed drugs from the endosomal compartments to the cytoplasm. The pH of an endosome is lower than that of the cytosol by one to two pH units, depending on the stage of endosomal development. This pH gradient is a key factor in the design of membrane-disruptive polymers which could enhance the endosomal release of drugs. Such polymers should disrupt lipid bilayer membranes at pH 6.5 and below, but should be non-lytic at pH 7.4. We have designed and synthesized pH-sensitive synthetic polymers which efficiently disrupt red blood cells within a sharply defined pH range. One of these polymers, poly(ethyl acrylic acid) (PEAAc) has been previously shown to disrupt synthetic vesicles in a pH-dependent fashion [6]. PEAAc hemolyzes red blood cells with an activity of 10 7 molecules per red blood cell, which is as efficient on a molar basis as the peptide melittin. The mechanism of RBC hemolysis by PEAAc is consistent with the colloid osmotic mechanism. PEAAc’s hemolytic activity rises rapidly as the pH decreases from 6.3 to 5.0, and there is no hemolytic activity at pH 7.4. A related polymer, poly(propyl acrylic acid) (PPAAc), was synthesized to test whether making the pendant alkyl group more hydrophobic by adding one methylene group would increase the hemolytic activity. PPAAc was found to disrupt red blood cells 15 times more efficiently than PEAAc at pH 6.1. PPAAc was also not active at pH 7.4 and displayed a pH-dependent hemolysis that was shifted toward higher pH’s. Random 1:1 copolymers of ethyl acrylate (EA) and acrylic acid (AAc) (which contain random –COOH and –C 2 H 5 groups that are present and regularly repeat in PEAAc) also displayed significant hemolytic activity, with an efficiency close to PEAAc. These results demonstrate that pH-sensitive synthetic polymers can be molecularly engineered to efficiently disrupt eukaryotic membranes within defined and narrow pH ranges. Thus, these polymers might serve as endosomal disruptive agents with specificities for early or late endosomes.  1999 Elsevier Science B.V. All rights reserved. Keywords: Gene therapy; Drug delivery; Endosomal release; Hemolysis; pH-sensitive polymers

*Corresponding author. Tel.: 11-206-543-9423; fax: 11-206543-6124. E-mail address: [email protected] (A.S. Hoffman)

1. Introduction There are several challenges which currently limit gene therapy. One of the most important of these is

0168-3659 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00114-5

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the inefficiency of DNA delivery to the appropriate cellular compartments. The most common method for gene therapy is through viral delivery. Viral carriers are very efficient at DNA delivery, but clinical use is potentially limited by their antigenicity and toxicity [1]. Non-viral methods for delivering DNA have also been developed. Polycationic carriers have shown considerable clinical potential, but suffer from low transfection efficiencies. A principal reason for these low efficiencies is related to intracellular trafficking problems; e.g., DNA-polycation complexes are trafficked to lysosomes, where they are degraded [2]. Major goals in developing efficient non-viral delivery systems for DNA transfection are to increase the release of endocytosed DNA from the endosome into the cytoplasm, and from there into the nucleus. Several groups have attempted to increase the release of DNA from endosomes by designing endosomal-disrupting peptides (EDPs) [2,3]. EDPs are peptides that are membrane disruptive at endosomal / lysosomal pH’s, which are ca. pH 5.0 to 6.5, but not at physiological pH. The majority of EDPs have been designed to mimic naturally-occurring viral fusion peptide sequences. Two principal approaches have been used to incorporate EDPs into gene delivery systems. The first is by direct conjugation to the polycationic carrier [3], and the second is in a physical mixture with the polycationic carrier [2]. In both cases significant increases in transfections were reported, demonstrating that disrupting endosomes leads to significant increases in transfection levels. However, transfection levels with EDPs are still far below those of viral infections. The majority of membrane-disruptive agents that have been evaluated to date are peptides. Synthetic polymers such as PEAAc are exceptionally efficient at disrupting liposomes and synthetic vesicles [5,6,10,12]. Lipid bilayer disruption by PEAAc occurs only below a pH of 6.3 [6]. In this report, we have investigated the ability of specially designed synthetic polymers to disrupt eukaryotic cell membranes using a standard hemolysis assay. The hemolysis assay was used to assess the potential of our polymers to disrupt endosomes because it had been previously shown that there is a correlation between peptide hemolytic efficiency and endosomal disruption [2]. We demonstrate in this paper that synthetic polymers can disrupt red cell membranes

with higher efficiencies than the bee venom peptide melittin and with an appropriate pH sensitivity.

2. Materials and methods

2.1. Materials Diethyl ethyl malonate and diethyl propyl malonate were purchased from Acros chemicals. Diethyl amine was purchased from Sigma and formalin was purchased from Baker chemicals. Acrylic acid and ethyl acrylate were purchased from Aldrich and were distilled prior to use. AIBN was purchased from Aldrich and recrystallized before use.

2.2. Monomer and polymer synthesis Ethyl and propyl acrylic acid monomers were synthesized according to the procedure outlined by Ferrito et al. [7]. The syntheses of the monomers were verified by NMR (Bruker 200 Mhz) and FTIR. PEAAc, PPAAc and the random copolymer of ethyl acrylate and acrylic acid were synthesized by free radical polymerization, using AIBN as the initiator. Their reactivity ratios are r 1 51.18 and r 2 50.81 (where AAc is monomer 1 and EA is monomer 2), indicating that the copolymer should be random [13]. The sketch below shows the repeating structures of these three polymers.

The polymerizations were carried out in bulk, at 608C for 24 h, with 2 mol% AIBN. The polymers were isolated by precipitation in ethyl acetate, and the precipitate was filtered and dried under vacuum overnight. The resulting polymer was then dissolved in a small amount of methanol and precipitated in a large excess of ether. The molecular weights of the ether precipitated PPAAc and PEAAc were determined by GPC using two 250 Waters Ultrahydrogel

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columns connected to a Waters 500 Ultrahydrogel column. The running buffer was 0.1 M sodium phosphate buffer (pH 8.5). PEG standards of Mw 4.2, 10, 20 and 72 kD (TSK) were used as the calibration curve. The polydispersities were estimated by manual integration of the GPC curves using Simpson’s rule [14]. This yielded for PEAAc a Mw522 kD, Mn518.5 kD, with a polydispersity index (PI) of 1.18, and for PPAAc a Mw524 kD, Mn518 kD, with a PI of 1.33. The ionization of PPAAc as a function of pH was determined by dissolving 25 mg of the polymer in 50 ml of 0.01 N NaOH and titrating with 0.05 N HCl.

2.3. Hemolysis assay Fresh human red blood cells (RBC) were isolated in vacutainers containing EDTA, centrifuged in a clinical centrifuge, washed three times with 150 mM NaCl and counted with a hemocytometer. The RBCs (10 8 ) were then suspended in either 1 ml of 0.1 M sodium phosphate buffer (dibasic), or 0.1 M MES buffer at the appropriate pH. For the investigation of the colloid osmotic mechanism, the buffers also contained either 10 kD dextran, 5 kD PEG or glucose at a 10 mM concentration. The polymers were dissolved in 0.1 N sodium hydroxide, at a concentration of 10 mg / ml. Melittin was dissolved in deionized water at a concentration of 1 mg / ml. The hemolysis assay was performed by adding the polymer solution to 10 8 RBCs suspended in 1 ml of the appropriate pH buffer. The RBCs were inverted several times for mixing, and incubated in a 378C water bath for 60 min. The cells were then centrifuged at 13 500 g for 5 min, the absorbance of the supernatant was then measured at 541 nm. To determine 100% hemolysis, 10 8 RBCs were lysed by suspending them in distilled water. The control was 10 8 RBCs in buffer. All hemolysis experiments were done in triplicate.

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cules to hemolyze a red blood cell [4]. The utility of better EDPs has been experimentally verified by recent work of Plank et al. [2], who demonstrated a correlation between the hemolytic efficiency of a peptide and its ability to increase transfection efficiencies. To determine the relative ability of PEAAc to disrupt cell membranes in comparison to other membrane-disruptive peptides, a hemolysis assay comparing PEAAc to melittin was performed. Melittin has been shown to be considerably more efficient at hemolysis than EDPs such as the viral fusion peptide mimic GALA [2]. Fig. 1 represents a hemolysis assay in which the effect of PEAAc concentration on RBC hemolysis is measured. Approximately 35 mg of PEAAc (Mw522 kD) is required to cause 50% hemolysis of 10 8 RBCs. An estimate of the number of polymer molecules (or MDP molecules) needed to disrupt a single red blood cell can be obtained by dividing the number of polymer molecules (or MDP molecules) added by the number of red blood cells hemolysed. This calculation yields approximately 10 7 molecules of PEAAc or 10 7 molecules of melittin [9] (data not shown) are required to disrupt a single red blood cell.

3. Results and discussion

3.1. RBC hemolysis by PEAAc vs. melittin EDPs are relatively inefficient at disrupting cell membranes, requiring between 10 8 and 10 9 mole-

Fig. 1. Concentration dependence of RBC hemolysis by PEAAc at pH 5.5. Error bars represent standard deviations of three samples.

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3.2. Comparison of PPAAc vs. PEAAc in RBC hemolysis The chemical composition of a typical endosomal disruptive peptide includes amino acids having carboxyl side chains intermixed with alkyl side chains. This composition is similar to that found in PEAAc, and we reasoned that varying the alkyl component in analogy to the biological EDP’s might result in more efficient membrane disruptive agents. While the precise molecular mechanism by which PEAAc disrupts lipid bilayers at pHs below 6.0 is not known, this activity is most likely related to its increased hydrophobic character when the carboxylate ions become protonated at low pHs. The partitioning of PEAAc into the cell membrane should increase significantly at low pH. This should lead to disruption of the lipid bilayer packing because the shape of PEAAc is considerably different from that of biological lipids. The polymer, PPAAc, was synthesized to investigate the effect on RBC hemolysis of increasing the length of the alkyl group by one methylene group. PPAAc’s membrane-disruptive efficiency is compared to that of PEAAc in Fig. 2. At

Fig. 2. Concentration dependence of RBC hemolysis by PEAAc (unfilled square) vs. PPAAc (filled square) at pH 6.1. Error bars represent standard deviations of three samples.

pH 6.1, PPAAc is approximately 15 times more effective than PEAAc in causing hemolysis. The molecular weights and polydispersities of the PPAAc and PEAAc used in this experiment were similar, suggesting that the increased effectiveness of PPAAc is due mainly to its extra methylene group.

3.3. Effect of pH on RBC hemolysis by PEAAc vs. PPAAc To compare the effect of pH on the membrane disruptive ability of PEAAc vs. PPAAc, a hemolysis assay was performed at several different pHs between 7.4 and 5.0 (Fig. 3). The results demonstrate that PPAAc and PEAAc are not hemolytic at pH 7.4, and that PPAAc becomes hemolytic approximately one pH unit higher than PEAAc. PPAAc’s maximum hemolysis is achieved at pH 6.0 and below, whereas the maximum for PEAAc is reached at pH 5.0 and below. These results suggest that it may be possible to rationally design efficient membrane disruptive polymers for early vs. late endosomal release. The different pH sensitivity of PPAAc is most likely due to its increased hydrophobicity in comparison to

Fig. 3. pH dependence of RBC hemolysis by PEAAc (100 mg / ml) (unfilled square) vs. PPAAc (10 mg / ml) (filled square). Error bars represent standard deviations of three samples.

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PEAAc. Free PEAAc is 12 to 15% ionized at pH 6.0 [11], whereas PPAAc is only 4% ionized at pH 6.0. It is likely that the different pKa’s of PEAAc and PPAAc also contribute to the different pH-sensitivity for hemolysis of the two polymers. The higher pKa value of PPAAc in comparison to PEAAc was expected, since increasing the hydrophobicity of a polymer generally decreases its acidity. This trend has been previously observed with the pKa values of poly(acrylic acid) vs. poly(methacrylic acid), and poly(methacrylic acid) vs. PEAAc. These experiments suggest that it is possible to molecularlyengineer the pH-sensitivity of synthetic polymers for membrane disruption by changing their hydrophobic / hydrophilic balance.

3.4. RBC hemolysis by random copolymers of ethyl acrylate and acrylic acid The synthetic polymer compositions of PEAAc and PPAAc mimic those of EDP’s such as LEAL, EALA (the latter is also known as ‘GALA’) [3] and other peptides designed to mimic viral fusion peptides; all of these compositions have similar pendant aliphatic and carboxyl groups. To study further the effects of polymer structure on hemolysis, a 1:1 random copolymer of ethyl acrylate and acrylic acid (EA–AAc) was made that combines the aliphatic and carboxylate groups in a random fashion rather than as regularly repeating groups as in PEAAc. This copolymer should have a similar overall hydrophilic–hydrophobic balance to that of PEAAc, since it has a 1 / 1 ratio of ethyl and carboxyl groups and should also be a random copolymer based on the reactivity ratios of the two monomers with each other (see above). As seen in Fig. 4, the EA–AAc copolymer has a hemolytic efficiency which is close to that of PEAAc at pH 5.5. These results suggest that random copolymers with varying contents of pendant alkyl and carboxyl groups could be molecularly engineered to optimize membrane disruptive capacity.

3.5. Mechanism of hemolysis by synthetic polymers Membrane disruptive peptides often cause hemolysis through the colloid osmotic mechanism [9]. Colloid osmotic hemolysis occurs when aqueous

Fig. 4. Concentration dependence of RBC hemolysis by PEAAc (filled square) vs. EA–AAc (unfilled square) random copolymer at pH 5.5. Error bars represent standard deviation of three samples.

pores are formed in the RBC membrane. These pores permit the equilibration of the extracellular buffer solutes with the solutes in the RBC cytosol. While the low molecular weight solutes in the cytoplasm can diffuse out of the cell, the macromolecules in the RBC cytoplasm cannot permeate through the aqueous pores. Hence, a net influx of solutes occurs, leading to an osmotic imbalance that draws water into the cell and causes red blood cell lysis. PEAAc has been demonstrated to form aqueous pores in lipid bilayers at relatively low polymer concentrations [8]. The conductance of these pores was comparable to that of the acetylcholine receptor, a protein ion channel found in cell membranes. To test for the colloid osmotic mechanism [9], we added high molecular weight solutes to the buffer solution at concentrations far below the range where osmotic pressures could induce hemolysis without pore formation. The high molecular weight osmolytes added to the buffer solution should be unable to diffuse through the pores caused by the polymers, and thereby balance the osmotic imbalance caused by the macromolecules within the RBC cytosol. Thus, these osmolytes should protect against hemolysis. Fig. 5

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Fig. 5. Effect of osmolytes on PEAAc-induced RBC hemolysis. PEAAc (50 mg) with 10 mM dextran (10 kD); PEAAc (50 mg) with 10 mM dextran (5 kD); PEAAc (50 mg) with 10 mM glucose; PEAAc (50 mg). Error bars represent standard deviation of three samples.

represents a hemolysis assay by PEAAc, in which dextran (10 kD), PEG (5 kD), and glucose were added separately to the RBC buffer solution at a concentration of 10 mM. The 10 kD dextran and 5 kD PEG inhibited hemolysis by PEAAc substantially, but glucose had no significant effect. These results support a colloid osmotic mechanism where the polymers create pores in the red blood cell membrane. It is not known if such a mechanism would occur within endosomes.

4. Conclusions The release of endocytosed DNA from the endosome to the cytosol is a major barrier to transfection. To address this problem, we have designed pHsensitive synthetic polymers that mimic the membrane-disruptive peptides of viruses, and investigated their hemolytic activities. The synthetic polymers studied have high hemolytic activity at pHs similar to those in endosomes. They are easy to synthesize and can be rationally engineered to have a range of pH-induced membrane-disrupting profiles by varying the polymer composition. We have shown that PEAAc is as effective in disrupting RBCs as mel-

litin, a well known membrane disruptive peptide, and that the membrane-disrupting efficiency can be improved significantly by designing slightly more hydrophobic polymers such as PPAAc. Hemolysis induced by PEAAc and PPAAc is pH-sensitive and neither polymer is membrane lytic at pH 7.4. PPAAc displays maximum hemolysis at pH 6.0 and below, while PEAAc displays a maximum at pH 5.0 and below. Future work in this area will focus on understanding the key compositional and structural factors which govern the lipid membrane interactions of pH-sensitive synthetic polymers, in order to design even more efficient membrane disruptive polymers.

Acknowledgements This work was mainly supported by the NIH (NIGMS Grant No. R01-GM53771-02). We are also grateful for support from the UW Center for Nanotechnology (graduate fellowship to N.M.), the UW Office of Technology Transfer, the Washington Technology Center at UW, and the Washington Research Foundation.

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