Multivalent Presentation of Peptide Targeting. Groups Alters Polymer Biodistribution to Target

Transcription

1 Supporting Information 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, Department of Orthopaedics, Center for Oral Biology, Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, New York 14642, United States * S1

2 Table of Contents Peptide Synthesis and Functionalization (1, 2, and 4)... S4 MALDI Supplemental Figure 1A-D... S4 S-2-hydroxyethyl-O-ethyl Dithiocarbonate Monomer Synthesis (6)... S4 1 H NMR Supplemental Figure 2... S4 1 H NMR Supplemental Figure 3... S4 Random Copolymer Precursor Synthesis (7)... S5 1 H NMR Supplemental Figure 4... S5 Random Copolymer Synthesis (9)... S5 1 H NMR Supplemental Figure 5... S5 Fluorescent Labeling... S5 Gradient Polymer Properties Supplemental Table 1... S5 Random Polymer Properties Supplemental Table 2... S6 Peptide Incorporation Determination... S6 Absorbance Spectra Supplemental Figure 6A-B... S6 Incorporation vs Feed Supplemental Figure 7... S6 Molecular Weight Determination... S7 dn/dc Equation 1... S7 GPC Traces Supplemental Figure 8... S7 Monomer Reactivity... S7 Incorporation vs Time Supplemental Figure 9... S7 Circular Dichroism and Secondary Structure... S8 3D Modeling and Ellipticity Supplemental Figure 10A-B... S8 Mesenchymal Stem Cell Cytocompatibility... S9 MSC Phenotype Supplemental Figure 11A-H... S9 Polymer Biodistribution in Fractured Mice... S9 Fracture X-ray Supplemental Figure S9 24 h and 1 week Statistics Per Organ Supplemental Figure S10 TRAP Affinity... S10 SPR Response for TBP Supplemental Figure S11 Dissociation Constants Supplemental Figure S11 Linear Regression for PAV/SBV/EDV of Gradient Copolymers Supplemental Table 4... S11 SPR Response for Random and Control Polymers Supplemental Figure 16A-C... S11 Linear Regression for PAV/SBV/EDV of Random Copolymers Supplemental Table 5... S12 Linear Regression for % Drop of Random Copolymers Supplemental Table 6... S13 S2

3 References... S13 S3

4 Supplemental Figure 1: Peptide synthesis and functionalization (1, 2, and 4). MALDI-TOF verifies correct synthesis of (A) TRAP-binding peptide (TBP) 1, (B) TBP-methacrylamide 2, and (C) TBPacrylamide 4. To ensure peptides are stable in resorption pits, which are acidic, TBP was dissolved in ph 5 acetate buffer for 1 week, dialyzed against dh 2 O for 3 days, frozen, lyophilized, and assessed by MALDI-TOF to verify no change in molecular weight (D). High-intensity peaks beyond the expected molecular weight (MW) are due to electrostatically-bound sodium (23 Da) and potassium (39 Da) ions. Supplemental Figure 2: S-2-hydroxyethyl-O-ethyl dithiocarbonate synthesis (5). NMR spectrum of 5. 1 H NMR (400 MHz, CDCl 3, 25 C): = 4.65 (q, J = 7.12 Hz, 2 H; CH 2 ), 3.87 ppm (t, J = 6.3 Hz, 2 H; CH 2 ), 3.35 (t, J = 6.3 Hz, 2 H; CH 2 ), 1.41 (t, J = 7.12 Hz, 3 H; CH 3 ). [1] Supplemental Figure 3: S-2-hydroxyethyl-O-ethyl dithiocarbonate monomer synthesis (6). NMR spectrum of 6. 1 H NMR (400 MHz, CDCl 3, 25 C): = 6.09 (m, J = 1 Hz, 1 H; CH), 5.56 (quint, J = 1.6 Hz, 1 H; CH), 4.63 (q, J = 7.12 Hz, 2 H; CH 2 ), 4.35 ppm (t, J = 6.44 Hz, 2 H; CH 2 ), 3.41 (t, J = 6.44 Hz, 2 H; CH 2 ), 1.91 (dd, J = 1 Hz and J = 1 Hz, 3 H; CH 3 ), 1.40 (t, J = 7.12 Hz, 3 H; CH 3 ). [1] S4

5 Supplemental Figure 4: Random peptide-functionalized OEG brush copolymer precursor polymer synthesis (7). NMR spectrum of 7. 1 H NMR (400 MHz, CDCl 3, 25 C). Supplemental Figure 5: Random peptide-functionalized OEG copolymer synthesis (9). NMR spectrum of 8. Arrow indicates removal of thiol protecting group peak. 1 H NMR (400 MHz, CDCl 3, 25 C). Supplemental Table 1: Gradient polymer properties used for biodistribution studies Polymer M n PDI Percent Peptide Peptides per Polymer TBP-LM n 6.1 kda % 4 TBP-IM n 19.5 kda 1.1 6% 4 TBP-HM n 63.4 kda % 4 SCP-IM n 22.7 kda % 4 S5

6 Supplemental Table 2: Random polymer properties used for biodistribution studies Polymer M n PDI Percent Peptide Peptides per Polymer Interligand Distance* TBP-LM n 10.1 kda % 4 10 angstroms TBP-IM n 23.6 kda 1.1 5% 4 40 angstroms TBP-HM n 62.3 kda 1.3 2% angstroms SCP-IM n 23.6 kda 1.1 5% 4 40 angstroms *OEG units =([M n 4(1550)]/300)/4. ILD = 2.52(OEG units +1), where 2.52 angstroms separate two adjacent monomer side chains in the oligomer backbone Supplemental Figure 6: Peptide incorporation determination. Ultraviolet-visible light absorbance spectra of (A) random p(oeg-co-tbp) 9, gradient p(oeg-co-tbp) 3, and homopolymerized OEG methyl ether methacrylate (p(oeg)). The absorbance peak at 280 nm corresponds to the tyrosine ring in 1. All polymers are 3 mg ml -1 in ddh 2 O. Both copolymers contain 20% peptide. (B) Similar to Figure S1, polymers were dissolved in ph 5 acetate buffer to investigate stability in acidic resorption pits. Dialyzed and lyophilized polymers were reconstituted at 1 mg ml -1 in ddh 2 O. Polymers exposed to low ph exhibited less than 10% change in absorbance at 280 nm, suggesting stability of peptide bonds. Supplemental Figure 7: Monomer incorporation is controlled by monomer feed, as calculated by ultraviolet-visible light absorbance spectrophotometry. Data±SD, n=3. S6

7 Molecular weight (M n ) determination A dn dc -1 value of ml g -1 was used for homopolymerized OEG methyl ether methacrylate, and a dn dc -1 value of ml g -1 was used for 7. A dn dc -1 value was calculated for 3 using % peptide incorporation (y) according to Equation (1), determined through experimentation (R 2 = , p = ). dn dc -1 [ml g -1 ] = 6.8x10-4 y + 7.6x10-2 (1) Supplemental Figure 8: Molecular weight (M n ) determination. Example plots of Rayleigh ratios for polymers synthesized during time course experiments for gradient (A) and random (B) polymers. Supplemental Figure 9: Monomer reactivity. Peptide incorporation over time for 3 and 9. (A) 3 exhibits preferential peptide incorporation before 4 h, with minimal or no incorporation between 4 and 24 h. (B) 9 exhibits consistent peptide incorporation from the beginning to the end of polymerization. As 3 and 7 were polymerized to achieve similar M n, 9 exhibited greater numbers of peptides per chain than 3. Reducing 6 incorporation and M n of 7 would reduce numbers of peptides per chain. S7

8 Supplemental Figure 10: Circular dichroism and simulated modeling of peptide secondary structure. (A) Predicted 3D secondary structure of TBP and SCP. [2] Black to white gradient represents N- to C-terminus. (B) Circular dichroism of random copolymer, gradient copolymer, TBP, and SCP. Both copolymers contain 20% peptide. S8

9 Supplemental Figure 11: Mesenchymal stem cell (MSC) phenotype is preserved after polymer treatment, as identified by marker analysis (A-F). MSCs are described as CD90+, CD105+, CD44+, and CD45-. No significant differences were detected (p>0.05) by one-way ANOVA. Compared to control (red, blue, and purple traces), MSCs (shaded grey) treated with polymers for 24 hours (G) and 1 week (H) exhibited no differences in marker expression. Supplemental Figure 12: Right tibia fractures in mice were used to evaluate biodistribution of polymers. S9

10 Supplemental Figure 13: 24 h and 1 week biodistribution for individual organs in support of Figure 2. Gradient copolymers are solid bars (left) and random copolymers are striped bars (right). Significance determined by one-way ANOVA using p<0.05: & vs. targeted low molecular weight (T-LM n ), % vs. targeted intermediate molecular weight (T-IM n ), # vs. targeted high molecular weight (T-HM n ), and * vs. scrambled intermediate molecular weight (S-IM n ). ^, p<0.05 between fracture and naïve by twoway ANOVA. TRAP affinity The peak association value (PAV) represents the amount of polymer that reaches the TRAPfunctionalized surface during injection. The stable binding value (SBV) represents the amount of S10

11 polymer that is stably bound to the TRAP-functionalized surface immediately following injection. The end dissociation value (EDV) represents the amount of polymer that is strongly bound to the TRAPfunctionalized surface following 60 seconds of dissociation, when sensorgram data has plateaued to a nearly unchanging value. The percent change between PAV and SBV (% Drop) represents the relative amount of polymer that reaches the TRAP-functionalized surface but is not stably bound. Supplemental Figure 14: SPR response for TBP. (1) PAV. (2) SBV. (3) EDV. (4) % Drop. Supplemental Figure 15: Dissociation constants (K D ) for gradient and random copolymers, as well as soluble TBP. Lower K D indicates greater affinity. Supplemental Table 4: Linear regression for gradient copolymers (Figure 5C). r 2 Linear regression RU y, # TBP x PAV 0.99 y = 1.5E-4x + 6.8E-5 SBV 0.97 y = 9.5E-5x + 1.1E-4 EDV 0.99 y = 4.6E-5x + 6.4E-5 S11

12 Supplemental Figure 16: SPR response of (A) 10% TBP and (B) 40% TBP random copolymers, as well as (C) p(oeg) and p(oeg-co-scp) gradient and random controls. Supplemental Table 5: Linear regression for random copolymers (Figure 6D-F). 10% 15% 20% 40% r 2 Linear regression RU y, # TBP x PAV 0.97 y = -1.0E-5x + 4.2E-4 SBV 0.63 y = 1.3E-6x + 1.1E-4 EDV 0.81 y = 3.3E-6x + 3.2E-5 PAV 1.0 y = -7.8E-6x + 5.3E-4 SBV 0.99 y = -7.4E-6x + 4.2E-4 EDV 0.99 y = -6.8E-6x + 3.7E-4 PAV 0.89 y = 6.2E-6x + 7.8E-5 SBV 0.91 y = 7.4E-6x 4.2E-5 EDV 0.97 y = 9.3E-6x 1.5E-4 PAV 0.94 y = 2.5E-6x + 8.6E-6 SBV 0.98 y = 2.3E-6x 7.5E-5 EDV 1.0 y = 2.4E-6x 1.2E-4 S12

13 Supplemental Table 6: Linear regression for random copolymers (Figure 6G). r 2 Linear regression % Drop y, # TBP x 10% 0.96 y = -3x % 1.0 y = x % 0.98 y = -0.9x % 0.97 y = -0.6x + 96 References [1] R. Nicolay, Macromolecules 2012, 45, 821. [2] Y. Shen, J. Maupetit, P. Derreumaux, P. Tuffery, J Chem Theory Comput 2014, 10, S13