Biomimetic Hydrogels Incorporating Polymeric Cell-Adhesive Peptide To Promote the 3D Assembly of Tumoroids

Toward the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of <i>tert</i>-butyl methacrylate (<i>t</i>BMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of <i>tert</i>-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ∼95 μm by day 28. Cells in these structures were viable, formed cell–cell contacts through E-cadherin (E-CAD), and displayed cortical organization of F-actin. Compared with the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids, and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD are a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs

Interfacial Bioorthogonal Cross-Linking

Described herein is interfacial bioorthogonal cross-linking, the use of bioorthogonal chemistry to create and pattern biomaterials through diffusion-controlled gelation at the liquid-gel interface. The basis is a rapid (<i>k</i>₂ 284000 M^–1 s^–1) reaction between strained <i>trans</i>-cyclooctene (TCO) and tetrazine (Tz) derivatives. Syringe delivery of Tz-functionalized hyaluronic acid (HA-Tz) to a bath of bis-TCO cross-linker instantly creates microspheres with a cross-linked shell through which bis-TCO diffuses freely to introduce further cross-linking at the interface. Tags can be introduced with 3D resolution without external triggers or templates. Water-filled hydrogel channels were prepared by simply reversing the order of addition. Prostate cancer cells encapsulated in the microspheres have 99% viability, proliferate readily, and form aggregated clusters. This process is projected to be useful in the fabrication of cell-instructive matrices for in vitro tissue models

Tuning the Properties of Elastin Mimetic Hybrid Copolymers via a Modular Polymerization Method

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly­(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)₂ (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX­(AKAAAKA)₂X (AK2-CBD1) and X­(AKAAAKA)₂XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering

Hyaluronic Acid-Based Hydrogels Containing Covalently Integrated Drug Depots: Implication for Controlling Inflammation in Mechanically Stressed Tissues

Synthetic hydrogels containing covalently integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically cross-linkable block copolymer micelles (<i>x</i>BCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly­(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly­(<i>n</i>-butyl acryclate) (P<i>n</i>BA) were employed as the drug depots and the microscopic cross-linkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HA<i>x</i>BCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of <i>x</i>BCMs. When micelles prepared from the parent PAA-<i>b</i>-P<i>n</i>BA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HA<i>p</i>BCM) were obtained. The addition of <i>x</i>BCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HA<i>x</i>BCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled <i>x</i>BCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of <i>x</i>BCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded <i>x</i>BCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HA<i>x</i>BCM gels was accelerated by intermittently applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HA<i>x</i>BCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds to initiate tissue repair

Sequence and Conformational Analysis of Peptide–Polymer Bioconjugates by Multidimensional Mass Spectrometry

The sequence and helical content of two alanine-rich peptides (AQK18 and GpAQK18, Gp: l-propargylglycine) and their conjugates with poly­(ethylene glycol) (PEG) have been investigated by multidimensional mass spectrometry (MS), encompassing electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) interfaced with tandem mass spectrometry (MS²) fragmentation and shape-sensitive separation via ion mobility mass spectrometry (IM-MS). The composition, sequence, and molecular weight distribution of the peptides and bioconjugates were identified by MS and MS² experiments, which also confirmed the attachment of PEG at the C-terminus of the peptides. ESI coupled with IM-MS revealed the existence of random coil and α-helical conformers for the peptides in the gas phase. More importantly, the proportion of the helical conformation increased substantially after PEG attachment, suggesting that conjugation adds stability to this conformer. The conformational assemblies detected in the gas phase were largely formed in solution, as corroborated by independent circular dichroism (CD) experiments. The collision cross sections (rotationally averaged forward moving areas) of the random coil and helical conformers of the peptides and their PEG conjugates were simulated for comparison with the experimental values deduced by IM-MS in order to confirm the identity of the observed architectures and understand the stabilizing effect of the polymer chain. C-terminal PEGylation is shown to increase the positive charge density and to solvate intramolecular positive charges at the conjugation site, thereby enhancing the stability of α-helices, preserving their conformation and increasing helical propensity

Regulation of Epithelial-to-Mesenchymal Transition Using Biomimetic Fibrous Scaffolds

Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis, and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. Although the molecular mechanism leading to EMT has been well-established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the meshlike structure of the basement membrane with an average fiber diameter of 0.5 and 5 μm were produced via the electrospinning of poly­(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 μm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 μm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon the addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 μm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell–cell adhesion, and up-regulation of mesenchymal markers. In contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 μm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression

CD19-Targeted Nanodelivery of Doxorubicin Enhances Therapeutic Efficacy in B‑Cell Acute Lymphoblastic Leukemia

Nanomedicine has advanced to clinical trials for adult cancer therapy. However, the field is still in its infancy for treatment of childhood malignancies such as acute lymphoblastic leukemia (ALL). Nanotherapy offers multiple advantages over conventional therapy. It facilitates targeted delivery and enables controlled release of drugs to reduce treatment-related side effects. Here, we demonstrate that doxorubicin (DOX) encapsulated in polymeric nanoparticles (NPs) modified with targeting ligands against CD19 (CD19-DOX-NPs) can be delivered in a CD19-specific manner to leukemic cells. The CD19-DOX-NPs were internalized via receptor-mediated endocytosis and imparted cytotoxicity in a CD19-dependent manner in CD19-positive ALL cells. Leukemic mice treated with CD19-DOX-NPs survived significantly longer and manifested a higher degree of agility, indicating reduced apparent systemic toxicity during treatment compared to mice treated with free DOX. We suggest that targeted delivery of drugs used in childhood cancer treatment should improve therapeutic efficacy and reduce treatment-related side effects in children

Hyaluronan (HA) Interacting Proteins RHAMM and Hyaluronidase Impact Prostate Cancer Cell Behavior and Invadopodia Formation in 3D HA-Based Hydrogels

<div><p>To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, “invadopodia”, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.</p> </div

Cells cultured in the HA hydrogel show differences in morphology compared to an agar gel of matched stiffness.

<p>Images of C4-2 cells cultured for zero, two, or five days with 5% FBS in the HA hydrogel or in 0.3% LMP agar. Scale bars represent 200 µm (A). Average elastic (G’) and loss (G”) moduli across one hour time for HA hydrogel (B) or 0.3% LMP agar (C). Error bars = SEM, n = 3.</p

HA hydrogel culture changes expression and localizes RHAMM to the cell surface.

<p>C4-2 cells with 5% FBS on 2D (A) or in 3D HA hydrogel for 3 days (B) immunostained for RHAMM (green) and nuclei (blue). Arrows indicate differences in nuclear RHAMM between the two culture conditions. A quantification method was utilized that measured RHAMM staining intensity across a single line both inside and outside of the nucleus (C). Arrows indicate marked points to average staining intensity between and outside of as depicted in the chart to the right. Results of the quantification method show nuclear and cytoplasmic or membrane (cyto/mem)-bound RHAMM for both culture conditions (D). Western blotting for RHAMM expression after five days of 2D or 3D culture with β-actin used as a load control (E).</p