Ribonuclease A modification with poly[N‐(2‐hydroxypropyl)methacrylamide] copolymers: new route of synthesis and purification

Abstract

BACKGROUND N‐(2‐Hydroxypropyl)methacrylamide (HPMA), a synthetic polymer, has biocompatible features that allow it to be used as a drug carrier and used in drug delivery systems; however, its potential as a protein drug carrier has not been completely exploited. This study provides a new conjugation pathway to obtain HPMA–protein conjugates and, the use of hydrophobic interaction chromatography (HIC) to separate polymer–protein conjugates exploiting the hydrophobicity change on the protein surface due to the conjugation. In this work, Ribonuclease A (RNase A, EC 3.1.27.5), with recognized therapeutic properties, was used as a model protein. RESULTS HPMA was copolymerized with N‐(3‐aminopropyl)methacrylamide (APMA) and then activated with glutaraldehyde. The activation process raises copolymer hydrophobicity. Additionally, the HPMA–RNase A conjugation reaction was carried out at pH 5.1 through reductive amination. At higher copolymer molecular weight (> 50 kDa), the conjugation does not occur whereas molecular weights between 30 and 50 kDa promote the conjugation reaction. The native protein was separated from modified protein using HIC and conjugates with a narrow molecular weight were obtained. CONCLUSIONS RNase A can be conjugated with HPMA–APMA copolymer, activated with glutaraldehyde, through reductive amination. This represents an alternative to previous conjugation reactions. Moreover, the molecular size of the copolymer impacts strongly in the conjugation reaction. The HPMA–RNase A conjugates can be separated employing HIC and, it is possible to obtain conjugates with a narrow molecular weight dispersion. This work proposes a new conjugation pathway and purification process in route to developing chromatographic alternatives to efficiently separate HPMA–protein conjugates. © 2020 Society of Chemical Industry

Ribonuclease A–polymer conjugates via in situ growth for cancer treatment

“…To increase the stability and delivery efficiency of proteins, several strategies have been developed to complex proteins via electrostatic interaction or construct protein–polymer conjugates using cationic polymers. 14–16 However, the efficiency of cationic polymer-mediated protein delivery may be restricted when it comes to delivering positively charged proteins since the charges on both proteins and cationic polymers can repel each other, making the formation and internalization of the complex more challenging. Meanwhile, the conjugation of proteins and polymers is usually carried out under special conditions such as reductive amination, 16 which may influence the conformation and activity of proteins.…”

“…14–16 However, the efficiency of cationic polymer-mediated protein delivery may be restricted when it comes to delivering positively charged proteins since the charges on both proteins and cationic polymers can repel each other, making the formation and internalization of the complex more challenging. Meanwhile, the conjugation of proteins and polymers is usually carried out under special conditions such as reductive amination, 16 which may influence the conformation and activity of proteins. Alternatively, to overcome the limitation of cationic polymers in delivering positively-charged proteins, a promising strategy of “ in situ growth (SIG) of a cationic polymer from protein” has been successfully developed.…”