Morphological control of bundled actin networks subject to fixed-mass depletion.

Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells.

Photothermal Modulation of Dynamic Covalent Poly(ethylene glycol)/PEDOT Composite Hydrogels for On-Demand Drug Delivery.

Hydrogels are cross-linked three-dimensional polymer networks that have tissue-like properties. Dynamic covalent bonds (DCB) can be utilized as hydrogel cross-links to impart injectability, self-healing ability, and stimuli responsiveness to these materials. In our research, we utilized dynamic thiol-Michael bonds as cross-links in poly(ethylene glycol) (PEG)-based hydrogels. Because the equilibrium of the reversible, exothermic thiol-Michael reaction can be modulated by temperature, we investigated the possibility of using thermal and photothermal stimuli to modulate the gel-to-sol transition of these materials with the aim of developing an on-demand pulsatile cargo release system. For this purpose, we incorporated poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles within the hydrogel to facilitate photothermal modulation using near-infrared light. PEDOT nanoparticles of 50 nm in diameter and with strong near-infrared absorption were prepared by oxidative emulsion polymerization. We then used Michael addition of thiol-ene pairs from 4-arm PEG-thiol (PEG-SH) and 4-arm PEG-benzylcyanoacetamide (PEG-BCA) to form dynamically cross-linked hydrogels. PEDOT nanoparticles were entrapped in situ to form Gel/PEDOT composites. Rheology and inverted tube test studies showed that the gel-to-sol transition occurred at 45-50 °C for 5 wt % gels and that this transition could be tailored by varying the wt % of the polymer precursors. The hydrogels were found to be capable of self-healing and being injected with a clinically relevant injection force. Bovine serum albumin-fluorescein isothiocyanate (BSA-FITC), a fluorescently labeled protein, was then loaded into the Gel/PEDOT as a therapeutic mimic. Increased release of BSA-FITC upon direct thermal stimulation and photothermal stimulation with an 808 nm laser was observed. Pulsatile release of BSA-FITC over seven cycles was demonstrated. MTS and live-dead assays demonstrated that Gel/PEDOT was cytocompatible in MDA-MB-231 breast cancer and 3T3 fibroblast cell lines. Further studies demonstrated that the encapsulation and laser-triggered release of the chemotherapeutic agent doxorubicin (DOX) could also be achieved. Altogether, this work advances our understanding of the temperature-dependent behavior of a dynamic covalent hydrogel, Gel/PEDOT, and leverages that understanding for application as a photothermally responsive biomaterial for controlled release.

Shear Thickening Behavior in Injectable Tetra-PEG Hydrogels Cross-Linked via Dynamic Thia-Michael Addition Bonds.

Injectable poly(ethylene glycol) (PEG)-based hydrogels were reversibly cross-linked through thia-conjugate addition bonds and demonstrated to shear thicken at low shear rates. Cross-linking bond exchange kinetics and dilute polymer concentrations were leveraged to tune hydrogel plateau moduli (from 60 to 650 Pa) and relaxation times (from 2 to 8 s). Under continuous flow shear rheometry, these properties affected the onset of shear thickening and the degree of shear thickening achieved before a flow instability occurred. The changes in viscosity were reversible whether the shear rate increased or decreased, suggesting that chain stretching drives this behavior. Given the relevance of dynamic PEG hydrogels under shear to biomedical applications, their injectability was investigated. Injection forces were found to increase with higher polymer concentrations and slower bond exchange kinetics. Altogether, these results characterize the nonlinear rheology of dilute, dynamic covalent tetra-PEG hydrogels and offer insight into the mechanism driving their shear thickening behavior.

Biodegradable microneedle patch for delivery of meloxicam for managing pain in cattle.

Microneedle patches are a promising source for transdermal diffusion of macromolecules and are designed to painlessly penetrate the skin. In this study, a biodegradable chitosan microneedle patch to deliver meloxicam for managing pain in cattle was tested. The potential of reuse of the polymeric solution to fabricate the patches, optimization of fabrication, morphological analysis of the microneedle patch and analysis of preservation of the chemical composition after sterilization were evaluated. In-vitro analysis consisted of studying in-vitro penetration mechanical properties, compression testing analysis of microneedle patch, and in-vitro drug release analysis. In-vivo studies were performed to analyze the dissolution capability of the microneedle patch. Results regarding the physical characteristics, chemical composition, and mechanical properties confirmed that rheological properties of the chitosan solution, present significant differences over time, demonstrating that reusing the solution on the fourth day results in failure patches. Morphological characteristics and chemical composition studies revealed that the process of sterilization (ethylene oxide gas) needed for implanting the patches into the skin did not affect the properties of microneedle patches. In-vitro studies showed that approximately 33.02 ± 3.88% of the meloxicam was released over 7 days. A full penetration of the microneedles into the skin can be obtained by applying approximately 3.2 N. In-vivo studies demonstrated that microneedle patches were capable of swelling and dissolving, exhibiting a dissolution percentage of more than 50% of the original height of microneedle after 7 days. No abnormal tissue, swelling, or inflammation was observed in the implanted area. The results of this work show that chitosan biodegradable microneedle patches may be useful to deliver meloxicam to improve pain management of cattle with positive effects for commercial manufacturing.

Electro-hydrodynamic extraction of DNA from mixtures of DNA and bovine serum albumin.

We report separation of genomic DNA (48 kbp) from bovine serum albumin (BSA) by the electro-hydrodynamic coupling between a pressure-driven flow and a parallel electric field. Electro-hydrodynamic extraction exploits this coupling to trap DNA molecules at the entrance of a microfluidic contraction channel, while allowing proteins and salts to be flushed from the device. Samples (10 µL) containing λ-DNA (1 ng) and BSA (0.3 mg) were injected directly into the device and convected to the contraction channel entrance by a flowing buffer solution. The DNA remains trapped in this region essentially indefinitely, while proteins and salts are eluted. The effectiveness of the concept has been assessed by fluorescence measurements of DNA and BSA concentrations. Electro-hydrodynamic extraction in a single-stage device was found to enhance the concentration of DNA 40-fold, while reducing the BSA concentration by four orders of magnitude. The relative concentrations of DNA to BSA at the contraction channel entrance can be as large as 1.5 : 1, corresponding to an A260/280 ratio of 1.9. The maximum yield of DNA from a salt-free solution is 50%, while salted (150 mM) solutions have a lower yield (38%).