Multi-arm Poly Ethylene Glycol Research Articles

Biomimetic Spider Silk by Crosslinking and Functionalization with Multiarm Polyethylene Glycol

Biomimetic Spider Silk by Crosslinking and Functionalization with Multiarm Polyethylene Glycol

Quantitation of polyethylene glycol by size exclusion chromatography with charged aerosol, differential refractive index, and multi-angle light scattering detectors

Polyethylene glycol (PEG) has found tremendous applications in pharmaceutical products and has played a critical role in PEGylated drug modalities to improve pharmacokinetic properties and biological efficacy. The characterization and quantitation of PEGs are essential to control manufacture process and drug product quality. However, the assay value of PEG could change dramatically depending on the structures of the PEG and the detection techniques used. In this study, we developed a size exclusion chromatographic (SEC) method for quantitative PEG analysis, and we systematically evaluated the performance of three online detectors with different operating principles: a charged aerosol detector (CAD), a differential refractive index (dRI) detector, and a multi-angle light scattering detector (MALS). Fourteen PEG compounds covering a wide range of molecular weight (MW, 1 – 40 kDa) and molecular architectures (linear, branched, Y-shaped and multi-arm geometries) were evaluated by these three detection techniques. Our study revealed that the dRI showed the most universal responses among all the PEGs regardless of their molecular weight or geometries. In the contrast, CAD and MALS detector showed MW-dependent and semi-universal geometry-dependent responses. Another key finding is that the relative response factor for each multi-arm PEG in the CAD and the MALS were inversely correlated, suggesting both can be applied to qualitatively assess polymers of different architectures, including the ones with subtle differences in their core structures. The comparison of the three detectors not only provides the fundamental and comprehensive understanding of PEG quantitation but also enables the process development and control of high-quality PEGs in producing PEGylated therapeutics in the pharmaceutical industry.

P41: 4D Manufacturing: Immunoprotective Materials for Islet Tissue Patches in Cell-Based Diabetes Treatments

Purpose: In order to cure diabetes on a large scale, researchers are working to develop insulin-producing cells differentiated from human pluripotent stem cells, encapsulating the tissues to protect the cells from immune attack, and implanting the cells for regulated insulin secretion. This project explored the synthesis and in vivo performance of a 4D biomaterial intended to protect implanted insulin-producing cells in the body. 3D printing of insulin-producing cells encapsulated within the biomaterial with additional timed-release anti-inflammatory agents enabled complex, personalized shapes and a 4D functional material. Methods: Anti-inflammatory agents were functionalized with varying permanent and degradable linkages to achieve a range of timed-release therapeutics to prevent immune system-mediated rejection. The molecules were crosslinked with a multi-arm polyethylene glycol (PEG) material in the subcutaneous space of immunocompetent C57BL/6 mice. After 30 days, tissue slices of the explant were examined using H&E staining revealing the thickness of biological encapsulation compared to alginate controls. Results: PEG-based implants with acetal linkages enabling the slow release (~30 days) of anti-inflammatory agents achieved the thinnest layer of biological encapsulation compared to the controls. Summary: Collectively, the described 4D biomaterial with other novel approaches and tools will enable the investigation of survival and function of a stem cell-based therapy for diabetes that could ultimately allow patients to live a diabetes-free lifestyle.

Design and characterization of matrix metalloproteinase-responsive hydrogels for the treatment of inflammatory skin diseases.

Enzyme-responsive hydrogels, formed by step growth photopolymerization of biscysteine peptide linkers with alkene functionalized polyethylene glycol, provide interesting opportunities as biomaterials and drug delivery systems. In this study, we developed stimuli-responsive, specific, and cytocompatible hydrogels for delivery of anti-inflammatory drugs for the treatment of inflammatory skin diseases. We designed peptide linkers with optimized sensitivity towards matrix metalloproteinases, a family of proteolytic enzymes overexpressed in the extracellular matrix of the skin during inflammation. The peptide linkers were crosslinked with branched 4-arm and 8-arm polyethylene glycols by thiol-norbornene photopolymerization, leading to the formation of a hydrogel network, in which the anti-inflammatory Janus kinase inhibitor tofacitinib citrate was incorporated. The hydrogels were extensively characterized by physical properties, in vitro release studies, cytocompatibility with fibroblasts, and anti-inflammatory efficacy testing in both an atopic dermatitis-like keratinocyte assay and an activated T-cell assay. The drug release was studied after single and multiple-time exposure to matrix metalloproteinase 9 to mimic inflammatory flare-ups. Drug release was found to be triggered by matrix metalloproteinase 9 and to depend on type of crosslinker and of the polyethylene glycol polymer, due to differences in architecture and swelling behavior. Moreover, swollen hydrogels showed elastic properties similar to those of extracellular matrix proteins in the dermis. Cell studies revealed limited cytotoxicity when fibroblasts and keratinocytes were exposed to the hydrogels or their enzymatic cleavage products. Taken together, our results suggest multi-arm polyethylene glycol hydrogels as promising matrix metalloproteinase-responsive drug delivery systems, with potential in the treatment of inflammatory skin disease. STATEMENT OF SIGNIFICANCE: Smart responsive drug delivery systems such as matrix metalloproteinase-responsive hydrogels are excellent candidates for the treatment of inflammatory skin diseases including psoriasis. Their release profile can be optimized to correspond to the patient's individual disease state by tuning formulation parameters and disease-related stimuli, providing personalized treatment solutions. However, insufficient cross-linking efficiency, low matrix metalloproteinase sensitivity, and undesirable drug release kinetics remain major challenges in the development of such drug delivery systems. In this study, we address shortcomings of previous work by designing peptide linkers with optimized sensitivity towards matrix metalloproteinases and high cross-linking efficiencies. We further provide a proof-of-concept for the usability of the hydrogels in inflammatory skin conditions by employing a drug release set-up simulating inflammatory flare-ups.

Preparation and properties of a fast curing carboxymethyl chitosan hydrogel for skin care

Fast curing hydrogels are highly valuable in the skin care industry. Multi-arm polyethylene glycol (PEG) is a commonly used crosslinking agent for the preparation of fast curing hydrogels. However, the hydrogel preparation using multi-arm PEG crosslinking agents usually requires the end group modifications of reactants, which makes the preparation process complicated and produces hydrogels with low curing rates. In the present study, a fast in situ curing hydrogel, 4-arm-PEG-succinimidyl carbonate/carboxymethyl chitosan (PEG-SC/CMCS), was prepared using 4-arm-PEG-SC as the crosslinking agent and carboxymethyl chitosan (CMCS), a derivative of natural polysaccharide with excellent comprehensive properties, as the framework without any end groups modified. The structure and properties of the prepared hydrogel were characterized by FTIR, XRD, SEM, rheology and swelling kinetics. The effects of reaction conditions on the structure and properties of hydrogel were investigated. The PEG-SC/CMCS hydrogel exhibited pH-sensitive swelling behaviors. Its application potential in skin care products is demonstrated with its excellent sustained-release performance of Vitamin C, a model drug for skin treatment, and high adsorption capacity of heavy metal ions that are easily deposited on skin surface. Our work has provided a new strategy for building multifunctional gel materials for skin care and offered a PEG-SC/CMCS hydrogel with great application potentials.

Rational Design of Hydrogel Networks with Dynamic Mechanical Properties to Mimic Matrix Remodeling.

Engineered hydrogels are increasingly used as extracellular matrix (ECM) surrogates for probing cell function in response to ECM remodeling events related to injury or disease (e.g., degradation followed by deposition/crosslinking). Inspired by these events, this work establishes an approach for pseudo-reversible mechanical property modulation in synthetic hydrogels by integrating orthogonal, enzymatically triggered crosslink degradation, and light-triggered photopolymerization stiffening. Hydrogels are formed by a photo-initiated thiol-ene reaction between multiarm polyethylene glycol and a dually enzymatically degradable peptide linker, which incorporates a thrombin-degradable sequence for triggered softening and a matrix metalloproteinase (MMP)-degradable sequence for cell-driven remodeling. Hydrogels are stiffened by photopolymerization using a flexible, MMP-degradable polymer-peptide conjugate and multiarm macromers, increasing the synthetic matrix crosslink density while retaining degradability. Integration of these tools enables sequential softening and stiffening inspired by matrix remodeling events within loose connective tissues (Young's modulus (E) ≈5 to 1.5 to 6kPa with >3x ΔE).The cytocompatibility and utility of this approach is examined with breast cancer cells, where cell proliferation shows a dependence on the timing of triggered softening. This work provides innovative tools for 3D dynamic property modulation that are synthetically accessible and cell compatible.

Customizing biohybrid cryogels to serve as ready-to-use delivery systems of signaling proteins

Macroporous cryogels have recently gained increasing interest for the controlled administration of signaling proteins in tissue engineering due to an advantageous combination of material properties. However, most of the previously reported cryogel systems did not allow for tunable, sustained protein release. We therefore designed a set of ready-to-use multi-armed polyethylene glycol (starPEG)-heparin cryogel systems containing different amounts of the protein-affine glycosaminoglycan component heparin to enable systematically tunable long-term delivery of different signaling proteins without affecting other cell-instructive properties. Experimental data and mathematical modeling indicate that the macroporous structure causes local differences in the concentration of proteins released into the pores and in the surrounding of the cryogels. As a proof-of-concept for their ready-to-use potential, cryogels pre-functionalized with signaling proteins and cell adhesion-peptides were demonstrated to induce the neuronal differentiation of colonizing pheochromocytoma cells. The elaborated approach opens up new perspectives for cryogels as easily storable and applicable systems for the precision delivery of signaling proteins.

Thiol-Thioester Exchange Reactions in Precursors Enable pH-Triggered Hydrogel Formation.

Bio-interactive hydrogel formation in situ requires sensory capabilities toward physiologically relevant stimuli. Here, we report on pH-controlled in situ hydrogel formation relying on latent cross-linkers, which transform from pH sensors to reactive molecules. In particular, thiopeptolide/thio-depsipeptides were capable of pH-sensitive thiol-thioester exchange reactions to yield α,ω-dithiols, which react with maleimide-functionalized multi-arm polyethylene glycol to polymer networks. Their water solubility and diffusibility qualify thiol/thioester-containing peptide mimetics as sensory precursors to drive in situ localized hydrogel formation with potential applications in tissue regeneration such as treatment of inflamed tissues of the urinary tract.

Self-Assembled Folic Acid-Targeted Pectin-Multi-Arm Polyethylene Glycol Nanoparticles for Tumor Intracellular Chemotherapy.

Ursolic acid is widely used as an effective anticancer drug for the treatment of various cancers. However, its poor water solubility, short circulation time in vivo, and lack of targeting have made it a burden for clinical applications. We report a self-assembled folate-modified pectin nanoparticle for loading ursolic acid (HCPT@F-Pt-PU NPs) and embed the anticancer drug hydroxycamptothecin to achieve synergistic treatment with ursolic acid. In addition, the galactose residue of the pectin molecule can be recognized by the asialoglycoprotein receptor on the surface of the liver cancer cell, promoting the rapid penetration and release of HCPT@F-Pt-PU NPs intracellularly. In particular, the introduction of multiarm polyethylene glycol can improve the uniformity (106 nm) and concealment of the nanoparticles and avoid the early release of the drug or the toxicity to normal cells. HCPT@F-Pt-PU NPs have a high drug loading (7.27 wt %) and embedding efficiency (19.84 wt %) and continuous circulation up to 80 h, leading to more apoptosis (91.61%). HCPT@F-Pt-PU NP intracellular drug delivery will be a promising strategy.

Investigation of Cancer Cell Migration and Proliferation on Synthetic Extracellular Matrix Peptide Hydrogels.

Chemical and mechanical properties of a tumor microenvironment are essential players in cancer progression, and it is important to precisely control the extracellular conditions while designing cancer in vitro models. The study investigates synthetic hydrogel matrices from multi-arm polyethylene glycol (PEG) functionalized with collagen-like peptide (CLP) CG(PKG)4(POG)4(DOG)4 alone and conjugated with either cell adhesion peptide RGD (mimicking fibronectin) or IKVAV (mimicking laminin). Human glioblastoma HROG36, rat C6 glioma cells, and A375 human melanoma cells were grown on the hydrogels and monitored for migration, proliferation, projected cell area, cell shape index, size and number, distribution of focal contacts in individual cells, and focal adhesion number. PEG-CLP-RGD induced migration of both glioma cell lines and also stimulated proliferation (assessed as metabolic activity) of HROG36 cells. Migration of C6 cells were also stimulated by PEG-CLP-IKVAV. These responses strongly correlated with the changes in adhesion and morphology parameters of individual cells – projected cell area, cell shape index, and focal contact number. Melanoma A375 cell proliferation was increased by PEG-CLP-RGD, and this was accompanied by a decrease in cell shape index. However, neither RGD nor IKVAV conjugated to PEG-CLP stimulated migratory capacity of A375 cells. Taken together, the study presents synthetic scaffolds with extracellular matrix (ECM)-mimicking peptides that allow for the exploration of the effect of ECM signaling to cancer cells.

Crosslinker length dictates step-growth hydrogel network formation dynamics and allows rapid on-chip photoencapsulation

Hydrogels formed via free radical-mediated thiol-ene step-growth photopolymerization have been developed for a broad range of tissue engineering and regenerative medicine applications. While the crosslinking mechanism of thiol-ene hydrogels has been well-described, there has been only limited work exploring the physical differences among gels arising from variations in crosslinker properties. Here, we show that the character of linear polyethylene glycol (PEG) dithiols used to crosslink multi-arm polyethylene glycol norbornene (PEGNB) can be used as a facile strategy to tune hydrogel formation kinetics, and therefore the equilibrium hydrogel network architecture. Specifically, we report the dramatic effect of crosslinker length on PEGNB hydrogel formation kinetics and the formed hydrogel properties. It is shown that the hydrogel formation kinetics and formed hydrogel properties can be tuned by solely varying the crosslinker length. It was hypothesized that under identical reaction conditions, a more accessible 3.5 k PEG dithiol crosslinker would improve network ideality relative to a shorter 1.5 k crosslinker. Longer linkers consequently promote significantly more rapid macromer crosslinking and therefore gelation. Accelerated gel formation satisfies an urgent unmet need for rapid polymerization in droplet microfluidics. Using long linkers, we demonstrate the ability to photopolymerize PEGNB microgels under flow on a microfluidic chip, with reliable control over microgel size and shape in a high-throughput manner. To further validate the potential of this platform to produce novel, microstructured cell carrier vehicles, 3T3 fibroblasts were successfully encapsulated and cultured over 14 days with excellent cell viability. This study demonstrates that PEGNB hydrogel dynamics could be readily customized to fulfill a variety of needs in tissue engineering, controlled cell delivery, or drug release applications.

Near-infrared-IIb probe affords ultrahigh contrast inflammation imaging.

Deep tissue imaging in the near-infrared II (NIR-II) window with significantly reduced tissue autofluorescence and scattering provides an important modality to visualize various biological events. Current commercially used contrast agents in the near-infrared spectrum suffer from severe photobleaching, high tissue scattering, and background signals, hampering high-quality in vivo bioimaging, particularly in small animals. Here, we applied a NIR-IIb quantum dot (QD) probe with greatly suppressed photon scattering and zero autofluorescence to map inflammatory processes. Two-layer surface modification by a combination of amphiphilic polymer and mixed linear and multi-armed polyethylene glycol chains prolonged probe circulation in vivo and improved its accumulation in the inflammation sites. Compared to indocyanine green, a widely applied dye in the clinic, our QD probe showed greater photostability and capacity for deeper tissue imaging with superior contrast. The longer circulation of QDs also improved vessel imaging, which is vital for better understanding of biological mechanisms of the inflammation microenvironment. Our proposed NIR-IIb in vivo imaging modality proved effective for the visualization of inflammation in small animals, and its use may be extended in future to studies of immunity and cancer.

Octopus-like Flexible Vector for Noninvasive Intraocular Delivery of Short Interfering Nucleic Acids.

Gene therapy is promising for chronic posterior ocular diseases, which are causal factors for severe vision impairment and even blindness worldwide. However, the inherent absorption barriers of the eye restrict intraocular delivery of therapeutic nucleic acids via topical instillation. Safe and efficient nonviral vectors for ocular gene therapy are still unmet clinical desires. Herein, an octopus-like flexible multivalent penetratin (MVP) was designed to facilitate condensation and delivery of therapeutic nucleic acids using multiarm polyethylene glycol (PEG) as a core and conjugating penetratin at each end of the PEG arms as outspread tentacles. Among the MVPs, 8-valent penetratin (8VP) stably compacted nucleic acids into positively charged polyplexes smaller than 100 nm, promoting cellular uptake efficiency (approaching 100%) and transfection rate (over 75%). After being instilled into the conjunctival sac, 8VP enabled rapid (<10 min) and prolonged (>6 h) distribution of nucleic acids in the retina via a noncorneal pathway. In a retinoblastoma-bearing mice model, topical instillation of 8VP/siRNA efficiently inhibited the protein expression of intraocular tumor without toxicity. MVP is advantageous over the commercial transfection reagent in safety and efficiency, and therefore provides a promising vector for noninvasive intraocular gene delivery.

Enhanced Bone Defect Repair by Polymeric Substitute Fillers of MultiArm Polyethylene Glycol-Crosslinked Hyaluronic Acid Hydrogels.

Bone regeneration is still one of the greatest challenges for the treatment of bone defects since no current clinical approach has been proven effective. To develop an alternative biodegradable bone graft material, multiarm polyethylene glycol (PEG) crosslinked hyaluronic acid (HA) hydrogels are synthesized and applied to promote osteogenesis of mesenchymal stem cells (MSCs) with the ultimate goal for bone defect repair. The multiarm PEG-HA hydrogels provide a significant improvement of alkaline phosphatase (ALP) activity and calcium mineralization of the in vitro encapsulated MSCs under osteogenic condition after 3, 7, and 28 days. In addition, the multiarm PEG-HA hydrogels also facilitate healing of the cranial bone defects more effectively in a Sprague Dawley rat model after 10 weeks of implantation based on histological evaluations and microcomputed tomography analysis. These promising results set the stage for the development of innovative biodegradable hydrogels to provide a more effective and versatile treatment option for bone regeneration.

Development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo

Polyrotaxane (PRX) is a promising supramolecular carrier for gene delivery. Classic PRX exhibits a linear structure in which the amine-functionalized α-cyclodextrin (CD) is threaded along the entire polyethylene glycol (PEG) backbone. While promising in vitro, the absence of free PEG moieties after CD threading compromised the in vivo implementation, due to the unfavorable pharmacokinetics (PK) and biodistribution profile. Herein, we developed a multi-arm PRX nanocarrier platform, which has been designed for protective nucleic acid encapsulation, augmented biodistribution and PK, and suitable for intravenous (IV) administration. A key design was to introduce cationic CD rings onto a multi-arm PEG backbone in a spatially selective fashion. The optimal structural design was obtained through iterative rounds of experimentation to determine the appropriate type and density of cationic charge on CD ring, the degree of PEGylation, the size and structure of polymer backbone, etc. This allowed us to effectively deliver large size reporter and therapeutic plasmids in cancer mouse models. Post IV injection, we demonstrated that our multi-arm polymer design significantly enhanced circulatory half-life and PK profile compared to the linear PRX. We continued to use the multi-arm PRX to formulate a therapeutic plasmid encoding an immunomodulatory cytokine, IL-12. When tested in a colon cancer syngeneic mouse model with same background, the IL-12 plasmid was protected by the multi-arm PRX and delivered through the tail vein to the tumor site, leading to a significant tumor inhibition effect. Moreover, our delivery system was devoid of major systemic toxicity.

Covalently functionalized amide cross-linked hydrogels from primary amines and polyethylene glycol acyltrifluoroborates (PEG-KATs).

A new method for the rapid preparation of chemically cross-linked hydrogels based on a multi-arm polyethylene glycol (PEG) bearing potassium acyl trifluoroborate (KAT) functional groups with multi-dentate amines is described. These scaffolds - prepared in aqueous buffer - give strong, transparent hydrogels. At pH 3, the gel formation is complete within seconds, and the reaction rate can be tuned by modulating the pH. Rheology measurements show that the hydrogel properties can be tuned as a function of both the weight percent of solids in the gel and the denticity of amine cross-linker, allowing for predictable formation of gels with desired traits. This process relies on a rapid amide-forming reaction of KATs and in situ generated N-chloroamines. Numerous commercially available amines, including di-, tri- and tetra-functional amines as well as peptides and carbohydrates serve as effective cross-linkers. Monodentate amines included in the gelation mixture are covalently linked into the gel matrix by amide-bonds, allowing gels containing immobilized molecules including dyes, sensors, or biotin amine, to be prepared in a single step from simple starting materials. The ability to induce gelation only upon addition of equimolar amounts of an inexpensive inducer (N-chlorosuccinimide) allows premixed components to be stored as an aqueous solution for weeks and converted to gels on demand.

Design of electrohydrodynamic sprayed polyethylene glycol hydrogel microspheres for cell encapsulation

Electrohydrodynamic spraying (EHS) has recently gained popularity for microencapsulation of cells for applications in cell delivery and tissue engineering. Some of the polymers compatible with EHS are alginate, chitosan, and other similar natural polymers, which are subject to ionotropic or physical gelation. It is desirable to further extend the use of the EHS technique beyond such polymers for wider biofabrication applications. Here, building upon our previous work of making PEG microspheres via EHS, we utilized the principles of EHS to fabricate cell-laden polyethylene glycol (PEG) hydrogel microspheres. The gelation of PEG hydrogel microspheres was achieved by forming covalent crosslinks between multiarm PEG acrylate and dithiol crosslinkers via Michael-type addition. We conducted a detailed investigation of the critical parameters of EHS, such as the applied voltage, inner needle diameter (i.d. needle), and flow rate, to obtain PEG microspheres with high cell viability and tightly-controlled diameters in the range of 70–300 μm. The polydispersity of cell-laden PEG hydrogel microspheres as measured by % coefficient of variation was between 6% and 23% for all conditions tested. We established that our method was compatible with different cell types and that all tested cell types could be encapsulated at high densities of 106–109 and ≥90% encapsulation efficiency. We observed cell aggregation within the hydrogel microspheres at applied voltage >5 kV. Since PEG is a synthetic polymer devoid of cell attachment sites, we could overcome this limitation by tethering Arg-Gly-Asp-Ser (RGDS) peptide to the PEG hydrogel microspheres; upon RGDS tethering, we observed uniform cell dispersion. The microencapsulated cells could be cultured in the PEG hydrogel microspheres of different sizes for up to one week without significant loss in cell viability. In conclusion, the EHS technique developed here could be used to generate cell-laden PEG hydrogel microspheres of controlled sizes for potential applications in cell delivery and organoid cultures.

Dual-crosslinked hydrogel microwell system for formation and culture of multicellular human adipose tissue-derived stem cell spheroids.

Three-dimensional (3D) multicellular spheroids of human adipose tissue-derived stem cells (hASCs) are an attractive system for basic science studies and tissue engineering applications, as they can resemble cellular condensations present in developmental and healing processes. The purpose of this study was to engineer a hydrogel-based microwell platform by capitalizing on the differential swelling behavior of micropatterned dual-crosslinked oxidized, methacrylated alginate (OMA)/multi-arm polyethylene glycol (PEG) hydrogels for rapid formation of uniform multicellular hASC spheroids with controllable size and evaluation of the capacity of the system to be used to drive osteogenic differentiation of the spheroids. By changing the micropattern size, the size of the hydrogel microwells was easily controllable. Microwell-seeded hASCs generated spheroids of relatively uniform size and high cell viability. hASC spheroids exhibited rapid mineralization in osteogenic media, which was faster than that of conventional two-dimensionally cultured hASCs. This new hydrogel microwell system has great potential for controlled multicellular spheroid formation and defined signal presentation from the hydrogel material to the cell aggregates to regulate tissue formation.

Cell-mediated mineralization of synthetic polyethylene glycol hydrogels to create bone tissue engineering constructs

Event Abstract Back to Event Cell-mediated mineralization of synthetic polyethylene glycol hydrogels to create bone tissue engineering constructs Çiğdem Demirkaya1, Evita Willems2, Yoke C. Chai3, 4, Frank P. Luyten3, 4 and Jennifer Patterson2, 3 1 Ege University, Department of Bioengineering, Türkiye 2 KU Leuven, Department of Materials Engineering, Belgium 3 KU Leuven, Prometheus, Division of Skeletal Tissue Engineering, Belgium 4 KU Leuven, Tissue Engineering Laboratory, Skeletal Biology and Engineering Research Center, Belgium Introduction: A key principle of tissue engineering is that the appropriate combination of a biomaterial scaffold, cells, and biological signals can be used to create a living implant, a tissue engineering construct, that will help a damaged tissue to regenerate its lost function[1]. To make a bone tissue engineering construct, we chose to combine: an enzymatically degradable, synthetic hydrogel scaffold based on the Michael-type addition reaction between end-functionalized, multi-arm polyethylene glycol (PEG) and protease-sensitive peptides[2]; human periosteum-derived cells (hPDCs), a progenitor cell type with mesenchymal stem cell characteristics[3]; and in vitro culture with calcium (Ca) and phosphate (P) supplementation in the medium, which has been shown to promote hPDC proliferation, collagen deposition, and mineralization[4]. Interestingly, culture of these hydrogels in the Ca- and P-rich medium led to the deposition of mineral inside the scaffold only when cells were encaspulated in the hydrogels, suggesting a cell-mediated mineratization process. In this study, we examined the behavior of hPDCs throughout the culture period and characterized the mineral that was formed. Materials and Methods: hPDCs were encapsulated in degradable PEG hydrogels that were modified with RGD. The hydrogels, with and without cells, were cultured in growth medium that was or was not supplemented with Ca and P for between 21 and 42 days. In some cases, a two-step procedure was followed where culture in growth medium was followed by Ca- and P-supplementation. Constructs were analyzed to measure cell proliferation (Presto blue), morphology (phalloidin/DAPI), viability (Live/Dead), and differentiation (alkaline phosphatase). To characterize the deposited mineral, nanofocus computed tomography (nanoCT) was used to examine the distribution and volume of mineral, X-ray diffraction (XRD) was used to identify the type of mineral, and a dissolution study was performed at 37°C. Results and Discussion: Ca- and P-supplementation led to the precipitation of a layer of mineral on the surface of the hydrogels, both with and without cells. Intriguigingly, nanoCT analysis (Fig. 1) revealed a punctate, radio-opaque phase inside the hydrogels, but only for samples that contained cells and were incubated in Ca- and P-supplemented medium. Quantitatively, this mineral volume was significantly higher in samples with cells than those without cells. In addition, XRD spectra showed peaks that were consistent with the presence of hydroxyapatite. Moreover, a dissolution study showed that release of Ca and P reached a plateau after 10 days for the cell-free samples whereas hydrogels with cells continued to release Ca and P for over 21 days. Importantly, cells within the hydrogels not only remained viable over the entire culture period, as indicated by a Live/Dead assay (Fig. 2), but they also proliferated, as seen by an increase in Presto blue readings. Additionally, the cells adopted a spread morphology, which was expected given the use of a protease-sensitive and RGD-functionalized PEG hydrogel. Conclusion: We have demonstrated that synthetic hydrogels based on PEG can not only support the proliferation of hPDCs, an osteoprogenitor cell, but can promote cell-mediated mineralization when cultured in medium supplemented with Ca and P. These hydrogels are particularly exciting for tissue engineering because they can be degraded and cleared during the tissue regeneration process. This work is part of Prometheus, the R&D Division of Skeletal Tissue Engineering of the KU Leuven: http://www.kuleuven.be/prometheus.; This work was supported by Research Programme G.0982.11 of the Research Foundation - Flanders (FWO) and the special research fund of the KU Leuven, grant number CREA/13/017.; Yoke Chin Chai was supported by a postdoctoral mandate (1.5.172.13N-Interdisc.) from the Research Foundation - Flanders (FWO).; We acknowledge Abhijith Kudva for assistance with cell culture and Yujing He for assistance with data analysis.

Novel multiarm polyethylene glycol-dihydroartemisinin conjugates enhancing therapeutic efficacy in non-small-cell lung cancer.

The clinical application of dihydroartemisinin (DHA) has been hampered due to its poor water-solubility. To overcome this hurdle, we devised a novel polymer-drug conjugate, multiarm polyethylene glycol-dihydroartemisinin (PEG-DHA), made by linking DHA with multiarm polyethylene glycol. Herein, we investigated PEG-DHA on chemical structure, hydrolysis, solubility, hemolysis, cell cytotoxicity in vitro, and efficacy in vivo. The PEG-DHA conjugates have showed moderate drug loadings (2.82 ~ 8.14 wt%), significantly good water-solubilities (82- ~ 163-fold of DHA), excellent in vitro anticancer activities (at concentrations ≥8 μg/ml, showed only 15–20% cell viability) with potency similar to that of native DHA, and long blood circulation half-time (5.75- ~ 16.75-fold of DHA). Subsequent tumor xenograft assays demonstrated a superior therapeutic effect of PEG-DHA on inhibition of tumor growth compared with native DHA. The novel PEG-DHA conjugates can not only improve the solubility and efficacy of DHA but also show the potential of scale-up production and clinical application.