Subscriber access provided by READING UNIV
Article
Multivalent Presentation of Peptide Targeting Groups Alters Polymer Biodistribution to Target Tissues Maureen R Newman, Steven G Russell, Christopher S Schmitt, Ian A Marozas, Tzong-Jen Sheu, J. Edward Puzas, and Danielle S. W. Benoit Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01193 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
Multivalent Presentation of Peptide Targeting Groups Alters Polymer Biodistribution to Target Tissues Maureen R. Newman†,§, Steven G. Russell‡, Christopher S. Schmitt‡, Ian A. Marozas†, Tzong-Jen Sheu§,‖, J. Edward Puzas§,‖, and Danielle S. W. Benoit*,†,‡,§,‖ †
Biomedical Engineering, ‡Chemical Engineering, University of Rochester, Rochester, New
York 14627, United States §
Center for Musculoskeletal Research, ǁDepartment of Orthopaedics, University of Rochester
Medical Center, Rochester, New York 14642, United States KEYWORDS: peptide targeting, polymer therapeutics, drug delivery, tartrate-resistant acid phosphatase, oligo(ethylene glycol)
ABSTRACT: Drug delivery to bone is challenging, whereby drug distribution is commonly 99% reaction efficiency to form random copolymers 9. Using these two arrangements in peptide-functionalized polymers, the effect of molecular weight (Mn), which is inherently controlled via RAFT, on biodistribution was analyzed. Scheme 2. Synthesis and Functionalization of TBP-acrylamide 4 and Xanthate Monomer 6 to Give p(OEG-co-TBP) Random Brush Copolymers 9
ACS Paragon Plus Environment
18
Page 19 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
3.2 Characterization of copolymers. Peptide incorporation was controlled by monomer feed of 2 and 6 (Figure S6-7). Copolymer Mn exhibited narrow distributions (polydispersity ≤1.1) and were well-controlled by varying the degree of polymerization (DP, i.e. the stoichiometric ratio of monomer and RAFT chain transfer agent) (Figure 1A-B, Figure S8). Mn of 25-100 kDa and 25125 kDa were achieved by varying DP from 100-600 for 3 and 100-400 for 7, respectively. Mn increased non-linearly over time for 3 (Figure 1C), likely due to high initial 2 incorporation, which has been previously demonstrated as a result of peptide monomer aggregation.56 After 6h, incorporation of 2 reduces precipitously (Figure 1D), which can be attributed to disparate reactivity ratios (see SI). Monomer reactivity mismatch may also explain why higher DP is required to achieve higher Mn for 3.57 In contrast, Mn evolution for 7 was uniform (Figure 1E) due to consistent monomer incorporation (Figure 1F) resulting from similar monomer reactivity (see SI). Thus, 3 exhibited peptide gradients, and 9 exhibited random (uniform) peptide presentation (Figure S9).
ACS Paragon Plus Environment
19
Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 20 of 52
Figure 1. Degree of polymerization controls molecular weight (Mn) of (A) gradient copolymer 3 and (B) random copolymer 7. (C) Mn of 3 increases non-linearly over time. (D) Incorporation of TBP-methacrylamide 2 decreases over time, indicating formation of gradient copolymer 3. (E) Mn of 7 increases linearly over time. (F) Incorporation of xanthate monomer 6 is consistent over time, indicating formation of random copolymer 7. Peptide secondary structure was evaluated to determine the effects of polymer incorporation and architecture. Unaltered 1 contains mixed secondary structure, with an alpha helix located central to disordered N- and C-termini (Figure S10A).49, 58 This structure was largely preserved in gradient 3 and random 9 copolymers, as the circular dichroism ellipticity ratio of 222/208 nm
ACS Paragon Plus Environment
20
Page 21 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
was consistent and less than 1.25, indicating disordered structures59 (Figure S10B). It is possible the change in ellipticity observed at ~200 nm indicates shorter alpha helix length caused by elongation of the unstructured N-terminus region when juxtaposed to the polymer backbone.60 To ensure cytocompatibility of copolymers, mesenchymal stem cells (MSCs) and osteoclasts, relevant cell types within the bone microenvironment, were exposed to a range of concentrations of peptides 1, gradient copolymers 3, and random copolymers 9. Normalizing to untreated cells, as per ISO 10993-5,51 all treatments resulted in greater than 70% viable MSCs and osteoclasts, the threshold for cytocompatible biomaterials (Figure 2). After 24 hours and 1 week of MSC treatment, there were neither significant differences in viability between treated and untreated MSCs, nor concentration-dependent effects. Similarly for osteoclasts, treatments neither resulted in significantly different viability relative to untreated osteoclasts, nor were dose dependent. Finally, MSC phenotype was conserved with polymer treatment, as there were no significant differences in CD90, CD105, CD44, or CD45 expression relative to untreated controls (Figure S11).
Figure 2. Cytocompatibility of peptides 1, p(OEG), peptide-functionalized gradient copolymers (3), unfunctionalized random copolymers p(OEG-co-6) (7), and peptide-functionalized random copolymers (9). Mesenchymal stem cells (MSCs) were treated for 24 h (A) and 1 week (B), then evaluated using alamarBlue. Osteoclasts were treated for 24 h (C), then stained with TRAP and enumerated. All treatments were significantly greater than 70% viability relative to untreated cells by two-way ANOVA with Bonferroni post-hoc testing, p
