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Engineered Polymer-Transferrin Conjugates as Self-Assembling Targeted Drug Delivery Systems Hiteshri Makwana, Francesca Mastrotto, Johannes Pall Magnusson, Darrell Sleep, Joanna Hay, Karl J. Nicholls, Stephanie Allen, and Cameron Alexander Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00101 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Engineered Polymer-Transferrin Conjugates as SelfAssembling Targeted Drug Delivery Systems. Hiteshri Makwana,1 Francesca Mastrotto,1 Johannes P. Magnusson,1 Darrell Sleep,2 Joanna Hay,2 Karl J Nicholls,2 Stephanie Allen1* and Cameron Alexander.1* 1-School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD

2- Albumedix Ltd, 59 Castle Boulevard, NG7 1FD Nottingham UK.

KEYWORDS Transferrin, transferrin receptor (TfR) drug delivery, polymer-protein conjugates, therapeutics, self-assembly, supramolecular, cancer.

Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface and the polymer conjugates grown from these variants were compared with polymer conjugates grown from nonselectively derivatised sites on native Tf. The resulting synthetic biopolymer hybrids were evaluated for self-assembly properties, size and topology, ability to carry an anti-cancer drug

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(paclitaxel) and cytotoxicity with and without a drug payload in a representative human colon cancer cell line. The results demonstrated that the engineered Tf variant polymer conjugates formed better-defined self-assembled nanoparticles than the non-selectively derivatised conjugates and showed greater efficacy in paclitaxel delivery. A polymer conjugate grown from a specific Tf variant, S415C was found to be taken up rapidly into cancer cells expressing the Tfreceptor, and, while tolerated well by cells in the absence of drugs, was as cytotoxic as free paclitaxel, when loaded with the drug. Importantly, the S415C conjugate polymer was not the most active variant in Tf-receptor binding, suggesting that the nanoscale self-assembly of the polymer-protein hybrid is also a key factor in delivery efficacy. The data overall suggest new design rules for polymer-biopolymer hybrids and therapeutic delivery systems which include engineering specific residues for conjugation which mediate nanoscale assembly as well as control of ligand-receptor interactions to target specific cell types.

1. INTRODUCTION

Polymer protein conjugates constitute one of the most successful classes of nanomedicines, with many conjugates now in regular clinical use, such as Oncaspar® for lymphoblastic leukemia and Neulasta® for treating neutropenia caused by chemotherapy.1 The proteins used in the conjugates can be used both for their target specificity and therapeutic efficacy, with an important advantage compared to many small molecule synthetic drugs of good patient tolerance due to known excretory pathways. While some proteins exhibit direct therapeutic effects, other proteins control functions such as transport and signalling behaviour. Consequently, proteins have been explored not only as therapeutics, but also as drug carriers and targeting agents for nanomedicine applications.2-5


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The combination of other polymers with proteins enables ‘hybrid’ materials to be prepared that exhibit properties of significant importance for therapeutic use.6-9 Conjugation of proteins to other natural macromolecules or synthetic polymers confers increases in solution volume, surface masking, charge modification and epitope shielding.10 These modifications can reduce losses of injected proteins due to renal filtration and can also promote the accumulation of the conjugate via the “EPR effect”.11, 12 Shielding of the exposed functionalities at a protein surface can reduce phagocytosis by the RES and liver cells, as well as reducing proteolysis and immune system recognition, which are major routes of elimination.13 In addition to improving the biopharmaceutical properties of the protein, the conjugated polymer can introduce specific functions of value in biomedical application, such as stimulus response and control of solution architecture. In particular, the wealth of knowledge gained through studying the supramolecular structures of synthetic amphiphilic block co-polymers in aqueous solutions14, 15 has informed the preparation of self-assembling polymer-protein conjugates. A number of examples now exist in which hydrophobic polymers have been coupled to proteins in order to generate micellar or nanoparticulate structures, with the protein components at the surface of the supramolecular assembly.16, 17 Furthermore, conformational changes in synthetic polymers which undergo coilto-globule phase transitions in response to solution temperature, have been used to reversibly assemble and disassemble protein conjugates into supramolecular objects which in turn alter the activities of the biopolymer.18-21 The ability to switch ‘on’ or ‘off’ the specific function of the protein thus offers the possibility to control biological recognition, signalling and, in the medical field, therapeutic efficacy.22-25 However, the fine control of activity in polymer-protein conjugates remains challenging, as minor changes in solution architecture and self-assembly can alter key parameters such as


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substrate accessibility, macromolecular crowding and allosteric response. Also, while there are many chemistries in development for selective conjugation of proteins,

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investigated as extensively as site-specific mutagenesis in polypeptides. Accordingly, we sought to explore conjugates of some specific variants of a protein widely used in drug delivery system targeting, in this case the iron-binding and transport protein transferrin,30 with amphiphilic polymers known to self-assemble in aqueous solutions. In addition, we sought to utilise the resulting hybrids as carriers of the widely-used anti-cancer drug paclitaxel, exploiting the expected self-assembling and self-delivering properties of the conjugate to improve delivery to a selected cancer cell line. We report here the synthesis, characterisation, drug encapsulation and release experiments of a series of polymer-transferrin conjugates, and show enhanced cytotoxic activity of engineered biopolymer hybrids compared to non-selectively derivatised protein conjugates in HCT-116 colon cancer cells.

2. MATERIALS AND METHODS All solvents and reagents were of analytical or HPLC grade and purchased from Sigma Aldrich or Fisher Scientific unless otherwise stated. 2-(2-Methoxyethoxy) ethyl methacrylate (MEO2MA, Mn = 188 g/mol) was purified before use by passing through a column filled with neutral alumina. Tris (2-pyridyl) methylamine (TPMA) was prepared as described elsewhere.31 Dialysis membrane (MWCO 6-8 kDa, regenerated cellulose) was used as received from Spectrapor. For AFM studies, mica discs and specimen discs were purchased from Agar Scientific (Stansted, UK), SNL-10 (Sharp Nitride Lever-10) AFM probes were purchased from Bruker (Cambridge, UK). RPMI (1640) and DMEM-F12 cell culture medium, foetal bovine serum (FBS), NuPAGE Novex 4-12% Bis-Tris Protein Gels and other cell culture media were purchased from Life


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Technologies. Dulbecco’s phosphate buffered saline (DPBS, without Ca2+ and Mg2+), anhydrous DMSO (>99.5%, 100 hours). The in vitro toxicities of the Thio-Tf conjugates loaded with PTX varied over the IC50 range of 6.8 x 10-9 M to 4.7 x 10-8 M across the 3 time points (24, 48 and 72 hours), with S415C-P-PTX and N611C-PPTX assemblies demonstrating the highest level of potency after 72 h of incubation (Figure 8). It was thus established that modification of the S415 and N611 residues in the C-lobe of transferrin did not result in loss of drug delivery efficacy in the selected cell line. However, the complexity surrounding the binding and internalisation of Tf-conjugates was highlighted by the fact that variant S28C, in which the cysteine was introduced distal to the CD71 binding site and surface exposed, did not show superior cytotoxic activity as a polymer conjugate loaded PTX. This suggests that other factors such as conformational changes to proteins when conjugated to polymers, and their overall supramolecular structures may have been important additional contributors to cell entry and delivery of the cytotoxic payload. Indeed it was apparent across the whole range of Tf-conjugates, for both ‘randomly’ grafted polymers and those in which the


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polymers were grown from engineered Thio-Tf variants, that cytotoxicities via delivered PTX were rather variable. The two most effective conjugates for delivery of PTX, as judged by their cytotoxicity to HCT-116 cells at 72hr, were the single polymer-chain conjugates Tf-P1 and S415C-P (IC50 = 6.3 x 10 9 M-1and 6.8 x 10 9 M-1, respectively). However, as noted above, not all the Thio-Tf single-chain polymer conjugates were as toxic when loaded with PTX, with a 6-fold decrease in potency of the S28C-P PTX formulation compared to the S415C-P PTX system. The latter difference cannot be explained solely by self-assembly and particle size of the conjugates, as S28C-P complexes were similar in diameter to the S415C-P and Tf-P1 micelles (~ 20 nm). Total drug incorporated was expected to be a strong contributory factor, as shown by Tf-P1 and Tf-P2, which were able to accommodate PTX to the highest extent (~10 wt %, Table 3). Intriguingly, the most potent Thio-Tf polymer conjugate, S415C-P contained 6.0 wt % PTX, i.e. ~30 % less PTX, but its IC50 value was only 8 % higher than that of Tf-P1. Similar kinetic drug release profiles were observed for all the Thio-Tf conjugates, with sustained release over 72 hr, whereas for the ‘random’ Tf-polymer conjugates, only Tf-P1 and Tf-P2 were able to prolong the release time beyond 48 hr. These data indicated that neither drug incorporation or release kinetics were the most dominant factors in determining cytotoxicities. It should also be noted that uptake of Tf-polymer complexes via the Tf-receptor mediated pathway was variable across the set of materials tested. As apparent from Figures 4 and 5, the binding of Tf-P1 to the Tfreceptor CD71 occurred to a reduced extent compared to Tf-P2 and Tf-P3 (~ 75% relative binding). For S415C-P, binding to CD71 was ~40 % of the value for S28C-P-CD71 binding, and similar to the other Thio-Tf conjugates. Thus it is likely that the conjugates entered cells by more than one pathway, with a passive endocytic mechanism for conjugates in their assembled form and an active Tf-mediated pathway for Tf-polymer ‘unimers’. It has recently been shown that


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‘clustered’ Tf-biotin-streptavidin conjugates traffic to lysosomal compartments, whereas single Tf-biotin conjugates are very rapidly recycled.43 It is possible therefore that the unimer-micelle balance in the Tf-polymer conjugates controlled the delivery efficacy, as any PTX bound strongly to the protein in Tf-polymer unimers would have been exported from the cells very quickly, whereas PTX entrapped in micelles would have trafficked to lysosomal compartments where digestion of protein would result in release of PTX and accumulation in the cytosol. Release of PTX in this way and prolonged retention in the cell interior would be expected to enhance proximity to microtubules and thus promote binding and microtubule stabilization. It was not possible from the microscopy data alone to verify this assertion , but experiments to test the hypotheses relating to single- or multiple receptor interactions and intracellular trafficking of nanoparticulates are ongoing.

Conclusions A range of new transferrin-polymer conjugates were prepared, characterised and evaluated for delivery of paclitaxel to cancer cells. A series of recombinant Tf variants with cysteine residues engineered to enable site-specific conjugation were used as macroinitiators from which polymers were grown. These conjugates were compared with Tf-polymer conjugates in which polymers were grown from residues reactive to NHS-esters. A range of self-assembled structures formed when these conjugates were added to aqueous buffer solutions, and well-defined micelles (~ 20 nm diameter) were produced for one of the ‘random’ Tf-polymer conjugates and all of the ThioTf single polymer chain conjugates. Binding of Tf to its cognate receptor CD71 in selected cell lines was shown for the polymer conjugates, but while a correlation was observed for a reduction


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in binding to CD71 as the number of polymer chains attached to the protein increased in the “random” conjugates, the changes in CD71 binding incurred as the cysteine residue position was altered were all within one order of magnitude. The conjugates were effective in delivering PTX to selected cells, and the most potent conjugates were those with only one polymer chain attached, i.e. Tf-P1 and the specific variant S415C-P. Considering the regulatory complexities associated with multiple attachment points in polymer-protein conjugates, the data suggest that S415C-P might have distinct advantages if selected as a targeted drug delivery system. We are currently developing further variants of these conjugates, with a view to initial experiments to test in vivo efficacy.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank BBSRC (Industrial CASE BB/H016279/1), Albumedix Ltd, the University of Nottingham and EPSRC (Grants EP/H005625/1) for funding. Paul Cooling, Tom Booth and Christine Grainger-Boultby are thanked for expert technical assistance. Esben Friis and Christopher Finnis are thanked for their insight in designing and developing the thiotransferrin engineered transferrin variants. Data access statement All raw data created during this research are openly available from the corresponding author ([email protected]) and at the University of Nottingham Research Data


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Management Repository (https://rdmc.nottingham.ac.uk/) and all analysed data supporting this study are provided as supplementary information accompanying this paper.

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TOC Graphic

Engineered transferrin variants with introduced cysteine residues can initiate growth of amphiphilic polymers to form self-assembled structures which are different to those of analogous conjugates grown from native transferrin. Specific engineered constructs show enhanced activity in delivery of the cytotoxic drug paclitaxel in vitro.


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