Universal Surface-Initiated Polymerization of Antifouling Zwitterionic Brushes Using a Mussel-Mimetic Peptide Initiator

We report a universal method for the surface-initated polymerization (SIP) of an antifouling polymer brush on various classes of surfaces, including noble metals, metal oxides, and inert polymers. Inspired by the versatility of mussel adhesive proteins, we synthesized a novel bifunctional tripeptide bromide (Br<u>Y</u>K<u>Y</u>) that combines atom-transfer radical polymerization (ATRP) initiating alkyl bromide with l-3,4-dihydroxyphenylalanine (DOPA) and lysine. The simple dip-coating of substrates with variable wetting properties and compositions, including Teflon, in a Br<u>Y</u>K<u>Y</u> solution at pH 8.5 led to the formation of a thin film of cross-linked Br<u>Y</u>K<u>Y</u>. Subsequently, we showed that the Br<u>Y</u>K<u>Y</u> layer initiated the ATRP of a zwitterionic monomer, sulfobetaine methacrylate (SBMA), on all substrates, resulting in high-density antifouling pSBMA brushes. Both Br<u>Y</u>K<u>Y</u> deposition and pSBMA grafting were unambiguously confirmed by ellipsometry, X-ray photoelectron spectroscopy, and goniometry. All substrates that were coated with Br<u>Y</u>K<u>Y</u>/pSBMA dramatically reduced bacterial adhesion for 24 h and also resisted mammalian cell adhesion for at least 4 months, demonstrating the long-term stability of the Br<u>Y</u>K<u>Y</u> anchoring and antifouling properties of pSBMA. The use of Br<u>Y</u>K<u>Y</u> as a primer and polymerization initiator has the potential to be widely employed in surface-grafted polymer brush modifications for biomedical and other applications

Enzymatically Degradable Mussel-Inspired Adhesive Hydrogel

Mussel-inspired adhesive hydrogels represent innovative candidate medical sealants or glues. In the present work, we describe an enzyme-degradable mussel-inspired adhesive hydrogel formulation, achieved by incorporating minimal elastase substrate peptide Ala-Ala into the branched poly­(ethylene glycol) (PEG) macromonomer structure. The system takes advantage of neutrophil elastase expression upregulation and secretion from neutrophils upon recruitment to wounded or inflamed tissue. By integrating adhesive degradation behaviors that respond to cellular cues, we expand the functional range of our mussel-inspired adhesive hydrogel platforms. Rapid (<1 min) and simultaneous gelation and adhesion of the proteolytically active, catechol-terminated precursor macromonomer was achieved by addition of sodium periodate oxidant. Rheological analysis and equilibrium swelling studies demonstrated that the hydrogel is appropriate for soft tissue-contacting applications. Notably, hydrogel storage modulus (<i>G</i>′) achieved values on the order of 10 kPa, and strain at failure exceeded 200% strain. Lap shear testing confirmed the material’s adhesive behavior (shear strength: 30.4 ± 3.39 kPa). Although adhesive hydrogel degradation was not observed during short-term (27 h) in vitro treatment with neutrophil elastase, in vivo degradation proceeded over several months following dorsal subcutaneous implantation in mice. This work represents the first example of an enzymatically degradable mussel-inspired adhesive and expands the potential biomedical applications of this family of materials

Two-photon luminescence imaging.

<p>Two-photon luminescence (TPL) confocal imaging of MCF-7 and MDA-MB-231 breast cancer cells 24 h post treatment with NRs. Brightfield (BF, grayscale) and AuNR TPL images (red) reveal specific targeting in MUC1⁺ MCF-7 cells. Scale bar = 50 μm.</p

Darkfield imaging.

<p>AuNR uptake by cells detected by darkfield scattering imaging of MUC1-overexpressing SCC15 oral or MCF-7 breast cancer cells following treatment with MUC1-modified NRs. Scale bar = 50 μm.</p

Photothermal therapy of breast and oral cancer cells.

<p>Photoablation of cells with NKT Photonics SuperK Versa near-infrared (NIR) light source (480–850 nm). Cells were imaged with calcein AM (live, green) and propidium iodide (dead, red) to visualize photoablation efficiency following treatment with increasing doses of AuNRs. Due to the delamination of dead cells, no red staining is present in the treated regions of interest. In the MCF-7 cell line, this dead staining was occasionally observed prior to delamination (Fig I in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.s001" target="_blank">S1 File</a>). Scale bar = 100 μm.</p

MUC1-Targeted Cancer Cell Photothermal Ablation Using Bioinspired Gold Nanorods

<div><p>Recent studies have highlighted the overexpression of mucin 1 (MUC1) in various epithelial carcinomas and its role in tumorigenesis. These mucins present a novel targeting opportunity for nanoparticle-mediated photothermal cancer treatments due to their unique antenna-like extracellular extension. In this study, MUC1 antibodies and albumin were immobilized onto the surface of gold nanorods using a “primer” of polydopamine (PD), a molecular mimic of catechol- and amine-rich mussel adhesive proteins. PD forms an adhesive platform for the deposition of albumin and MUC1 antibodies, achieving a surface that is stable, bioinert and biofunctional. Two-photon luminescence confocal and darkfield scattering imaging revealed targeting of MUC1-BSA-PD-NRs to MUC1⁺ MCF-7 breast cancer and SCC-15 squamous cell carcinoma cells lines. Treated cells were exposed to a laser encompassing the near-infrared AuNR longitudinal surface plasmon and assessed for photothermal ablation. MUC1-BSA-PD-NRs substantially decreased cell viability in photoirradiated MCF-7 cell lines vs. MUC1- MDA-MB-231 breast cancer cells (p < 0.005). Agents exhibited no cytotoxicity in the absence of photothermal treatment. The facile nature of the coating method, combined with targeting and photoablation efficacy, are attractive features of these candidate cancer nanotherapeutics.</p></div

Schematic illustration of MUC1 antibody-conjugated gold NRs (anti-MUC1-BSA-PD-NRs) with a polydopamine adlayer and BSA coating.

<p>MUC1 antibodies serve as novel targeting constructs in the application of plasmonic photothermal therapy (A). Probing MUC1 targeting at underglycosylated N- and C-terminal domains in various epithelial carcinomas will expand our cancer-targeting repertoire with the potential for synergistic therapeutic effects (B, adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.ref012" target="_blank">12</a>]).</p

Quantification of breast and oral cancer photothermal therapy.

<p>Analysis of normalized viable cell areas reveals MUC1-dependent therapy vs. non-targeted BSA-PD-NRs in MCF-7 and SCC15 cell lines (* p < 0.05, ** p < 0.01, *** p < 0.001). Two-way ANOVA reveals a statistically significant impact of dose (p < 0.0001) and treatment (p < 0.0001). The interaction of dose and treatment was found to be statistically significant only for MCF-7 cells (p < 0.001).</p

Bovine serum albumin (BSA) coating on polydopamine-primed gold NRs.

<p>(A) Electron microscopy of BSA-PD-NRs in secondary electron mode. Scale bar = 600 nm; Inset = 50 nm; (B) Circular dichroism of modified vs. unmodified gold NRs. BSA-modified NRs were modified with 10 or 20 mg/mL BSA, as indicated. The concentration of the BSA control was 0.25 mg/mL. Protein denaturation into β-sheet formation is indicated on PD-treated NRs modified with a sub-optimal concentration of BSA. Otherwise, BSA secondary structure is preserved, as quantified by the respective α-helical propensity of each modification.</p

Catechol Polymers for pH-Responsive, Targeted Drug Delivery to Cancer Cells

A novel cell-targeting, pH-sensitive polymeric carrier was employed in this study for delivery of the anticancer drug bortezomib (BTZ) to cancer cells. Our strategy is based on facile conjugation of BTZ to catechol-containing polymeric carriers that are designed to be taken up selectively by cancer cells through cell surface receptor-mediated mechanisms. The polymer used as a building block in this study was poly(ethylene glycol), which was chosen for its ability to reduce nonspecific interactions with proteins and cells. The catechol moiety was exploited for its ability to bind and release borate-containing therapeutics such as BTZ in a pH-dependent manner. In acidic environments, such as in cancer tissue or the subcellular endosome, BTZ dissociates from the polymer-bound catechol groups to liberate the free drug, which inhibits proteasome function. A cancer-cell-targeting ligand, biotin, was presented on the polymer carriers to facilitate targeted entry of drug-loaded polymer carriers into cancer cells. Our study demonstrated that the cancer-targeting drug–polymer conjugates dramatically enhanced cellular uptake, proteasome inhibition, and cytotoxicity toward breast carcinoma cells in comparison with nontargeting drug–polymer conjugates. The pH-sensitive catechol–boronate binding mechanism provides a chemoselective approach for controlling the release of BTZ in targeted cancer cells, establishing a concept that may be applied in the future toward other boronic acid-containing therapeutics to treat a broad range of diseases