Pharmaceutical Nanotechnology
Impact of polymer tacticity on the physico-chemical behaviour of polymers proposed as therapeutics

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Abstract

Although water-soluble polymers are finding increasing use as polymer therapeutics, there has been little consideration of the effect of polymer stereochemistry on their physico-chemical and biological properties. The aim of this study was to investigate these properties using polymethacrylic acids (PMAs) of similar molecular weights with a difference in syndiotacticity of about 20% of rr triad content. Experiments to characterize the solution behaviour were conducted at pHs encountered during the transport, endocytic uptake and intracellular trafficking (7.4–3.0). These showed that with increasing rr triads, the polymer become less hydrophobic, a stronger acid, displayed a locally ordered solution conformation at pH 5.5, and interacted more strongly with dodecyl trimethylammonium bromide (DTAB) micelles. Preliminary cytotoxicity experiments using B16F10 melanoma cells showed lower toxicity in the concentration range of 1–100 μg/mL with increased rr triads. These observations indicate that the higher content of rr triads could drive a chain organization that minimize the influence of negative charges and so underline the importance of further, systematic studies to investigate the effect of tacticity on the behaviour of polymers in respect of their pharmacokinetics, toxicity and efficacy.

Graphical abstract

Increasing rr triads, polymethacrylic acid results less hydrophobic, interacts more strongly with DTAB micelles and shows lower toxicity in the concentration range of 1–100 μg/mL.

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Introduction

Over the last three decades functional polymers have been finding increasing use as polymer-therapeutics (reviewed in Duncan, 2003, Vicent and Duncan, 2009). Polymers are being used as therapeutics with intrinsic activity, as platforms for delivery of drugs and proteins, and as non-viral vectors to promote cytosolic delivery of macromolecular drugs. Considering the increasing number of synthetic polymers that have entered clinical development there has been little consideration of the potential effect of polymer stereochemistry on their physico-chemical and biological properties and the implications for the safety/efficacy of the medicines to which they relate. The aim of this study was to investigate the effect of polymer tacticity on physico-chemical and biological properties relevant to the use of polymer therapeutics using poly(methacrylic acid) (PMA) as a model polymer.

Early studies involving the anionic polyelectrolyte PMA showed that, like other polyanions, it displays antiviral activity (De Somer et al., 1968). More recently, PMA has demonstrated the ability to reduce, and even prevent completely, protein thermo-aggregation. This is interesting as this phenomenon is very important for induction of neurodegenerative diseases like Alzheimer's disease, bovine spongiform encephalopathy and Huntington disease (Shalova et al., 2005). From the chemical perspective, the PMA backbone is characterized by the presence of quaternary carbon atoms bearing methyl and carboxylic groups, and thus it can be present with two stereoregular forms, syndiotactic and isotactic, as well as a non-stereoregular atactic form. The simplified Fisher projections (Scheme 1) show that placement of substituent groups in the isotactic structure corresponds to a meso or a m-placement of two consecutive monomer units (diad). The syndiotactic structure corresponds to a racemic or r-placement (Frisch et al., 1966). A sequence of three monomer units is called a triad, the isotactic triad is mm, the heterotactic triad is mr, and the syndiotactic one is rr. The prevalence of mm or rr triads along the polymer backbone confers the iso- or syndio-tacticity to the polymer chain respectively. PMA has been already reported to exhibit different solution properties in the isotactic and atactic form. For example, the isotactic PMA is a weaker acid than the atactic, it is insoluble in water below a critical degree of neutralization and shows an irreversible potentiometric behaviour. Furthermore, in the presence of a cationic surfactant such as cetylpyridinium chloride (CPC), the interactions of isotactic chains with CPC starts at a lower surfactant concentration (CAC) than in the case of atactic PMA (van den Bosch et al., 2004, Loebl and O’Neill, 1960, Leyte et al., 1972, Vlachy et al., 2006, Ruiz-Pérez et al., 2008).

The aim of this study was to investigate the physico-chemical behaviour in aqueous solution of atactic and syndiotactic PMAs obtained from hydrolysis of PMMAs of average molecular weight in the range of 43–48 kDa that had slightly increasing amounts (from 60% to 80%) of rr triads. In both cases, there were very low amounts of isotactic mm triads. Small-angle neutron scattering (SANS) was used to study polymer conformation in solution, whilst surface tension measurement, electron paramagnetic resonance (EPR) and SANS were used to investigate the interfacial interaction with the cationic surfactant DTAB in a unimolecular and micellar form, and consequently to define the effect of increasing stereoregularity on the aggregation behaviour of PMA. These characteristics have been interpreted in terms of the charge on the polymer, quantified by pH titration. Preliminary experiments were also undertaken to investigate the biological properties (cytotoxicity and cell uptake using B16F10 cells) of these PMAs.

Section snippets

Materials

Syndiotactic polymethylmethacrylate (PMMA) (Mw = 43 kDa, Mw/Mn = 1.02) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Gly-Gly-ONp (4.19 mol% activated groups, Mw = 30 kDa) were from Polymer Laboratories, UK, and used without further purification. Toluene (SigmaAldrich, Steinhem, Germany) was dried over sodium and distilled before use. Methylmethacrylate (MMA) (SigmaAldrich, Steinhem, Germany) was treated with calcium hydride for 4 h and distilled under vacuum. Cu(I)Br (Aldrich, Steinhem,

Results and discussion

Conformational behaviour of the polymers is strongly influenced by the nature and the net charge on the macromolecular chain. Moreover, the tacticity can play an important role in the conformational organization of the chain, such that the physico-chemical and biochemical properties of polymers with different stereochemistry can significantly change with pH (or ionic strength). It is thus surprising that the stereochemistry of polymeric excipients and polymer therapeutics is so rarely discussed.

Conclusions

These studies show that relatively small changes in PMA rr triad content cause changes in the solution and interfacial behaviour of the polymer that could impact on biological properties during use as a polymeric excipient or a polymer therapeutic. At pH 5.5 (α < 0.3) the syndiotactic PMA showed a pronounced maximum in the SANS over the Q range 0.05 < Q < 0.1 Å−1 not detectable for the atactic form, implying the presence of a possible ordered conformation and/or the presence of aggregates due to

Acknowledgements

EPSRC is gratefully acknowledged for the provision of a Platform grant (EP/C013220/1). STFC is thanked for provision of neutron beamtime.

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    1

    Current address: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK.

    2

    Current address: Wound Biology Group, School of Dentistry, Cardiff University, Heath Park, Cardiff CF14 4XY, UK.

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