Degradable Crosslinker Research Articles

Smart Polymeric Nanocarriers of Met-enkephalin.

This study describes a novel approach to polymeric nanocarriers of the therapeutic peptide met-enkephalin based on the aggregation of thermoresponsive polymers. Thermoresponsive bioconjugate poly((di(ethylene glycol) monomethyl ether methacrylate)-ran-(oligo(ethylene glycol) monomethyl ether methacrylate) is synthesized by AGET ATRP using modified met-enkephalin as a macroinitiator. The abrupt heating of bioconjugate water solution leads to the self-assembly of bioconjugate chains and the formation of mesoglobules of controlled sizes. Mesoglobules formed by bioconjugates are stabilized by coating with cross-linked two-layer shell via nucleated radical polymerization of N-isopropylacrylamide using a degradable cross-linker. The targeting peptide RGD, containing the fluorescence marker carboxyfluorescein, is linked to a nanocarrier during the formation of the outer shell layer. In the presence of glutathione, the whole shell is completely degradable and the met-enkephalin conjugate is released. It is anticipated that precisely engineered nanoparticles protecting their cargo will emerge as the next-generation platform for cancer therapy and many other biomedical applications.

Controlled release of anti-inflammatory peptides from reducible thermosensitive nanoparticles suppresses cartilage inflammation

Characterized by pain, cartilage degradation, and inflammation, osteoarthritis is often treated with anti-inflammatory therapies that provide short-term relief but can have adverse side effects; intra-articular drug delivery systems with controlled release of anti-inflammatory peptides using degradable poly(N-isopropylacrylamide) (pNIPAM) nanoparticles could prolong relief and minimize these side effects. Nanoparticles provide a biocompatible drug carrier that can protect encapsulated therapeutics from enzymatic degradation and increase payload delivery upon encountering a degradation stimulus. Here we demonstrate passive targeting of inflamed cartilage ex vivo by uptake of PEGylated pNIPAM nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS) into chondrocytes and subsequent intracellular release of an anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK). The KAFAK-loaded NGPEGSS treatment reduced ex vivo inflammation to a greater extent compared to its non-degradable counterparts. This study highlights a nanoparticle system that delivers therapeutics intracellularly with improved efficacy by triggered degradation and suppresses inflammation in multiple cell types within an inflamed joint.

Differential regulation of angiogenesis using degradable VEGF-binding microspheres

Vascular endothelial growth factor (VEGF) spatial and temporal activity must be tightly controlled during angiogenesis to form perfusable vasculature in a healing wound. The native extracellular matrix (ECM) regulates growth factor activity locally via sequestering, and researchers have used ECM-mimicking approaches to regulate the activity of VEGF in cell culture and in vivo. However, the impact of dynamic, affinity-mediated growth factor sequestering has not been explored in detail with biomaterials. Here, we sought to modulate VEGF activity dynamically over time using poly(ethylene glycol) microspheres containing VEGF-binding peptides (VBPs) and exhibiting varying degradation rates. The degradation rate of VBP microspheres conferred a differential ability to up- or down-regulate VEGF activity in culture with primary human endothelial cells. VBP microspheres with fast-degrading crosslinks reduced VEGF activity and signaling, while VBP microspheres with no inherent degradability sequestered and promoted VEGF activity in culture with endothelial cells. VBP microspheres with degradable crosslinks significantly reduced neovascularization in vivo, but neither non-degradable VBP microspheres nor bolus delivery of soluble VBP reduced neovascularization. The covalent incorporation of VBP to degradable microspheres was required to reduce neovascularization in a mouse model of choroidal neovascularization in vivo, which demonstrates a potential clinical application of degradable VBP microspheres to reduce pathological angiogenesis. The results herein highlight the ability to modulate the activity of a sequestered growth factor by changing the crosslinker identity within PEG hydrogel microspheres. The insights gained here may instruct the design and translation of affinity-based growth factor sequestering biomaterials for regenerative medicine applications.

Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. Statement of SignificanceHuman induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured in synthetic hydrogels self-assemble into capillary networks through mechanisms consistent with in vivo vascular morphogenesis.

Microgels as vehicles for effective delivery of hydrophilic drugs to the brain

Event Abstract Back to Event Microgels as vehicles for effective delivery of hydrophilic drugs to the brain Madeline Simpson1, Justin Piazza2, Yogesh K. Katare2, Sharnpreet K. Kooner2, Niels B. Smeets1, Ram K. Mishra2 and Todd Hoare1 1 McMaster University, Department of Chemical Engineering, Canada 2 McMaster University, Department of Psychiatry and Behavioural Neuroscience, Canada Introduction: The treatment of central nervous system (CNS) disorders is challenging due to difficulties in drug transport across the blood-brain barrier (BBB). Nanoparticle-based therapies are of interest as they can be designed for specific targets, to control drug release, and lower the required dosage for therapeutic intervention. Degradable microgels (hydrogel-based nanoparticles) composed of poly(oligoethylene glycol methacrylate) (POEGMA), are promising delivery vehicles which have demonstrated protein-repellent properties[1]. In comparison to “hard” nanoparticles widely cited to cross the BBB with diameters of <150 nm[2], we show that larger elastic microgels can be transported across the BBB, which can improve drug loading and biodistribution of the hydrophilic drug 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide) (PAOPA). This drug is a positive allosteric modulator that selectively targets the dopamine D2 receptor and has been demonstrated to reduce the negative symptoms of schizophrenia in animal models[3]. Materials and Methods: POEGMA-based microgels were synthesized by precipitation polymerization of diethylene glycol methacrylate, acrylic acid (AA) and 2-hydroxyethyl disulfide dimethacrylate (degradable crosslinker) with various quantities of sodium dodecyl sulfate (SDS). The AA groups were functionalized using carbodiimide chemistry with Solanum tuberosum lectin (STL) which enables binding to the nasal epithelium for intranasal delivery. Microgel characterisation was carried out by light scattering (size), transmission electron microscopy (morphology) and conductometric titration (functional group content). PAOPA was passively loaded, with excess drug removed by centrifugation. The in vitro drug release was assessed using HPLC, and microgel cytotoxicity was determined by an MTT assay using RPMI 2650 nasal septum and SH-SY5Y dopamine 2D receptor-transduced neuronal cells. Rhodamine-labelled microgels were injected into the peritoneum of male Sprague-Dawley rats to assess the in vivo biodistribution of different microgel sizes. Blood and tissues were recovered after one hour and analyzed using fluorescence to quantify microgel biodistribution. Results and Discussion: Microgels with sizes of 99±2 nm, 152±3 nm, and 249±4 nm can be prepared with low polydipsersity (<0.1). PAOPA was loaded with an encapsulation efficiency of 30±2%, independent of size. The diffusion of PAOPA from the two smallest microgels occured over four days, with 50% of the drug still retained after 24 hours (Figure 1) while the largest microgel prolonged release to five days. The biodistribution studies with the rhodamine-labelled microgels demonstrated that the smallest microgels were the most effective at crossing the BBB; however the largest microgels were transported in significant numbers despite their larger size than the 150 nm limit cited while also accumulating less in major non-target organs (Figure 2). In vivo efficacy studies as a function of microgel size in a rat schitzophrenia model will be presented. Conclusion: POEGMA microgels are effective delivery vehicles for the transport of hydrophilic drugs across the BBB. Due to their elastic nature, larger microgels may be advantageous as the compressibility may enable the delivery of bigger, longer circulating, and more highly drug loaded particles across tight biological barriers. Canadian Institutes for Health Research (CIHR); Natural Sciences and Engineering Research Council of Canada (NSERC)

Glycosaminoglycan-based hydrogels with tunable sulfation and degradation for anti-inflammatory small molecule delivery

Event Abstract Back to Event Glycosaminoglycan-based hydrogels with tunable sulfation and degradation for anti-inflammatory small molecule delivery Liane Tellier1*, Yifeng Peng1* and Johnna S. Temenoff1, 2* 1 Georgia Institute of Technology and Emory University, Coulter Department of Biomedical Engineering, United States 2 Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, United States Introduction: Crystal violet (CV) is a small molecule shown to have anti-bacterial, anti-fungal & more recently, anti-inflammatory properties in part by acting as a Nox & angiopoietin-2 (ang-2) inhibitor[1]. Though treatment for many inflammatory conditions would ideally be delivered with zero order release kinetics, this remains particularly challenging for small molecule drugs such as CV. Therefore, to tune CV release kinetics we have developed a CV-loaded glycosaminoglycan (GAG)-based hydrogel system with heparin (a highly sulfated GAG) of varying sulfation levels (affects CV affinity) & with varying amounts of degradable crosslinker (affects hydrogel degradation). It is hypothesized that more sulfated heparin hydrogels will release bioactive CV over a longer period than more desulfated heparin hydrogels & that varying hydrogel degradation will allow us to further tune the rate of CV release. Methods: Hydrogels were prepared via free radical polymerization with 90 wt% water, 9 wt% PEG diacrylate (PEGDA), 1 wt% heparin, & 0, 30, or 42 mol% hydrolytically degradable crosslinker (DTT) for non-, slow-, & fast-degrading hydrogels, respectively. N-desulfated (Hep-N), 6O,N-desulfated (Hep-N,-6O) & fully desulfated (Hep-) heparin were prepared & functionalized with methacrylamide groups. CV was preloaded (20mg) into the hydrogel precursor prior to crosslinking. To quantify CV release, hydrogel supernatant was removed & the amount of CV was measured with a plate reader (absorption at 590nm) every 2 days. CV bioactivity was assessed by incubating hydrogel supernatant diluted to ~20μM with bEnd.3 cells & quantifying bEnd.3 ang-2 gene expression via PCR. Results: CV release kinetics were dependent on heparin sulfation level, whereby more sulfated heparin hydrogels (Hep&Hep-N) exhibited a linear release profile, whereas more desulfated hydrogels (Hep-N,-6O&Hep-) released significantly more CV at Day 1, followed by more plateaued release over 9-15 days (Fig1). Moreover, slow-degrading hydrogels required at least 2 additional days to release an equivalent amount of CV as fast-degrading hydrogels with the same heparin derivative. Finally, after incubating hydrogel releasate with bEnd.3 cells, bEnd.3 ang-2 gene expression was significantly lower than the no treatment control & was not significantly different from the 20μM soluble CV, indicating that CV maintained its bioactivity after release from hydrogels (Fig2). Conclusion: We have fabricated GAG-based hydrogels with heparin derivatives of varying sulfation levels (alters CV release kinetics) & with varying concentrations of DTT (alters hydrogel degradation & CV release rate). In this way, we have developed a highly tailorable hydrogel platform & have achieved near zero-order release of bioactive CV in particular formulations. Thus, in the future, this system can be utilized in vivo with the unique advantage of releasing small molecule anti-inflammatories such as CV in a sustained manner over a period of weeks. We would like to acknowledge NIH R01 AR063692.

Integrin specific hydrogels as lymphoid tumors organoids for B cell receptor signaling and drug resistance

Event Abstract Back to Event Integrin specific hydrogels as lymphoid tumors organoids for B cell receptor signaling and drug resistance Ye F. Tian1, Haelee Ahn2, Rebecca Schneider3, Shao Ning Yang2, Lidia Roman-Gonzalez2, Leandro Cerchietti2, Ari M. Melnick2 and Ankur Singh1 1 Cornell University, Mechanical and Aerospace Engineering, United States 2 Weill Cornell Medical College, Division of Hematology and Oncology, Department of Medicine, United States 3 Cornell University, School of Chemical and Biomolecular Engineering, United States Introduction: Non-Hodgkin lymphomas (NHL) are a heterogeneous group of lymphoproliferative disorders of B and T cell origin. Pre-clinical research in NHL has relied on testing compounds with suspension cultures of human lymphoma cell lines, without taking into account the lymphoid niche in which these tumors arise and reside. This traditional approach for drug testing risks losing potential useful compounds by considering them as ineffective due to the conditions of the assays. This is the case for compounds with a mechanism of action that relies on suppressing survival signals from microenvironmental cues, such as integrin inhibitors. Our recent findings have demonstrated that knockdown or pharmacologic inhibition of the integrin αvβ3 abrogated the proliferation of malignant T cells in vitro and in patient-derived xenograft mice models, an effect partially mediated by defective angiogenesis[1]. Given the increasing importance of the ECM and the stromal cells to NHL biology and drug response, there is a need to develop 3D tissues that mimic the diseased lymphoid microenvironment and are adaptable to disease-specific needs. Method: Human B- and T-NHL cell lines were encapsulated in organoids of 4-arm Maleimide functionalized polyethylene glycol (PEG-MAL) and thiolated, enzymatically degradable crosslinker peptides along with human tonsil tissue derived follicular dendritic cells (FDC). Prior to organoid formation, PEGMAL was functionalized with thiolated peptides targeting αvβ3 and α4β1 on B and T-NHLs. The survival, proliferation, and drug studies were performed with confocal microscopy and biochemical assays. For drug-response analysis, organoids were cultured for 4 days before the 24 hr of exposure to Doxorubicin or Panobinostat, a histone deacetylate inhibitor and apoptosis studies were performed. Results: Our gene expression analysis shows that while αvβ3 integrin is consistently overexpressed in T-NHLs, these cells also express varying levels of α4β1[2]. B-NHLs on the other hand, do not have a predictable pattern of integrin expression. When NHLs were presented with integrin ligands in the organoids, both B and T-NHLs formed large clusters as a function of integrin ligands (αvβ3 ligand RGD > α4β1 ligand REDV). Importantly, REDV peptide significantly increased B-NHL proliferation while T-NHL proliferation was dependent on RGD signaling. Both B and T-NHLs, when cultured in organoids, demonstrated drug-resistance to Doxorubicin and the Panobinostat (Fig 1). We observed the 3D microenvironment to upregulate IgM B cell receptors BCR expression level (Fig. 2), which indicate enhanced survival and possibly explains the enhanced proliferation observed with B cell lymphomas. We predicted that activation of BCR is likely supporting the survival of B NHLs in 3D niches over 2D cultures by means of both up-regulating BCR and cross-talk with integrins. This hypothesis was supported by our findings with Syk inhibition that caused significantly higher apoptosis in the 3D group as compared to the 2D groups. Conclusions: Taken together, these results emphasize the role of biomaterials-based 3D tissues and the importance of integrin mediated signaling in malignant B and T cell tumors, as well as the role of FDCs in lymphoma proliferation. In the past, these factors have been largely ignored in drug evaluation or mechanistic studies of B and T cell lymphomas, ex vivo. Giorgio Inghirami

Gradually softening hydrogels for modeling hepatic stellate cell behavior during fibrosis regression.

The extracellular matrix (ECM) presents an evolving set of mechanical cues to resident cells. We developed methacrylated hyaluronic acid (MeHA) hydrogels containing both stable and hydrolytically degradable crosslinks to provide cells with a gradually softening (but not fully degradable) milieu, mimicking physiological events such as fibrosis regression. To demonstrate the utility of this cell culture system, we studied the phenotype of rat hepatic stellate cells, the major liver precursors of fibrogenic myofibroblasts, within this softening environment. Stellate cells that were mechanically primed on tissue culture plastic attained a myofibroblast phenotype, which persisted when seeded onto stiff (∼20 kPa) hydrogels. However, mechanically primed stellate cells on stiff-to-soft (∼20 to ∼3 kPa) hydrogels showed reversion of the myofibroblast phenotype over 14 days, with reductions in cell area, expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA), and Yes-associated protein/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) nuclear localization when compared to stellate cells on stiff hydrogels. Cells on stiff-to-soft hydrogels did not fully revert, however. They displayed reduced expression of glial fibrillary acidic protein (GFAP), and underwent abnormally rapid re-activation to myofibroblasts in response to re-stiffening of the hydrogels through introduction of additional crosslinks. These features are typical of stellate cells with an intermediate phenotype, reported to occur in vivo with fibrosis regression and re-injury. Together, these data suggest that mechanics play an important role in fibrosis regression and that integrating dynamic mechanical cues into model systems helps capture cell behaviors observed in vivo.

Influence of Novel Crosslinker on the Properties of the Degradable Thermosensitive Hydrogels

SummaryA novel degradable crosslinker, N, N′‐bis(acryloyl) cystamine (BACy), was synthesized with cystamine dihydrochloride (Cy) and acryloyl chloride. Then three series of degradable thermosensitive hydrogels with different crosslinker contents were prepared from N‐isopropyl acrylamide (NIPAAm), BACy (Bx‐series) or N, N′‐methylene‐bis‐acrylamide (NMBA)(Nx‐series) or equimolar BACy and NMBA (BNx‐series) as crosslinker, and diethoxyacetophenone (DEAP) as photoinitiator under UV‐irradiation polymerization. The effect of crosslinker content on the properties of the hydrogels such as swelling behavior and mechanical properties was investigated. At the same time, their degradabilities were investigated by dissolving them into different concentrations of cysteine solution. The results show that the swelling ratios for the Bx‐series hydrogels are lower than those for the Nx‐series hydrogels. But, after degradation in the cysteine solution, the swelling ratios for the Bx‐series hydrogels were greater than those for the Nx‐series hydrogels. When the crosslinker content was increased, all values of shear stress (τ), shear modulus (G), and effective crosslinking density (ρX) increased. But, the ρX values for the reduced Bx series and BNx series hydrogels were lower than those for original hydrogels. Results also showed that the ρX values for the Nx series hydrogel were not affected by cysteine. The disulfide bonds of BACy in these novel hydrogels were decomposed (reduced) in the cysteine solution and converted into thiol group. These thiol groups could be used to absorb some metal ions such as silver ion. Hence, the absorption of silver ion for the hydrogel with thiol group was also investigated in this research.

Thermosensitive Ionic Microgels with pH Tunable Degradation via in Situ Quaternization Cross-Linking

Degradable thermosensitive ionic microgels were synthesized via surfactant-free emulsion polymerization (SFEP) of N-isopropylacrylamide (NIPAm) and 1-vinylimidazole (VIM) at 70 °C with degradable 1,4-phenylene bis(4-bromobutanoate) or 1,6-hexanediol bis(2-bromopropionate) as quaternized cross-linkers. VIM could be quaternized by 1,4-phenylene bis(4-bromobutanoate) or 1,6-hexanediol bis(2-bromopropionate), leading to the formation of degradable cross-linking network and ionic microgels. Combined techniques of transmission electron microscopy (TEM), dynamic light scattering (DLS), electrophoretic light scattering (ELS), UV–vis spectroscopy, FT-IR spectra, and gel permeation chromatography (GPC) were employed to systematically investigate the sizes, morphologies, and properties of the obtained microgels before and after degradation as well as the degradation mechanism. The obtained microgels were spherical in shape with narrow size distribution and exhibited thermosensitive behavior and controllable degradat...

Release of anti-inflammatory peptides from thermosensitive nanoparticles with degradable cross-links suppresses pro-inflammatory cytokine production.

Pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) are mediators in the development of many inflammatory diseases. To demonstrate that macrophages take up and respond to thermosensitive nanoparticle drug carriers, we synthesized PEGylated poly(N-isopropylacrylamide-2-acrylamido-2-methyl-1-propanesulfonate) particles cross-linked with degradable disulfide (N,N'-bis(acryloyl)cystamine) (NGPEGSS). An anti-inflammatory peptide (KAFAK) was loaded and released from the thermosensitive nanoparticles and shown to suppress levels of TNF-α and IL-6 production in macrophages. Cellular uptake of fluorescent, thermosensitive, and degradable nanoparticles and therapeutic efficacy of free KAFAK peptide compared to that of KAFAK loaded in PEGylated degradable thermosensitive nanoparticles were examined. The data suggests that the degradable, thermosensitive nanoparticles loaded with KAFAK may be an effective tool to treat inflammatory diseases.

RAFT-mediated control of nanogel structure and reactivity: Chemical, physical and mechanical properties of monomer-dispersed nanogel compositions

ObjectiveThis study examines how nanogel structure correlates with photopolymerization and key polymer properties upon addition of nanogels with latent reactivity into a monomer dispersant to produce polymer/polymer composites. MethodsTwo nanogels that retained RAFT functionality based on the synthetic approach were prepared to have different branching densities. These reactive nanogels were dispersed in triethylene glycol dimethacrylate at 0–40wt%. Reaction kinetics, volumetric shrinkage and shrinkage stress associated with the photopolymerization of nanogel-modified formulations were measured in real time with mechanical properties of the polymers also evaluated. The basic structure of RAFT-derived nanogel particles was examined by the preparation of a separate nanogel constructed with degradable disulfide crosslinking groups. The model nanogel molecular weight and polydispersity were compared before and after degradation. ResultsDespite the controlled radical synthetic approach, the nanogels, which are composed of multiple interconnected, short primary chains, presented relatively high polydispersity. Through addition of the reactive nanogels to a monomer that both infiltrates and disperses the nanogels, the photopolymerization rate was moderately reduced with the increase of nanogel loading levels. Volumetric shrinkage decreased proportionally with nanogel concentration; however, a greater than proportional reduction of polymerization-induced stress was observed. Mechanical properties, such as flexural strength, storage modulus were maintained at the same levels as the control resin for nanogel systems up to 40wt%. SignificanceThis study demonstrated that beyond the use of RAFT functionality to produce discrete nano-polymeric structures, the residual chain end groups are important to maintain reactivity and mechanical properties of nanogel-modified resin materials.

CdS nanorods assisted thermal oxidation of polythiol segments of PS-b-polythiols to produce core cross-linking micellar clusters

In this paper, we described a new and simple method to produce degradable core cross-linking (CCL) micellar clusters that utilized CdS nanorods to assist thermal oxidation of the polythiol segment of a diblock copolymer, polystyrene-b-polythiol (PS-b-polythiol), at high concentration (2.3 wt%) in a common organic solvent (toluene). The CdS nanorods acted not only as catalysts, but also as templates. The diblock copolymer, PS-b-polythiol, was obtained by aminolysis of polystyrene-b-poly(2-(ethyl-xanthate)ethylmethacrylate) (PS-b-P1), a block copolymer synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization using a macroRAFT transfer agent, PS-CTA, and a methacrylate monomer that possessed a protected thiol group, in the presence of n-butylamine and tributyl phosphine. The CdS nanorods were synthesized conveniently by thermolysis of an alkyl xanthate, Cd-HDX. The TEM image clearly showed the formation of micellar clusters and the oxidation procedure was investigated by 1H-NMR and XPS. The formation of core cross-linking (CCL) micelles was confirmed to be reversible by reduction utilizing DTT as the reducing agent.

Mesenchymal stem cell and gelatin microparticle encapsulation in thermally and chemically gelling injectable hydrogels for tissue engineering.

In this work, we investigated the viability and osteogenic differentiation of mesenchymal stem cells encapsulated with gelatin microparticles (GMPs) in an injectable, chemically and thermally gelling hydrogel system combining poly(N-isopropylacrylamide)-based thermogelling macromers containing pendant epoxy rings with polyamidoamine-based hydrophilic and degradable diamine crosslinking macromers. Specifically, we studied how the parameters of GMP size and loading ratio affected the viability and differentiation of cells encapsulated within the hydrogel. We also examined the effects of cell and GMP co-encapsulation on hydrogel mineralization. Cells demonstrated long-term viability within the hydrogels, which was shown to depend on GMP size and loading ratio. In particular, increased interaction of cells and GMPs through greater available GMP surface area, use of an epoxy-based chemical gelation mechanism, and the tunable high water content of the thermogelled hydrogels led to favorable long-term cell viability. Compared with cellular hydrogels without GMPs, hydrogels co-encapsulating cells and GMPs demonstrated greater production of alkaline phosphatase by cells at all time-points and a transient early enhancement of hydrogel mineralization for larger GMPs at higher loading ratios. Such injectable, in situ forming hydrogels capable of delivering and maintaining populations of encapsulated mesenchymal stem cells and promoting mineralization in vitro offer promise as novel therapies for applications in tissue engineering and regenerative medicine.

A degradable polycyclic cross-linker for UV-curing nanoimprint lithography

In this paper, we design and synthesize a degradable polycyclic diacrylate cross-linker, 2,10-diacryloyloxymethyl-1,4,9,12-tetraoxaspiro[4.2.4.2] tetradecane (DAMTT), from easily available starting materials by bis-ketalization of 1,4-cyclohexanedione with glycerol, followed by esterification with acryloyl choride. The chemical structure of the degradable cross-linker is characterized by FTIR, 1H NMR and GC-MS. A UV-NIL resist based on DAMTT has a very low shrinkage (about 2.5%) and a high Young's modulus of 1.89 GPa after UV-curing, and is soluble in acidic media. Patterns with sub-10 nm resolution are faithfully imprinted into the DAMTT film. The degradable cross-linker is incorporated with a multifunctional acrylated siloxane to increase O2 RIE resistance as an etch barrier for high aspect ratio pattern transfer. The cross-linked film of the UV-curable resist remains soluble in acidic media even though its formulation contains acrylated siloxane up to 50% (w/w). Moreover, in contrast to most UV-NIL resists which are insoluble in solvents after curing, the DAMTT resist can independently achieve a lift-off process without the assistance of a thermoplastic polymer layer. That makes it possible to fabricate metal nanostructures on highly curved surfaces via a hybrid UV-curing nanoimprint technique. For example, metal gratings are successfully fabricated on the cylindrical surface of an optical fiber via a lift-off process.

Synthesis of hydrophilic intra-articular microspheres conjugated to ibuprofen and evaluation of anti-inflammatory activity on articular explants

The main limitation of current microspheres for intra-articular delivery of non-steroidal anti-inflammatory drugs (NSAIDs) is a significant initial burst release, which prevents a long-term drug delivery. In order to get a sustained delivery of NSAIDs without burst, hydrogel degradable microspheres were prepared by co-polymerization of a methacrylic derivative of ibuprofen with oligo(ethylene-glycol) methacrylate and poly(PLGA-PEG) dimethacrylate as degradable crosslinker. Microspheres (40-100 μm) gave a low yield of ibuprofen release in saline buffer (≈2% after 3 months). Mass spectrometry analysis confirmed that intact ibuprofen was regenerated indicating that ester hydrolysis occurred at the carboxylic acid position of ibuprofen. Dialysis of release medium followed by alkaline hydrolysis show that in saline buffer ester hydrolysis occurred at other positions in the polymer matrix leading to the release of water-soluble polymers (>6-8000 Da) conjugated with ibuprofen showing that degradation and drug release are simultaneous. By considering the free and conjugated ibuprofen, 13% of the drug is released in 3 months. In vitro, ibuprofen-loaded MS inhibited the synthesis of prostaglandin E2 in articular cartilage and capsule explants challenged with lipopolysaccharides. Covalent attachment of ibuprofen to PEG-hydrogel MS suppresses the burst release and allows a slow drug delivery for months and the cyclooxygenase-inhibition property of regenerated ibuprofen is preserved.

RNA replicon delivery via lipid-complexed PRINT protein particles.

Herein we report the development of a nonviral lipid-complexed PRINT (particle replication in nonwetting templates) protein particle system (LPP particle) for RNA replicon delivery with a view toward RNA replicon-based vaccination. Cylindrical bovine serum albumin (BSA) particles (diameter (d) 1 μm, height (h) 1 μm) loaded with RNA replicon and stabilized with a fully reversible disulfide cross-linker were fabricated using PRINT technology. Highly efficient delivery of the particles to Vero cells was achieved by complexing particles with a mixture of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids. Our data suggest that (1) this lipid-complexed protein particle is a promising system for delivery of RNA replicon-based vaccines and (2) it is necessary to use a degradable cross-linker for successful delivery of RNA replicon via protein-based particles.

Synthesis of degradable polyHIPEs by AGET ATRP

Conditions to synthesize a degradable polymerized high internal phase emulsion (PolyHIPE) by a controlled radical polymerization (CRP) method, atom transfer radical polymerization (ATRP), have been developed for the first time. Activators generated by electron transfer (AGET) ATRP were used to copolymerize 2-ethylhexyl methacrylate (EHMA) and bis(2-methacryloyloxyethyl) disulfide (DSDMA), a degradable cross-linker, in a HIPE system without the removal of oxygen. These conditions provided control over the polymerization and resulted in a fully degradable material. The degradation product had molecular weight Mn = 30,500, and relatively low dispersity, Mw/Mn = 1.6, indicating that a uniform cross-linked network was formed. Superior control over the polymerization resulted from the selection of a highly hydrophobic ATRP catalyst, CuBr2 with an N,N-bis(2-pyridylmethyl)octadecylamine (BPMODA) ligand, which is preferentially located in the organic phase. Alternate conditions with a less hydrophobic catalyst, CuBr2 with a 2,2′-bipyridine (bpy) ligand, result in a non-degradable polyHIPE. The CuBr2/bpy catalyst predominately partitions into the aqueous phase where it exerts little control over the polymerization. The polyHIPE synthesized using a CuBr2/BPMODA catalyst also exhibited a higher Young's modulus and was stiffer, due to its more uniform cross-linked network, than materials from almost identical HIPEs synthesized using a CuBr2/bpy catalyst.

Abstract 1397: BMP4 inhibits the proliferation of breast cancer cells in 3D and induces a migratory phenotype in MDA-MB-231 cells.

Abstract Bone morphogenetic protein 4 (BMP4) is an extracellular signaling molecule that belongs to the bone morphogenetic protein (BMP) family, which in turn is part of the transforming growth factor β (TGF-β) -superfamily. BMPs regulate cell proliferation, differentiation and motility, and have also been shown to be involved in cancer pathogenesis. We have previously shown that BMP4 is able to consistently reduce breast cancer cell proliferation through G1 cell cycle arrest and simultaneously to induce the migration in a subset of these cell lines. In order to study the effects of BMP4 in a more physiological setting, we used two different 3D culture systems: Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel with matrix metalloproteinase (MMP) degradable cross-links and RGD peptides. MCF-10A normal breast epithelial cells formed round acini with a hollow lumen and correct apicobasal localization of α-6 integrin in Matrigel whereas irregular structures without lumen formation or apicobasal integrin localization were seen in PEG gel. Thus the two matrices do not provide identical 3D environments. In Matrigel, BMP4 treatment reduced the proliferation of MCF-10A cells and T-47D breast cancer cells. In PEG gel, reduced growth was also seen in two other breast cancer cell lines, MDA-MB-231 and MDA-MB-361. To clarify the mechanisms behind the growth suppressive effects of BMP4, we first examined the expression levels of 11 known cell cycle regulators in T-47D and MDA-MB-361cells grown in 2D culture using Western blot. Based on its consistent upregulation in both cell lines, p21 was selected for detailed analyses. In 2D culture, BMP4 induced the expression of p21 in all three breast cancer cell lines but not in MCF-10A cells. Unexpectedly, there was no correlation between p21 expression and the BMP4 induced growth suppression in 3D. These data seem to indicate that different mechanisms are involved in BMP4 mediated cell growth regulation in 2D and 3D environments. Finally, in response to BMP4 treatment MDA-MB-231 cells formed large branching, stellate structures in Matrigel, suggestive of increased cell migration. This effect was not seen in PEG gel. Gremlin, a BMP antagonist, reversed the stellate phenotype, confirming that the effect was indeed dependent on BMP4. Similarly, a broad-spectrum MMP inhibitor, Batimastat, was also able to reverse the stellate appearance. These results imply that the actions of MMPs are required for this migratory phenotype. Taken together, our results show that BMP4 reduced the growth of breast epithelial cells and breast cancer cells in 3D culture. In addition, the highly metastatic breast cancer cell line MDA-MB-231 formed stellate, branching structures upon BMP4 treatment in Matrigel. Further research is needed to elucidate the molecular mechanisms behind these BMP4 induced phenotypes and their clinical relevance. Citation Format: Minna E. Ampuja, Riikka Jokimäki, Emma-Leena Alarmo, Kati Juuti-Uusitalo, Anne Kallioniemi. BMP4 inhibits the proliferation of breast cancer cells in 3D and induces a migratory phenotype in MDA-MB-231 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2013-1397

Cell-instructive hydrogels to assemble in biofluids

which mimic fundamental characteristics of naturally occurring extracellular microenvironments, is a key task in the rapidly progressing fields of tissue engineering and regenerative medicine (DOI: 10.1038/nature08602). Importantly, cell-instructive matrix signals – including matrix-associated growth factors, ligands of cellular adhesion receptors, intrinsic viscoelastic properties and susceptibility for cell-driven reorganization – have to be presented in a spatiotemporally adjusted manner (DOI: 10.1002/marc.201200382). For that purpose, the current practice in cell biology and biotechnology mostly relies on reconstituting non-covalently associating biopolymer meshworks (collagen fibrils, Matrigel). Although effective in numerous in vitro approaches to cell signaling, these preparations are hardly compatible with safety requirements for medical applications and are often not suitable for the mechanistic investigation of particular exogenous signals. Recent approaches have therefore aimed at forming covalent polymer hydrogel networks in the presence of cells in culture or even within living tissue. A range of poly(ethylene glycol) (PEG) conjugates with adhesive peptide ligands and/or degradable peptide crosslinkers were converted in cell-compatible crosslinking schemes, including Michael-type addition, triazole formation, enzymatic (Factor XIIIa/ transglutaminase-mediated) crosslinking, and photoinduced polymerization. Amongst these, the Michael-type addition reaction turned out to be particularly powerful, as it does not require a catalyst, has no side products, and proceeds rapidly under physiological conditions (DOI: 10.1002/adma.201103574). The thiol group of cysteine residues can participate as a nucleophile in this reaction, which massively extends the number of suitable substrates and simplifies the attachment of bioactive peptides or proteins. A very common synthetic scheme for the formation of PEG-peptide hydrogels utilizes dithiol-containing peptides to react with PEG-polymers carrying electron deficient double bonds at the terminal groups. Recently, this scheme was further extended by incorporating glycosaminoglycan building blocks for the in situ formation of biohybrid gel matrices, which allows for the versatile biomimetic functionalization of the obtained materials with a plethora of glycosaminoglycan-binding growth factors: Michaeltype reaction schemes were applied for the formation of customized, cell-embedding, PEG-glycosaminoglycan hydrogels with precisely adjusted polymer network properties and independently tunable signaling characteristics (DOI: 10.1002/adma. 201300691). These materials provide a means for the effective modulation of therapeutically relevant cell fate decisions in vivo and are thus expected to become instrumental in medical technologies aimed at regenerating diseased or injured tissues.