Research ArticleNanomedicine

Cartilage-penetrating nanocarriers improve delivery and efficacy of growth factor treatment of osteoarthritis

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Science Translational Medicine  28 Nov 2018:
Vol. 10, Issue 469, eaat8800
DOI: 10.1126/scitranslmed.aat8800
  • Fig. 1 Design and synthesis of bioactive PEGylated dendrimer–IGF-1 conjugates as cartilage penetrating nanocarriers.

    (A) Schematic of drug and carrier fates within joints after intra-articular injection based on size and charge. Material within the synovial fluid is rapidly cleared from the joint. Cationic material binds to anionic aggrecan within cartilage to avoid clearance. Small cationic materials can penetrate the aggrecan matrix to interact with chondrocytes deep in the tissue. ECM, extracellular matrix. (B) Chemical structure and key design characteristics of PAMAM dendrimers (Gen 4 shown) as cartilage-penetrating nanocarriers. (C) Synthetic scheme of a partially PEGylated dendrimer–IGF-1 formulation. For the Gen 4 35% PEG formulation, n = 64, x ≅ 18, y ≅ 4.4, and z ≅ 0.6. For the Gen 6 45% PEG formulation, n ≅ 224, x ≅ 90, y ≅ 9.4, and z ≅ 0.6. IGF-1 color code: orange, IGF-1 receptor binding site (Phe-Tyr-Phe); blue, lysine (alternative reaction site); cyan, matrix IGF-1 binding protein site (hinders IGF-1 transport); magenta, fluorescent tracer. (D) Rate of biosynthetic 35S sulfate incorporation from media into ex vivo bovine cartilage after treatment with no IGF-1, dendrimer–IGF-1, or free IGF-1. Data are means + 95% confidence interval (CI), n = 10 explants, and statistics by one-way analysis of variance (ANOVA) with Tukey post hoc tests. sGAG, sulfated glycosaminoglycan; ww, wet weight. (E) Percentage of NIH/3T3 cells in S phase after 24 hours of treatment with no IGF-1, dendrimer–IGF-1, or free IGF-1, as determined by cell cycle flow cytometry with 4′,6-diamidino-2-phenylindole and 5-ethynyl-2′-deoxyuridine staining. Data are means + 95% CI, n = 6 biological replicates, and statistics by one-way ANOVA with Tukey post hoc test. (F) Confocal image of interaction of dendrimer–IGF-1 conjugates with live bovine cartilage tissue. Binding to extracellular matrix and dense binding to cellular membrane (white arrows) are visible.

  • Fig. 2 Screen identifies optimal degree of dendrimer PEGylation for enhanced electrostatic cartilage binding without toxicity.

    (A) Synthesis (left) and characterization pipeline (right) of dendrimers with varying lengths and molar ratios of PEG grafted to end groups. (B) Schematic of fluorescent dendrimer uptake and viability experiments in bovine cartilage tissue explants (disks). (C) Bright-field image of fluorescent dendrimer uptake in bovine cartilage 24 hours after incubation. (D) Fluorescent dendrimer uptake in bovine tissue over 24 hours and human CHON-001 cell viability 48 hours after treatment with 10 μM partially PEGylated dendrimers (PEG Mn = 436 Da; xavg = 8). Theoretical surface charge is shown. Selected (shaded region) refers to the dendrimer formulation used for further study. Data are means + 95% CI, n = 15 explants for uptake, and n = 4 technical replicates for viability. ***P < 0.001 versus all others by one-way ANOVA with Tukey honestly significant difference (HSD) posttests. (E) Cell viability staining of sectioned bovine cartilage explants after 48 hours of incubation with PEGylated dendrimers. Cell death at edges is due to artifact from tissue harvest. Scale bars, 200 μm. (F) Histology [hematoxylin and eosin (H&E)] of rat knee synovium and cartilage 2 months after intra-articular injection of nanocarrier. Scale bars, 200 μm. B, bone; C, cartilage.

  • Fig. 3 PEGylated dendrimer nanocarriers extend joint residence, therapeutic time, and cartilage penetration of IGF-1 in rats.

    (A) Schematic of rat intra-articular injection and IVIS image of uninjected rat to establish background signal. The field of view for (B) is shown in the red box. (B) Representative IVIS images of rat knee joints over 28 days after injection of fluorescent IGF-1 formulations. Black circles represent the anatomical joint ROI used for quantification. Fluorescence scale: max = 9.0 × 107, min = 2.0 × 107 (units in Materials and Methods). (C) Time course of fluorescent radiant efficiency within joints. Data are fit to a one-phase exponential decay with a common plateau based on background signal. Data are means + 95% CIs of nonlinear fit, n = 8 joints per formulation. Half-lives were statistically different for each dataset (P < 0.0001) by extra sum of squares F test. (D) Estimated time at IGF-1 concentrations that saturate aggrecan biosynthesis activity for each delivery method, based on initial concentration after injection of ~6 μM IGF-1 (saturating concentration of 0.04 μM) and mean + 95% CI of carrier half-life. (E) 3D reconstruction of multiphoton microscopy images of full-thickness cartilage from intact rat femurs harvested 6 days after injection. Color code: gray, collagen II second-harmonic generation signal; red, aggrecan antibody; blue, IGF-1.

  • Fig. 4 Dendrimer–IGF-1 conjugates exhibit full penetration of human-thickness cartilage within window of residence time in joint.

    (A) Schematic of live bovine cartilage explant harvest and penetration assay. Fluorescent dendrimer distribution in cartilage was imaged after 2 or 6 days of incubation in Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS) + cartilage media supplements. Red box indicates field of view (FOV). (B) Confocal microscope images of fluorescently labeled IGF-1 (purple) across the diffusion gradient (right to left, −y) of the tissue (shown in bright field). Arrow indicates direction of dendrimer transport. Scale bars, 200 μm. (C) Quantification of IGF-1 fluorescence intensity across the explant section. Average (Avg.) fluorescence intensity over the entire tissue section is shown. All images were taken under the same laser power, intensity, and offset.

  • Fig. 5 Dendrimer–IGF-1 conjugate reduces cartilage degeneration 4 weeks after surgical traumatic joint injury.

    (A) Schematic of a rat knee frontal section illustrating the ACLT + MMx surgery. Dashed box outlines the primary zone of lesion formation. (B) Schematic of surgery timeline and tissue processing procedures. I.A., intra-articular. (C) Representative toluidine blue/fast green–stained frontal sections of the medial femur and tibia. Area of degeneration outlined in red. Total and significant widths of degeneration are outlined in black and yellow, respectively. Matrix loss is shown as black arrowheads. MF, medial femur; MT, medial tibia; MM, medial meniscus; AC, articular cartilage; L, lesion. Scale bars, 500 μm. (D) Quantified area of degenerated cartilage tissue of the medial tibia for each rat, as a percentage of total cartilage area in the section. Degenerated tissue was defined as >50% cell death and loss of toluidine blue staining. (E) Width of cartilage degeneration at the joint surface (0% depth). (F) Width of cartilage degeneration at 50% cartilage depth. (G) Representative H&E-stained frontal sections of rat joints across different treatment, displaying regions of the lateral synovium characteristic of the given score (in parentheses). Cyan arrow indicates increased number of synovial lining cells, and black arrows indicate subsynovial proliferation. Scale bars, 100 μm. (H) Synovial inflammation scores (0 to 4) for each treatment. All data are means + 95% CIs, n = 7 to 9 rats, statistics by one-way ANOVA with Tukey HSD posttest for (D) and (E), and by Kruskal-Wallis test with Dunn posttest for (F) and (H).

  • Fig. 6 Dendrimer–IGF-1 conjugate reduces osteophyte burden in surgically injured rats 4 weeks after surgery.

    (A) Representative 2D (top) and 3D (bottom) μCT images showing osteophytes in red arrows or red shading. ROIs were serially drawn around osteophytes in sequential frontal image stacks and reconstructed into 3D to generate bottom images and measure osteophyte volume. (B) Total osteophyte volume in each joint across the four treatment conditions. Data are means + 95% CI, n = 7 to 10 rats as shown, statistics by one-way ANOVA with Tukey HSD posttest.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/469/eaat8800/DC1

    Materials and Methods

    Fig. S1. Representative cell cycle flow cytometry scatter plots for NIH/3T3 cells after 24-hour treatment with IGF-1 formulations.

    Fig. S2. 1H NMR spectra of PEGylated dendrimer panel (without IGF-1).

    Fig. S3. MALDI-TOF mass spectra for partially PEGylated dendrimer screen.

    Fig. S4. Complete uptake data for partially PEGylated dendrimers into bovine cartilage explants.

    Fig. S5. Complete CHON-001 cellular viability data for partially PEGylated dendrimers at a range of doses of dendrimers.

    Fig. S6. Desorption of PEGylated dendrimers in solutions of different ionic strengths.

    Fig. S7. Quantification of ex vivo bovine cartilage tissue viability staining in Fig. 2.

    Fig. S8. Histology (H&E) of representative sections from likely contact organs for ~5- to 10-nm materials.

    Fig. S9. Histology (H&E) of rats treated with dendrimers having suboptimal percentage of end group PEGylation.

    Fig. S10. 3D reconstruction of multiphoton microscopy images of full-thickness cartilage from intact rat femurs harvested 2 days after injection of free IGF-1.

    Fig. S11. Penetration of cartilage explants in DMEM (no protein in media).

    Table S1. Characterization of percentage of PEGylation by NMR integral ratios.

    Table S2. Characterization of percentage of PEGylation by mass addition according to MALDI-TOF data.

    Table S3. Serum toxicology markers at 2 and 7 days after intra-articular injection of Gen 6 45% PEG–IGF-1.

    Table S4. Individual subject-level data for Fig. 2D and fig. S5.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Representative cell cycle flow cytometry scatter plots for NIH/3T3 cells after 24-hour treatment with IGF-1 formulations.
    • Fig. S2. 1H NMR spectra of PEGylated dendrimer panel (without IGF-1).
    • Fig. S3. MALDI-TOF mass spectra for partially PEGylated dendrimer screen.
    • Fig. S4. Complete uptake data for partially PEGylated dendrimers into bovine cartilage explants.
    • Fig. S5. Complete CHON-001 cellular viability data for partially PEGylated dendrimers at a range of doses of dendrimers.
    • Fig. S6. Desorption of PEGylated dendrimers in solutions of different ionic strengths.
    • Fig. S7. Quantification of ex vivo bovine cartilage tissue viability staining in Fig. 2.
    • Fig. S8. Histology (H&E) of representative sections from likely contact organs for ~5- to 10-nm materials.
    • Fig. S9. Histology (H&E) of rats treated with dendrimers having suboptimal percentage of end group PEGylation.
    • Fig. S10. 3D reconstruction of multiphoton microscopy images of full-thickness cartilage from intact rat femurs harvested 2 days after injection of free IGF-1.
    • Fig. S11. Penetration of cartilage explants in DMEM (no protein in media).
    • Table S1. Characterization of percentage of PEGylation by NMR integral ratios.
    • Table S2. Characterization of percentage of PEGylation by mass addition according to MALDI-TOF data.
    • Table S3. Serum toxicology markers at 2 and 7 days after intra-articular injection of Gen 6 45% PEG–IGF-1.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4 (Microsoft Excel format). Individual subject-level data for Fig. 2D and fig. S5.

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