Ultrapermeable Gel Membranes Enabling Superior Carbon Capture.

Membrane technology is rapidly gaining broad attraction as a viable alternative for carbon capture to mitigate increasingly severe global warming. Emerging CO2 -philic membranes have become crucial players in efficiently separating CO2 from light gases, leveraging their exceptional solubility-selectivity characteristics. However, economic and widespread deployment is greatly dependent on the boosted performance of advanced membrane materials for carbon capture. Here, we design a unique gel membrane composed of CO2 -philic molecules for accelerating CO2 transportation over other gases for ultrapermeable carbon capture. The molecular design of such soft membranes amalgamates the advantageous traits of augmented permeation akin to liquid membranes and operational stability akin to solid membranes, effectively altering the membrane's free volume characteristics validated by both experiments and molecular dynamics simulation. Surprisingly, gas diffusion through the free-volume-tuned gel membrane undergoes a 9-fold improvement without compromising the separation factor for the superior solubility selectivity of CO2 -philic materials, and CO2 permeability achieves a groundbreaking record of 5608 Barrer surpassing the capabilities of nonfacilitated CO2 separation materials and exceeding the upper bound line established in 2019 even by leading-edge porous polymer materials. Our designed gel membrane can maintain exceptional separation performance during prolonged operation, enabling the unparalleled potential of solubility-selective next-generation materials towards sustainable carbon capture.

MoleGear: A Java-Based Platform for Evolutionary De Novo Molecular Design.

A Java-based platform, MoleGear, is developed for de novo molecular design based on the chemistry development kit (CDK) and other Java packages. MoleGear uses evolutionary algorithm (EA) to explore chemical space, and a suite of fragment-based operators of growing, crossover, and mutation for assembling novel molecules that can be scored by prediction of binding free energy or a weighted-sum multi-objective fitness function. The EA can be conducted in parallel over multiple nodes to support large-scale molecular optimizations. Some complementary utilities such as fragment library design, chemical space analysis, and graphical user interface are also integrated into MoleGear. The candidate molecules as inhibitors for the human immunodeficiency virus 1 (HIV-1) protease were designed by MoleGear, which validates the potential capability for de novo molecular design.

Time-varying economic dominance in financial markets: A bistable dynamics approach.

By developing a continuous-time heterogeneous agent financial market model of multi-assets traded by fundamental and momentum investors, we provide a potential mechanism for generating time-varying dominance between fundamental and non-fundamental in financial markets. We show that investment constraints lead to the coexistence of a locally stable fundamental steady state and a locally stable limit cycle around the fundamental, characterized by a Bautin bifurcation. This provides a mechanism for market prices to switch stochastically between the two persistent but very different market states, leading to the coexistence and time-varying dominance of seemingly controversial efficient market and price momentum over different time periods. The model also generates other financial market stylized facts, such as spillover effects in both momentum and volatility, market booms, crashes, and correlation reduction due to cross-sectional momentum trading. Empirical evidence based on the U.S. market supports the main findings. The mechanism developed in this paper can be used to characterize time-varying economic dominance in economics and finance in general.

Mammalian PER2 regulates AKT activation and DNA damage response.

PER2 is a key mammalian circadian clock protein. It also has a tumor suppressive function. Down regulation of PER2 in the cultured cancer cells accelerates cell proliferation, while overexpression of PER2 inhibits cell growth and induces apoptosis. The Per2 mutant mice have a cancer prone phenotype and an altered DNA damage response. Here we report that PER2 regulates AKT activity. Cells with down-regulated PER2 expression have prolonged high levels of AKT T308 phosphorylation after growth factor stimulation or DNA damage. PER2 down-regulation delays DNA damage induced Chk2 activation and overrides DNA damage induced apoptosis and cell cycle arrest.

Combined effect of osteopontin and BMP-2 derived peptides grafted to an adhesive hydrogel on osteogenic and vasculogenic differentiation of marrow stromal cells.

The objective of this work was to investigate the combined effect of grafting the peptide corresponding to amino acid residues 162-168 of osteopontin (OPD peptide) and the peptide corresponding to amino acid residues 73-92 of bone morphogenetic protein-2 (BMP peptide) to an RGD-conjugated inert hydrogel on osteogenic and vasculogenic differentiation of bone marrow stromal (BMS) cells. RGD-conjugated three-dimensional (3D) porous hydrogel scaffolds with well-defined cylindrical pore geometry were produced from sacrificial wax molds fabricated by fused deposition modeling rapid prototyping system. Propargyl acrylate and 4-pentenal were conjugated to the hydrogel for orthogonal grafting of BMP and OPD peptides by click reaction and oxime ligation, respectively. The OPD peptide was grafted by the reaction between aminooxy moiety of aminooxy-mPEG-OPD (mPEG = mini-poly(ethylene glycol)) and the aldehyde moiety in the hydrogel. The BMP peptide was grafted by the reaction between the azide moiety of Az-mPEG-BMP and the propargyl moiety in the hydrogel. The hydrogels seeded with BMS cells were characterized by biochemical, immunocytochemical, and mRNA analyses. Groups included RGD control hydrogel (RGD), RGD and BMP peptides without OPD (RGD+BMP), RGD and BMP peptides with mutant OPD (RGD+BMP+mOPD), and RGD and BMP peptides with OPD (RGD+BMP+OPD) grafted hydrogels. The extent of mineralization of RGD, RGD+BMP, RGD+BMP+mOPD, and RGD+BMP+OPD groups after 28 days was 650 ± 70, 990 ± 30, 850 ± 30, and 1150 ± 40 mg/(mg of DNA), respectively, indicating that the BMP and OPD peptides enhanced osteogenic differentiation of the BMS cells. The BMS cells seeded on RGD+BMP+OPD grafted hydrogels stained positive for vasculogenic markers α-SMA, PECAM-1, and VE-cadherin while the groups without OPD peptide (RGD+BMP and RGD+BMP+mOPD) stained only for α-SMA but not PECAM-1 or VE-cadherin. These results were consistent with the significantly higher PECAM-1 mRNA expression for RGD+BMP+OPD group after 21 and 28 days, compared to the groups without OPD. These findings suggest that the RGD+BMP+OPD peptides provide a favorable microenvironment for concurrent osteogenic and vasculogenic differentiation of progenitor marrow-derived cells.

Gelation characteristics and osteogenic differentiation of stromal cells in inert hydrolytically degradable micellar polyethylene glycol hydrogels.

The use of poly(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly(ethylene glycol-co-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly(ethylene glycol-co-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, 100% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer (<10 wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of bone morphogenetic protein-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine.

Preparation and Characterization of Polyvinylalcohol/Polysulfone Composite Membranes for Enhanced CO2/N2 Separation.

The unique properties of polyvinyl alcohol (PVA) and polysulfone (PSf), such as good membrane-forming ability and adjustable structure, provide a great opportunity for CO2-separation membrane development. This work focuses on the fabrication of PVA/PSf composite membranes for CO2/N2 separations. The membranes prepared by coating a 7.5 wt% PVA on top of PSf substrate showed a relatively thin selective layer of 1.7 µm with an enhanced CO2/N2 selectivity of 78, which is a ca. 200% increase compared to the pure PSf membranes. The CO2/N2 selectivity decreases at a rapid rate with the increase of feed pressure from 1.8 to 5 bar, while the CO2 permeance shows a slight reduction, which is caused by the weakening of coupling transportation between water and CO2 molecules, as well as membrane compaction at higher pressures. Increasing operating temperature from 22 °C to 50 °C leads to a slight decrease in CO2 permeance, but a significant reduction in the CO2/N2 selectivity from 78 to 27.1. Moreover, the mass transfer coefficient of gas molecules is expected to increase at a higher velocity, which leads to the increase of CO2 permeance at higher feed flow rates. It was concluded that the CO2 separation performance of the prepared membranes was significantly dependent on the membrane operating parameters, and process design and optimization are crucial to bringing CO2-separation membranes for industrial applications in post-combustion carbon capture.

Synthesis and Gelation Characteristics of Photo-Crosslinkable Star Poly(ethylene oxide-co-lactide-glycolide acrylate) Macromonomers.

Viability of encapsulated cells in situ crosslinkable macromonomers depends strongly on the minimum concentration of polymerization initiators and monomers required for gelation. Novel 4-arm poly(ethylene oxide-co-lactide-glycolide acrylate) (SPELGA) macromonomers were synthesized and characterized with respect to gelation, sol fraction, degradation, and swelling in aqueous solution. SPELGA macromonomers were crosslinked in the absence of N-vinyl-2-pyrrolidone (NVP) monomer to produce a hydrogel network with a shear modulus of 27±4 kPa. The shear modulus of the gels increased by 170-fold as the macromonomer concentration was increased from 10 to 25 wt%. Sol fraction ranged between 8-18%. Addition of only 0.4 mol% NVP to the polymerization mixture increased modulus by 2.2-fold from 27±4 (no NVP) to 60±10 kPa. The higher modulus was attributed to the dilution effect of polymer chains in the sol, by delaying the onset of diffusion-controlled reaction, and cross-propagation of the growing chains with network-bound SPELGA acrylates. Degradation of SPELGA gels depended on water content and density of hydrolytically degradable ester groups.

Carbon hollow fiber membranes for a molecular sieve with precise-cutoff ultramicropores for superior hydrogen separation.

Carbon molecular sieve (CMS) membranes with rigid and uniform pore structures are ideal candidates for high temperature- and pressure-demanded separations, such as hydrogen purification from the steam methane reforming process. Here, we report a facile and scalable method for the fabrication of cellulose-based asymmetric carbon hollow fiber membranes (CHFMs) with ultramicropores of 3-4 Å for superior H2 separation. The membrane fabrication process does not require complex pretreatments to avoid pore collapse before the carbonization of cellulose precursors. A H2/CO2 selectivity of 83.9 at 130 °C (H2/N2 selectivity of >800, H2/CH4 selectivity of >5700) demonstrates that the membrane provides a precise cutoff to discriminate between small gas molecules (H2) and larger gas molecules. In addition, the membrane exhibits superior mixed gas separation performances combined with water vapor- and high pressure-resistant stability. The present approach for the fabrication of high-performance CMS membranes derived from cellulose precursors opens a new avenue for H2-related separations.

MoDoop: An Automated Computational Approach for COSMO-RS Prediction of Biopolymer Solubilities in Ionic Liquids.

An automated computational framework (MoDoop) was developed to predict the biopolymer solubilities in ionic liquids (ILs) on the basis of conductor-like screening model for real solvents calculations of two thermodynamic properties: logarithmic activity coefficient (ln γ) at infinite dilution and excess enthalpy (H E) of mixture. The calculation was based on the optimized two-dimensional structures of biopolymer models and ILs by searching the lowest-energy conformer and optimizing molecular geometry. Three lignin models together with one IL dataset were used to evaluate the prediction ability of the developed method. The evaluation results show that ln γ is a more reliable property to predict lignin solubilities in ILs and the p-coumaryl alcohol model is considered as the best model to represent lignin molecules. The developed MoDoop approach is efficient for rapid in silico screening of suitable ionic liquids to dissolve biopolymers.

Tissue-specific shRNA delivery: a novel approach for gene therapy in cancer.

A novel tissue-specific shRNA delivery system has been developed using cre-lox technology. Conditionally silenced pSico vector containing oligonucleotides of CD44shRNA and tissue-specific promoter-driven Cre-recombinase expression vector are packaged into transferrin-coated nanoparticles that can deliver shRNA into specific tumors. This system has strong potential in cancer therapy.

Process Simulation and Cost Evaluation of Carbon Membranes for CO2 Removal from High-Pressure Natural Gas.

Natural gas sweetening is required to remove the acid gas CO2 to meet gas grid specifications. Membrane technology has a great potential in this application compared to the state-of-the-art amine absorption technology. Carbon membranes are of particular interest due to their high CO2/CH4 selectivity of over 100. In order to document the advantages of carbon membranes for natural gas (NG) sweetening, HYSYS simulation and cost evaluation were conducted in this work. A two-stage carbon membrane process with recycling in the second stage was found to be technically feasible to achieve >98% CH4 with <2% CH4 loss. The specific natural gas processing cost of 1.122 × 10-2 $/m³ sweet NG was estimated at a feed pressure of 90 bar, which was significantly dependent on the capital-related cost. Future work on improving carbon membrane performance is required to increase the competitiveness of carbon membranes for natural gas sweetening.

Spinning Cellulose Hollow Fibers Using 1-Ethyl-3-methylimidazolium Acetate⁻Dimethylsulfoxide Co-Solvent.

The mixture of the ionic liquid 1-ethyl-3-methylimidazolium acetate (EmimAc) and dimethylsulfoxide (DMSO) was employed to dissolve microcrystalline cellulose (MCC). A 10 wt % cellulose dope solution was prepared for spinning cellulose hollow fibers (CHFs) under a mild temperature of 50 °C by a dry⁻wet spinning method. The defect-free CHFs were obtained with an average diameter and thickness of 270 and 38 µm, respectively. Both the XRD and FTIR characterization confirmed that a crystalline structure transition from cellulose I (MCC) to cellulose II (regenerated CHFs) occurred during the cellulose dissolution in ionic liquids and spinning processes. The thermogravimetric analysis (TGA) indicated that regenerated CHFs presented a similar pyrolysis behavior with deacetylated cellulose acetate during pyrolysis process. This study provided a suitable way to directly fabricate hollow fiber carbon membranes using cellulose hollow fiber precursors spun from cellulose/(EmimAc + DMSO)/H2O ternary system.

Fabrication of Defect-Free Cellulose Acetate Hollow Fibers by Optimization of Spinning Parameters.

Spinning of cellulose acetate (CA) with the additive polyvinylpyrrolidone (PVP) in N-methyl-2-pyrrolidone (NMP) solvent under different conditions was investigated. The spinning parameters of air gap, bore fluid composition, flow rate of bore fluid, and quench bath temperature were optimized based on the orthogonal experiment design (OED) method and multivariate analysis. FTIR and scanning electron microscopy were used to characterize the membrane structure and morphology. Based on the conjoint analysis in Statistical Product and Service Solutions (SPSS) software, the importance of these parameters was identified as: air gap > bore fluid composition > flow rate of bore fluid > quench bath temperature. The optimal spinning condition with the bore fluid (water + NMP (85%)), air gap (25 mm), flow rate of bore fluid (40% of dope rate), and temperature of quench bath (50 °C) was identified to make high PVP content, symmetric cross-section and highly cross-linked CA hollow fibers. The results can be used to guide the spinning of defect-free CA hollow fiber membranes with desired structures and properties as carbon membrane precursors.

Solid-phase synthesis of reactive peptide crosslinker by selective deprotection.

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.

Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization.

Purification and characterization of a cyclooxygenase-2 and angiogenesis suppressing factor produced by human fibroblasts.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays an important role in several pathophysiological processes, including inflammation, angiogenesis, and tumorigenesis. We have recently observed that COX-2 induction is restrained in proliferating fibroblasts. The mechanism by which this occurs is unclear. Here, we report the detection and isolation from the conditioned medium of proliferating fibroblasts a factor that suppressed COX-2 expression. This factor, which was named cytoguardin, suppressed COX-2 protein levels induced by phorbol 12-myristate 13-acetate, interleukin-1beta, tumor necrosis factor alpha, and lipopolysaccharide (LPS) in fibroblasts and LPS-induced COX-2 protein levels and promoter activities in human endothelial cells and murine RAW 264.7 cells in a comparable concentration-dependent manner. It inhibited COX-2 expression induced by angiogenic factors and endothelial tube formation induced by angiogenic factors and colon cancer cell medium. These findings provide evidence for the control of COX-2 transcription by an endogenous cellular factor.

Effect of grafting BMP2-derived peptide to nanoparticles on osteogenic and vasculogenic expression of stromal cells.

Bone morphogenetic protein-2 (BMP2) plays a major role in initiating the cascade of osteogenesis. However, high doses of exogenous BMP2 coupled with diffusion away from the intended site cause adverse side-effects. An alternative is to use biodegradable polymeric nanoparticles (NPs) grafted with peptides of the active domains of BMP2. NPs present a multivalent form of the peptide for stronger interaction with cell surface receptors, leading to a stronger activation of osteogenic signalling pathways. The objective of this work was to compare osteogenic activity of the BMP2 peptide (BMP2Pe), corresponding to residues 73-92 of BMP2 protein (BMP2Pr), grafted to biodegradable NPs with that of BMP2 protein (BMP2Pr). BMP2Pe was functionalized with a cysteine residue and grafted to poly(lactide fumarate) and poly(lactide-co-ethylene oxide fumarate) (PLAF/PLEOF) NPs via a thioether link. The calcium content of bone marrow stromal (BMS) cells cultured in osteogenic medium supplemented with BMP2 peptide/protein-grafted NPs (BMP2Pe-gNP and BMP2Pr-gNP) was slightly higher than other BMP2-treated groups, but all osteogenic groups showed similar levels of mineralization after 21 days. The expression pattern of master transcription factors Dlx5 and Runx2 indicated that BMP2 protein induced faster osteogenic signalling than the BMP peptide. The expression level of Osteopontin (OP), Osteocalcin (OC) and PECAM-1 in the NP-grafted BMP2 groups was significantly higher than those of ungrafted BMP2Pr and BMP2Pe groups, which may be due to a more effective presentation of the peptide/protein to cell surface receptors, thus leading to a stronger interaction of the peptide/protein with clustered cell surface receptors.

Drug release kinetics, cell uptake, and tumor toxicity of hybrid VVVVVVKK peptide-assembled polylactide nanoparticles.

An exciting approach to tumor delivery is encapsulation of the drug in self-assembled polymer-peptide nanoparticles. The objective of this work was to synthesize a conjugate of low molecular weight polylactide (LMW PLA) and V6K2 peptide and investigate self-assembly, drug release kinetics, cell uptake and toxicity, drug pharmacokinetics, and tumor cell invasion with Doxorubicin (DOX) or paclitaxel (PTX). The results for PLA-V6K2 self-assembled NPs were compared with those of polyethylene glycol stabilized PLA (PLA-EG) NPs. The size of PLA-V6K2 and PLA-EG NPs was 100 ± 20 and 130 ± 50 nm, respectively, with polydispersity index of 1.04 and 1.14. The encapsulation efficiency of DOX in PLA-V6K2 and PLA-EG NPs was 44 ± 9% and 55 ± 5%, respectively, and that of PTX was >90 for both NP types. The release of DOX and PTX from PLA-V6K2 was slower than that of PLA-EG, and the release rate was relatively constant with time. Based on molecular dynamic simulation, the less hydrophobic DOX was distributed in the lactide core as well as the peptide shell, while the hydrophobic PTX was localized mainly to the lactide core. PLA-V6K2 NPs had significantly higher cell uptake by 4T1 mouse breast carcinoma cells compared to PLA-EG NPs, which was attributed to the electrostatic interactions between the peptide and negatively charged moieties on the cell membrane. PLA-V6K2 NPs showed no toxicity to marrow stromal cells. DOX-loaded PLA-V6K2 NPs showed higher toxicity to 4T1 cells and the DNA damage response, and apoptosis was delayed compared to the free DOX. DOX or PTX encapsulated in PLA-V6K2 NPs significantly reduced invasion of 4T1 cells compared to those cells treated with the drug in PLA-EG NPs. Invasion of 4T1 cells treated with DOX in PLA-V6K2 and PLA-EG NPs was 5 ± 1% and 30 ± 5%, respectively, and that of PTX was 11 ± 2% and 40 ± 7%. The AUC of DOX in PLA-V6K2 NPs was 67% and 21% higher than those of free DOX and PLA-EG NPs, respectively. DOX-loaded PLA-V6K2 NPs injected in C3HeB/FeJ mice inoculated with MTCL syngeneic breast cancer cells displayed higher tumor toxicity than PLA-EG NPs and lower host toxicity than the free DOX. Cationic PLA-V6K2 NPs with higher tumor toxicity than the PLA-EG NPs are potentially useful in chemotherapy.

Three-dimensional-engineered matrix to study cancer stem cells and tumorsphere formation: effect of matrix modulus.

Maintenance of cancer stem cells (CSCs) is regulated by the tumor microenvironment. Synthetic hydrogels provide the flexibility to design three-dimensional (3D) matrices to isolate and study individual factors in the tumor microenvironment. The objective of this work was to investigate the effect of matrix modulus on tumorsphere formation by breast cancer cells and maintenance of CSCs in an inert microenvironment without the interference of other factors. In that regard, 4T1 mouse breast cancer cells were encapsulated in inert polyethylene glycol diacrylate hydrogels and the effect of matrix modulus on tumorsphere formation and expression of CSC markers was investigated. The gel modulus had a strong effect on tumorsphere formation and the effect was bimodal. Tumorsphere formation and expression of CSC markers peaked after 8 days of culture. At day 8, as the matrix modulus was increased from 2.5 kPa to 5.3, 26.1, and 47.1 kPa, the average tumorsphere size changed from 37±6 µm to 57±6, 20±4, and 12±2 µm, respectively; cell number density in the gel changed from 0.8±0.1×105 cells/mL to 1.7±0.2×105, 0.4±0.1×105, and 0.2±0.1×105 cells/mL after initial encapsulation of 0.14×105 cells/mL; and the expression of CD44 breast CSC marker changed from 17±4-fold to 38±9-, 3±1-, and 2±1-fold increase compared with the initial level. Similar results were obtained with MCF7 human breast carcinoma cells. Mouse 4T1 and human MCF7 cells encapsulated in the gel with 5.3 kPa modulus formed the largest tumorspheres and highest density of tumorspheres, and had highest expression of breast CSC markers CD44 and ABCG2. The inert polyethylene glycol hydrogel can be used as a model-engineered 3D matrix to study the role of individual factors in the tumor microenvironment on tumorigenesis and maintenance of CSCs without the interference of other factors.