Research ArticleAPPLIED SCIENCES AND ENGINEERING

Synthetic networks with tunable responsiveness, biodegradation, and molecular recognition for precision medicine applications

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Science Advances  27 Sep 2019:
Vol. 5, no. 9, eaax7946
DOI: 10.1126/sciadv.aax7946
  • Fig. 1 Overview of the P(AAm-co-MAA) nanogel platform and the use of its derivatives for precision medicine applications.

    Nanoscale networks of acrylamide (AAm) and methacrylic acid (MAA), cross-linked with methylenebisacrylamide (BIS) or its degradable disulfide analog [N,N′-bis(acryloyl)cystamine], were synthesized by inverse emulsion polymerization and modified via carbodiimide chemistry with tyramine (Tyr), N,N-dimethylethylenediamine (DMED), proteins, or peptides. In an additional post-synthesis step, gold nanoparticles (AuNP) were precipitated within DMED-modified (DMOD) nanogels. Here, we document the synthesis and modification of this nanogel platform and demonstrate the impact of nanogels’ modification on their ability to respond to the pH environment, load and release a model cationic drug, target cells, act as a functional enzyme, and transduce green light for photothermal therapy. Because of its tunability and the variety of therapeutic modalities enabled, we believe that this platform is suitable for precision medicine applications. DTT, dithiothreitol; TMB, 3,3′,5,5′-tetramethylbenzidine.

  • Fig. 2 Nanogel biodegradation analysis by DLS and QCM.

    (A) N,N′-bis(acryloyl)cystamine cross-linked nanogels degrade via reduction of the disulfide. The diagram demonstrates how, after an initial period of surface erosion, the nanogels experience bulk degradation, leading to simultaneous network swelling. (B) DLS analysis of nanogel degradation. While bisacrylamide cross-linked nanogels did not degrade under reducing conditions, those cross-linked with a disulfide cross-linker were digested by both reducing agents (n = 4, mean ± SD). (C) QCM analysis demonstrated the kinetic decomposition of nanogels under reducing conditions and flow. While the mass of nondegradable nanogels was relatively unaffected by reducing conditions, the mass of degradable gels declined rapidly (n = 3, mean ± SD). (D) Hydrodynamic diameter analysis by DLS supported the degradation mechanism of initial surface erosion followed by bulk degradation. While the normalized count rate declined steadily throughout the extended measurement, the hydrodynamic diameter decreased initially (surface erosion) and then increased for the remainder of the experiment (i.e., decrease in cross-links led to a reduction in the total number of nanoparticles but swelling of the remaining intact nanogels) (n = 3, mean ± SD).

  • Fig. 3 Characterization of small molecule–modified nanogels.

    (A) FTIR spectra of TMOD and DMOD nanogels, as compared with the unmodified formulation. The peaks at 1700 and 1200 cm−1 correspond to the carboxylic acid, at 1660 and 1590 cm−1 correspond to the amide, and at 800 cm−1 correspond to the aromatic groups, confirming the incorporation of each small-molecule ligand through covalent coupling. FTIR analysis of all formulations is presented in fig. S1. (B) Nanogel modification proceeded with approximately 60% efficiency when the ligand concentration did not exceed the carboxylic acid concentration (stoichiometric ratios less than 1). (C) Potentiometric titrations were used to quantify the carboxylic acid content of all formulations, elucidating the extent of small-molecule coupling. (D) Modified nanogels exhibited a pH-responsive zeta potential transition (anionic to cationic), whereas unmodified nanogels were anionic across all pH values tested (n = 3, mean ± SD). (E) Unmodified and TMOD nanogels exhibited a pH-responsive collapse with a critical transition point at pH ~ 4.8. DMOD nanogels did not undergo substantial pH-responsive swelling.

  • Fig. 4 Model drug (methylene blue) release from modified and unmodified nanogels.

    (A) Methylene blue experienced complementary electrostatic interactions with unmodified nanogels at both pH 4.5 and 7.4, leading to sustained release in both conditions. (B) TMOD nanogels exhibited an initial burst release of methylene blue, where the quantity of that release was greater in acidic than neutral conditions. (C) DMOD nanogels exhibited a burst release of greater than 50% the loaded payload in each pH condition, with more rapid release in acidic than neutral conditions. (D) DMOD and TMOD nanogels exhibited similar methylene blue release behavior in acidic conditions, while unmodified gels exhibited a more sustained-release profile. (E) DMOD nanogels released methylene blue rapidly in 1× PBS (pH 7.4), while unmodified nanogels exhibited sustained-release and TMOD gels displayed intermediate behavior. The results in (D) and (E) indicated that the nanogels’ zeta potential is largely predictive for their release profile [all panels: n = 4, mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001, two-way analysis of variance (ANOVA) with Tukey posttest].

  • Fig. 5 Relative uptake of unmodified, TMOD, and DMOD nanogels by murine fibroblasts, murine macrophages, and human colon epithelial carcinoma cells.

    The relative uptake was computed by normalizing the green fluorescent protein (GFP) (nanoparticle) signal to the slope of the calibration curve and then normalizing that value to the 4′,6-diamidino-2-phenylindole (DAPI) (cell nucleus) signal. Note that the y axis quantities differ between plots, as the DMOD nanogels were uptaken in significantly greater quantity than TMOD or unmodified nanogels. (A to C) Relative uptake of unmodified, TMOD, or DMOD fluorescent nanogels by each cell line, as a function of dose (24-hour exposure). (D to F) Kinetic uptake of unmodified, TMOD, and DMOD nanogels (400 μg/ml dose). Representative images for each plot are given in fig. S10 (all panels, n = 4, mean ± SEM).

  • Fig. 6 Gold nanoparticle precipitation and photothermal therapy.

    Gold nanoparticles were precipitated in DMOD nanogels. DMOD gels with a 0.39:1 ratio of N,N-dimethylethylenediamine:methacrylic acid or less were unable to facilitate gold nanomaterial formation. (A) Absorbance spectra of composite nanogels containing gold nanoparticles. (B) Transmission electron micrographs of gold nanomaterials within 3.1:1 DMOD nanogels. Arrows point to gold nanoparticles. (C) Proof of concept for the composite nanogels’ ability to transduce visible light (λ = 532 nm) into heat. DMOD (3.1:1) nanogels with gold nanoparticles effectively and rapidly heated a 1× PBS suspension. (D) Concentration-dependent photothermal activity of 3.1:1 DMOD–gold nanoparticle composites (n = 4, mean ± SD).

  • Fig. 7 Coupling efficiency and bioactivity of peptide and protein-nanogel conjugates.

    (A) A thiol-maleimide click reaction effectively conjugated cysteine-containing peptides to the nanogel network. (B) A carboxylic acid–amine reaction linked the peptides’ N terminus with the carboxylic acid–containing nanogels. (C) Differential incorporation of diverse peptides was explained by their net charge at physiological pH. (D) Nanogel conjugation at 2 wt % did not significantly alter the nanogel diameter or zeta potential. (E) Peptide content in the final conjugate product can be readily tuned by altering the peptide feed concentration via reaction scheme (B). (F) Horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) were incorporated into nanogels with 56.4 and 81% efficiency, respectively. (G) HRP retained 66.5 ± 33% of its activity upon conjugation to the nanogel platform, as evidenced by the ability of HRP-nanogel conjugates to convert TMB substrate. (H) WGA-NP conjugates retained native WGA activity, as they bound and stained the cell membrane of L929 murine fibroblasts (blue, DAPI stain of nucleus; red, WGA-NP conjugates) (C to F, n = 3, mean ± SD; G and H, n = 3, representative data).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax7946/DC1

    Fig. S1. Proof of similarity for degradable and nondegradable P(AAm-co-MAA) nanogels.

    Fig. S2. Nanogel swelling and degradation analysis with QCM.

    Fig. S3. FTIR analysis of N,N-dimethylethylenediamine– or tyramine-conjugated nanogels.

    Fig. S4. Potentiometric titration analysis of DMOD and TMOD nanogels.

    Fig. S5. Full data of pH-responsive, modified nanogel swelling, including data for aggregated nanogels.

    Fig. S6. Methylene blue loading in DMOD and TMOD nanogels.

    Fig. S7. Nanogel cytotoxicity to murine fibroblasts, as determined by MTS and LDH assays.

    Fig. S8. Cytotoxicity of degradable, nondegradable, and degraded nanogels to fibroblasts, macrophages, and colon epithelial cells.

    Fig. S9. Cytotoxicity of fluorescent TMOD, DMOD, or unmodified (Fluor) nanogels.

    Fig. S10. Representative images for dose-response and kinetic nanogel uptake.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Proof of similarity for degradable and nondegradable P(AAm-co-MAA) nanogels.
    • Fig. S2. Nanogel swelling and degradation analysis with QCM.
    • Fig. S3. FTIR analysis of N,N-dimethylethylenediamine– or tyramine-conjugated nanogels.
    • Fig. S4. Potentiometric titration analysis of DMOD and TMOD nanogels.
    • Fig. S5. Full data of pH-responsive, modified nanogel swelling, including data for aggregated nanogels.
    • Fig. S6. Methylene blue loading in DMOD and TMOD nanogels.
    • Fig. S7. Nanogel cytotoxicity to murine fibroblasts, as determined by MTS and LDH assays.
    • Fig. S8. Cytotoxicity of degradable, nondegradable, and degraded nanogels to fibroblasts, macrophages, and colon epithelial cells.
    • Fig. S9. Cytotoxicity of fluorescent TMOD, DMOD, or unmodified (Fluor) nanogels.
    • Fig. S10. Representative images for dose-response and kinetic nanogel uptake.

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