Polymeric Microbubble Shell Engineering: Microporosity as a Key Factor to Enhance Ultrasound Imaging and Drug Delivery Performance.

Microbubbles (MB) are widely used as contrast agents for ultrasound (US) imaging and US-enhanced drug delivery. Polymeric MB are highly suitable for these applications because of their acoustic responsiveness, high drug loading capability, and ease of surface functionalization. While many studies have focused on using polymeric MB for diagnostic and therapeutic purposes, relatively little attention has thus far been paid to improving their inherent imaging and drug delivery features. This study here shows that manipulating the polymer chemistry of poly(butyl cyanoacrylate) (PBCA) MB via temporarily mixing the monomer with the monomer-mimetic butyl cyanoacetate (BCC) during the polymerization process improves the drug loading capacity of PBCA MB by more than twofold, and the in vitro and in vivo acoustic responses of PBCA MB by more than tenfold. Computer simulations and physisorption experiments show that BCC manipulates the growth of PBCA polymer chains and creates nanocavities in the MB shell, endowing PBCA MB with greater drug entrapment capability and stronger acoustic properties. Notably, because BCC can be readily and completely removed during MB purification, the resulting formulation does not include any residual reagent beyond the ones already present in current PBCA-based MB products, facilitating the potential translation of next-generation PBCA MB.

Engineering poly(dehydroalanine)-based gels via droplet-based microfluidics: from bulk to microspheres.

Biomedical applications such as drug delivery, tissue engineering, and functional surface coating rely on switchable adsorption and desorption of specialized guest molecules. Poly(dehydroalanine), a polyzwitterion containing pH-dependent positive and negative charges, shows promise for such reversible loading, especially when integrated into a gel network. Herein, we present the fabrication of poly(dehydroalanine)-derived gels of different size scales and evaluate them with respect to their practical use in biomedicine. Already existing protocols for bulk gelation were remodeled to derive suitable reaction conditions for droplet-based microfluidic synthesis. Depending on the layout of the microfluidic chip, microgels with a size of approximately 30 µm or 200 µm were obtained, whose crosslinking density can be increased by implementing a multi-arm crosslinker. We analyzed the effects of the crosslinker species on composition, permeability, and softness and show that the microgels exhibit advantageous properties inherent to zwitterionic polymer systems, including high hydrophilicity as well as pH- and ionic strength-sensitivity. We demonstrate pH-regulated uptake and release of fluorescent model dyes before testing the adsorption of a small antimicrobial peptide, LL-37. Quantification of the peptide accommodated within the microgels reveals the impact of size and crosslinking density of the microgels. Biocompatibility of the microgels was validated by cell tests.

Transferrin Receptor-Targeted Nonspherical Microbubbles for Blood-Brain Barrier Sonopermeation.

Microbubbles (MB) are widely used for ultrasound (US) imaging and drug delivery. MB are typically spherically shaped, due to surface tension. When heated above their glass transition temperature, polymer-based MB can be mechanically stretched to obtain an anisotropic shape, endowing them with unique features for US-mediated blood-brain barrier (BBB) permeation. It is here shown that nonspherical MB can be surface-modified with BBB-specific targeting ligands, thereby promoting binding to and sonopermeation of blood vessels in the brain. Actively targeted rod-shaped MB are generated via 1D stretching of spherical poly(butyl cyanoacrylate) MB and via subsequently functionalizing their shell with antitransferrin receptor (TfR) antibodies. Using US and optical imaging, it is demonstrated that nonspherical anti-TfR-MB bind more efficiently to BBB endothelium than spherical anti-TfR-MB, both in vitro and in vivo. BBB-associated anisotropic MB produce stronger cavitation signals and markedly enhance BBB permeation and delivery of a model drug as compared to spherical BBB-targeted MB. These findings exemplify the potential of antibody-modified nonspherical MB for targeted and triggered drug delivery to the brain.

Cellular Architects at Work: Cells Building their Own Microgel Houses.

Microporous annealed particle (MAP) scaffolds are investigated for their application as injectable 3D constructs in the field of regenerative medicine and tissue repair. While available MAP scaffolds provide a stable interlinked matrix of microgels for cell culture, the infiltration depth and space for cells to grow inside the scaffolds is pre-determined by the void fraction during the assembly. In the case of MAP scaffolds fabricated from interlinked spherical microgels, a cellularity gradient can be observed with the highest cell density on the scaffold surface. Additionally, the interlinked microgel network limits the ability of cells to remodel their environment, which contradicts native tissue dynamics. In this work, a cell-induced interlinking method for MAP scaffold formation is established, which avoids the necessity of chemical crosslinkers and pre-engineered pores to achieve micro- or macropores in these 3D frameworks. This method enables cells to self-organize with microgels into dynamic tissue constructs, which can be further controlled by altering the microgel properties, the cell/microgel ratio, and well shape. To form a cell-induced interlinked scaffold, the cells are mixed with dextran-based microgels and function as a glue between the microgels, resulting in a more homogenous cell distribution throughout the scaffold with efficient cell-cell interactions.

Varying the Stiffness and Diffusivity of Rod-Shaped Microgels Independently through Their Molecular Building Blocks.

Microgels are water-swollen, crosslinked polymers that are widely used as colloidal building blocks in scaffold materials for tissue engineering and regenerative medicine. Microgels can be controlled in their stiffness, degree of swelling, and mesh size depending on their polymer architecture, crosslink density, and fabrication method-all of which influence their function and interaction with the environment. Currently, there is a lack of understanding of how the polymer composition influences the internal structure of soft microgels and how this morphology affects specific biomedical applications. In this report, we systematically vary the architecture and molar mass of polyethylene glycol-acrylate (PEG-Ac) precursors, as well as their concentration and combination, to gain insight in the different parameters that affect the internal structure of rod-shaped microgels. We characterize the mechanical properties and diffusivity, as well as the conversion of acrylate groups during photopolymerization, in both bulk hydrogels and microgels produced from the PEG-Ac precursors. Furthermore, we investigate cell-microgel interaction, and we observe improved cell spreading on microgels with more accessible RGD peptide and with a stiffness in a range of 20 kPa to 50 kPa lead to better cell growth.

Engineering the Acoustic Response and Drug Loading Capacity of PBCA-Based Polymeric Microbubbles with Surfactants.

Gas-filled microbubbles (MB) are routinely used in the clinic as ultrasound contrast agents. MB are also increasingly explored as drug delivery vehicles based on their ultrasound stimuli-responsiveness and well-established shell functionalization routes. Broadening the range of MB properties can enhance their performance in both imaging and drug delivery applications. This can be promoted by systematically varying the reagents used in the synthesis of MB, which in the case of polymeric MB include surfactants. We therefore set out to study the effect of key surfactant characteristics, such as the chemical structure, molecular weight, and hydrophilic-lipophilic balance on the formation of poly(butyl cyanoacrylate) (PBCA) MB, as well as on their properties, including shell thickness, drug loading capacity, ultrasound contrast, and acoustic stability. Two different surfactant families (i.e., Triton X and Tween) were employed, which show opposite molecular weight vs hydrophilic-lipophilic balance trends. For both surfactant types, we found that the shell thickness of PBCA MB increased with higher-molecular-weight surfactants and that the resulting MB with thicker shells showed higher drug loading capacities and acoustic stability. Furthermore, the higher proportion of smaller polymer chains of the Triton X-based MB (as compared to those of the Tween-based ones) resulted in lower polymer entanglement, improving drug loading capacity and ultrasound contrast response. These findings open up new avenues to fine-tune the shell properties of polymer-based MB for enhanced ultrasound imaging and drug delivery applications.

Functionalized Microgel Rods Interlinked into Soft Macroporous Structures for 3D Cell Culture.

In this work, a two component microgel assembly using soft anisometric microgels that interlink to create a 3D macroporous construct for cell growth is reported. Reactive microgel rods with variable aspect ratio are produced via microfluidics in a continuous plug-flow on-chip gelation method by photoinitiated free-radical polymerization of star-polyethylene glycol-acrylate with glycidyl methacrylate or 2-aminoethyl methacrylate comonomers. The resulting complementary epoxy- and amine-functionalized microgels assemble and interlink with each other via a ring opening reaction, resulting in macroporous constructs with pores up to several hundreds of micrometers. The level of crosslinking depends on the functionalization degree of the microgels, which also affects the stiffness and cell adhesiveness of the microgels when modified with the cell-adhesive GRGDS-PC peptide. Therefore, 3D spreading and growth of cells inside the macroporous structure is influenced not only by the presence of macropores but also by the mechanical and biochemical properties of the individual microgels.