Multi-arm PEG Research Articles

A nanoprodrug derived from branched poly (ethylene glycol) recognizes prostate-specific membrane antigen to precisely suppress prostate cancer progression

Prostate-specific membrane antigen (PSMA) is overexpressed in 80–90 % of prostate cancers (PCa) and is widely used as a diagnostic and therapeutic biomarker. Docetaxel (DTX), an FDA-approved anti-microtubule drug, is commonly employed to manage metastatic castration-resistant PCa; however, DTX therapy is often associated with severe side effects. One promising strategy to mitigate these side effects is the development of nanomedicine by loading small molecules into biocompatible vectors. Poly (ethylene glycol) (PEG) has been extensively used in clinical settings for this purpose, with PEGylated drugs demonstrating significant success. Compared to linear PEG, branched PEG (multi-arm PEG) provides enhanced stability for nanomedicines. In this study, we developed a novel nanoprodrug 4armPEG-Docetaxel DCL (4armPEG-DD) by conjugating a 4-arm PEG with DTX via a reduction-sensitive disulfide bond and further modifying it with 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (DCL), a PSMA-targeting ligand. Both in vitro and in vivo results demonstrated that the designed nanoprodrug specifically recognized PSMA-positive PCa cells and effectively released DTX in response to the intracellular reducing environment, leading to potent cytotoxic effects on PSMA-positive prostate tumors. Importantly, 4armPEG-DD exhibited improved in vivo safety compared to small-molecule DTX. Thus, we propose that 4armPEG-DD represents a promising candidate for the clinical treatment of PSMA-positive PCa.

A Novel Multiarmed Bifunctional PEG Derivative for the Preparation of Mass Spectrometry Ion Sources with Antifouling Property and High Selectivity.

It is challenging to prepare a highly selective mass spectrometry (MS) ion source for the rapid and highly sensitive detection of analytes, especially mycotoxins. In this study, an amino and tetrazine bifunctionalized multiarm PEG derivative (NH2HCl-4armPEG10K-(MTz)3), which can be easily immobilized on the substrate by the addition reaction between amino and polydopamine, was used for the preparation of MS ionization substrate. NH2HCl-4armPEG10K-(MTz)3 can also be used as a linker to immobilize sufficient streptavidin (SA) on the surface of the substrate by a click reaction. The process further promotes the immobilization of broad-spectrum antibodies (3D4), which were used as the recognition element for ZEN and its metabolites. The prepared SSS-Au-PDA-4armPEG10K-SA-3D4 not only can rapidly enrich ZEN and its metabolites with high selectivity but also shows good antifouling properties in the matrix. After simple sample preparation, the prepared SSS-Au-PDA-4armPEG10K-SA-3D4 can be directly coupled with MS to achieve high sensitivity (LODs: 0.18-0.66 ng/mL, LOQs: 0.5-1.0 ng/mL) and selective detection of ZEN and its metabolites in the matrix. At the same time, satisfactory recoveries (83.60-97.80%) and precision (RSD: 2.80-9.10%) can also be obtained. The prepared SSS-Au-PDA-4armPEG10K-SA-3D4 is expected to provide a powerful tool for the rapid and highly sensitive determination of multiple targets by MS.

Reversible Stabilization of Nanofiber-Polyplexes through Introducing Cross-Linkages.

Non-viral gene delivery systems are typically designed vector systems with contradictory properties, namely sufficient stability before cellular uptake and instability to ensure the release of nucleic acid cargoes in the transcription process after being taken up into cells. We reported previously that poly-(L-lysine) terminally bearing a multi-arm PEG (maPEG-PLL) formed nanofiber-polyplexes that suppressed excessive DNA condensation via steric repulsion among maPEGs and exhibited effective transcriptional capability in PCR amplification experiments and a cell-free gene expression system. In this study, the reversible stabilization of a nanofiber-polyplex without impairing the effective transcriptional capability was investigated by introducing cross-links between the PLL side chains within the polyplex using a cross-linking reagent with disulfide (SS) bonds that can be disrupted under reducing conditions. In the presence of dextran sulfate and/or dithiothreitol, the stability of the polyplex and the reactivity of the pDNA were evaluated using agarose gel electrophoresis and real-time PCR. We succeeded in reversibly stabilizing nanofiber-polyplexes using dithiobis (succinimidyl propionate) (DSP) as the cross-linking reagent. The effect of the reversible stabilization was confirmed in experiments using cultured cells, and the DSP-crosslinked polyplexes exhibited gene expression superior to that of polyethyleneimine polyplexes, which are typical polyplexes.

Scaffold-Free Multicellular 3D Tissue Constructs Utilizing Bio-orthogonal Click Strategy.

Multicellular 3D tissue constructs (MTCs) are important in biomedical research due to their capacity to accurately mimic the structure and variation found in real tissues. This study presents a novel bio-orthogonal engineering strategy (BIEN), a transformative scaffold-free approach, to create advanced MTCs. BIEN harnesses the cellular biosynthetic machinery to incorporate bio-orthogonal azide reporters into cell surface glycoconjugates, followed by a click reaction with multiarm PEG, resulting in rapid assembly of MTCs. The implementation of this cutting-edge strategy culminates in the formation of uniform, heterogeneous spheroids, characterized by a high degree of intercellular junction and pluripotency. Remarkably, MTCs simulate tumor features, ensure cell heterogeneity, and significantly improve the subcutaneous xenograft model after transplantation, thereby bolstering both in vitro and in vivo research models. In conclusion, the utilization of the bio-orthogonal engineering strategy as a scaffold-free method to generate superior MTCs holds promising potential for driving advancements in cancer research.

Structurally decoupled stiffness and solute transport in multi-arm poly(ethylene glycol) hydrogels

Synthetic hydrogels are widely used as artificial 3D environments for cell culture, facilitating the controlled study of cell-environment interactions. However, most hydrogels are limited in their ability to represent the physical properties of biological tissues because stiffness and solute transport properties in hydrogels are closely correlated. Resultingly, experimental investigations of cell-environment interactions in hydrogels are confounded by simultaneous changes in multiple physical properties. Here, we overcame this limitation by simultaneously manipulating four structural parameters to synthesize a library of multi-arm poly (ethylene glycol) (PEG) hydrogel formulations with robustly decoupled stiffness and solute transport. This structural design approach avoids chemical alterations or additions to the network that might have unanticipated effects on encapsulated cells. An algorithm created to statistically evaluate stiffness-transport decoupling within the dataset identified 46 of the 73 synthesized formulations as robustly decoupled. We show that the swollen polymer network model accurately predicts 11 out of 12 structure-property relationships, suggesting that this approach to decoupling stiffness and solute transport in hydrogels is fundamentally validated and potentially broadly applicable. Furthermore, the unprecedented control of hydrogel network structure provided by multi-arm PEG hydrogels confirmed several fundamental modeling assumptions. This study enables nuanced hydrogel design for uncompromised investigation of cell-environment interactions.

Facile Synthesis of Rapidly Degrading PEG-Based Thiol-Norbornene Hydrogels.

An alternate synthesis route was developed to prepare norbornene-functionalized poly(ethylene glycol) (PEG) from reacting multiarm PEG with carbic anhydride. The macromer, PEGNBCA, permits photo-cross-linking of thiol-norbornene hydrogels with kinetics comparable to conventional PEGNB macromer. In addition, PEGNBCA provides an additional carboxylate group for further conjugation with amine-bearing molecules. Interestingly, PEGNBCA thiol-norbornene hydrogels are highly susceptible to hydrolytic degradation through enhanced ester hydrolysis. The ester linkage is further weakened after the secondary conjugation, resulting in extremely rapid degradation of PEGNB hydrogels. More importantly, the degradation can be readily adjusted via tuning macromer compositions, with complete degradation time ranging from hours to weeks. The PEGNBCA hydrogels are also highly cytocompatible toward various cell types, providing opportunities for future applications in tissue engineering and advanced biofabrication.

An Injectable Hydrogel Platform for Sustained Delivery of Anti-inflammatory Nanocarriers and Induction of Regulatory T Cells in Atherosclerosis.

Chronic unresolved vascular inflammation is a critical factor in the development of atherosclerosis. Cardiovascular immunotherapy has therefore become a recent focus for treatment, with the objective to develop approaches that can suppress excessive inflammatory responses by modulating specific immune cell populations. A benefit of such immunomodulatory strategies is that low dosage stimulation of key immune cell populations, like antigen presenting cells, can subsequently propagate strong proliferation and therapeutic responses from effector cells. We have previously demonstrated that intravenous injections of anti-inflammatory nanocarriers provided atheroprotection that was mediated by regulatory T cells (Tregs) upregulated in lymphoid organs and atherosclerotic lesions. Here, we demonstrate an injectable filamentous hydrogel depot (FM-depot) engineered for low dosage, sustained delivery of anti-inflammatory nanocarriers. The bioactive form of vitamin D (aVD; 1, 25-Dihydroxyvitamin D3), which inhibits pro-inflammatory transcription factor NF-κB via the intracellular nuclear hormone receptor vitamin D receptor (VDR), was stably loaded into poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-b-PPS) filomicelles. These aVD-loaded filaments underwent morphological transitions to release monodisperse drug-loaded micelles upon oxidation. This cylinder-to-micelle transition was characterized in vitro by cryogenic transmission electron microscopy (CryoTEM) and small angle X-ray scattering (SAXS). Following crosslinking with multi-arm PEG for in situ gelation, aVD-loaded FM-depots maintained high levels of Foxp3+ Tregs in both lymphoid organs and atherosclerotic lesions for weeks following a single subcutaneous injection into ApoE−/− mice. FM-depots therefore present a customizable delivery platform to both develop and test nanomedicine-based approaches for anti-inflammatory cardiovascular immunotherapy.

Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture.

Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCs) and intestinal organoids. These matrices comprise a poly(ethylene glycol) (PEG) hydrogel backbone functionalized with minimal adhesion cues including RGD (Arg-Gly-Asp), which is sufficient for ISC expansion, and laminin-111, which is required for organoid formation. As such, the hydrogels present a defined and reproducible, but also tunable, environment, allowing researches to manipulate physical and chemical parameters, and examine their influence on ISC and organoid growth. Hydrogels are formed by an enzymatic cross-linking reaction of multiarm PEG precursors bearing glutamine- and lysine-containing peptides. PEG precursors containing either stable or hydrolytically degradable moieties are used to produce mechanically softening hydrogels, which are used for the expansion of ISCs or the formation of organoids, respectively. We also provide protocols for immunofluorescence analysis of cellular structures grown within these matrices, as well as for their dissociation and retrieval of cells for downstream use. Hydrogel precursors can be produced and their mechanical properties characterized to ascertain stiffness within 5-7 d. Hydrogel formation for ISC expansion or organoid formation takes 1-2 h. The materials described here can be readily adapted for the culture of other types of normal or transformed organoid structures.

Enhancement Effect of a Variable Topology of a Membrane-Tethered Anti-Poly(ethylene glycol) Antibody on the Sensitivity for Quantifying PEG and PEGylated Molecules.

Sensitive quantification of the pharmacokinetics of poly(ethylene glycol) (PEG) and PEGylated molecules is critical for PEGylated drug development. Here, we developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for PEG by tethering an anti-PEG antibody (AGP3) via tethers with different dimensions on the surface of 293T cells (293T/S-αPEG, short-type cells; 293T/L-αPEG, long-type cells; 293T/SL-αPEG, hybrid-type cells) to improve the binding capacity and detection limit for free PEG and PEGylated molecules. The binding capacity of hybrid-type cells for PEG-like molecules (CH3-PEG5K-FITC (FITC = fluorescein isothiocyanate) and eight-arm PEG20K-FITC) was at least 10-80-fold greater than that of 293T cells expressing anti-PEG antibodies with uniform tether lengths. The detection limit of free PEG (OH-PEG3K-NH2 and CH3-PEG5K-NH2) and PEG-like molecule (CH3-PEG5K-FITC, CH3-PEG5K-SHPP, and CH3-PEG5K-NIR797) was14-137 ng mL-1 in the hybrid-type cell-based sandwich ELISA. 293T/SL-αPEG cells also had significantly higher sensitivity for quantification of a PEGylated protein (PegIntron) and multiarm PEG macromolecules (eight-arm PEG20K-NH2 and eight-arm PEG40K-NH2) at 3.2, 16, and 16 ng mL-1, respectively. Additionally, the overall binding capacity of 293T/SL-αPEG cells for PEGylated macromolecules was higher than that of 293T/S-αPEG or 293T/L-αPEG cells. Anchoring anti-PEG antibodies on cells via variable-length tethers for cell-based sandwich ELISA, therefore, provides a sensitive, high-capacity method for quantifying free PEG and PEGylated molecules.

Fabrication of injectable high strength hydrogel based on 4-arm star PEG for cartilage tissue engineering.

Hydrogels prepared from poly(ethylene glycol) (PEG) are widely applied in tissue engineering, especially those derived from a combination of functional multi-arm star PEG and linear crosslinker, with an expectation to form a structurally ideal network. However, the poor mechanical strength still renders their further applications. Here we examined the relationship between the dynamics of the pre-gel solution and the mechanical property of the resultant hydrogel in a system consisting of 4-arm star PEG functionalized with vinyl sulfone and short dithiol crosslinker. A method to prepare mechanically strong hydrogel for cartilage tissue engineering is proposed. It is found that when gelation takes place at the overlap concentration, at which a slow relaxation mode just appears in dynamic light scattering (DLS), the resultant hydrogel has a local maximum compressive strength ∼20MPa, while still keeps ultralow mass concentration and Young's modulus. Chondrocyte-laden hydrogel constructed under this condition was transplanted into the subcutaneous pocket and an osteochondral defect model in SCID mice. The invivo results show that chondrocytes can proliferate and maintain their phenotypes in the hydrogel, with the production of abundant extracellular matrix (ECM) components, formation of typical chondrocyte lacunae structure and increase in Young's modulus over 12 weeks, as indicated by histological, immunohistochemistry, gene expression analyses and mechanical test. Moreover, newly formed hyaline cartilage was observed to be integrated with the host articular cartilage tissue in the defects injected with chondrocytes/hydrogel constructs. The results suggest that this hydrogel is a promising candidate scaffold for cartilage tissue engineering.

Hydrogels based on poly(ethylene glycol) as scaffolds for tissue engineering application: biocompatibility assessment and effect of the sterilization process.

Hydrogels are suitable materials to promote cell proliferation and tissue support because of their hydrophilic nature, porous structure and sticky properties. However, hydrogel synthesis involves the addition of additives that can increase the risk of inducing cytotoxicity. Sterilization is a critical process for hydrogel clinical use as a proper scaffold for tissue engineering. In this study, poly(ethylene glycol) (PEG), poly(ethylene glycol)-chitosan (PEG-CH) and multi-arm PEG hydrogels were synthesized by free radical polymerization and sterilized by gamma irradiation or disinfected using 70 % ethanol. The biocompatibility assessment in human fibroblasts and hemocompatibility studies (hemolysis, platelet aggregation, morphology of mononuclear cells and viability) in peripheral blood from healthy volunteers (ex vivo), were performed. The sterilization or disinfection effect on hydrogel structures was evaluated by FT-IR spectroscopy. Results indicated that hydrogels do not induce any damage to fibroblasts, erythrocytes, platelets or mononuclear cells. Moreover, there was no significant difference in the biocompatibility after the sterilization or disinfection treatment. However, after gamma irradiation, several IR spectroscopic bands were shifted to higher or lower energies with different intensity in all hydrogels. In particular, several bands associated to carboxyl or hydroxyl groups were slightly shifted, possibly associated to scission reactions. The disinfection treatment (70 % ethanol) and γ-irradiation at 13.83 ± 0.7 kGy did not induce morphological damages and yielded sterile and biocompatible PEG hydrogels potentially useful for clinical applications.

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure.

Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility; expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multiarm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multiarm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and cross-linking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, microheterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds.

Thiol-ene Photocrosslinking of Cytocompatible Resilin-Like Polypeptide-PEG Hydrogels.

A range of chemical strategies have been used for crosslinking recombinant polypeptide hydrogels, although only a few have employed photocrosslinking approaches. Here, we capitalize on the novel insect protein, resilin, and the versatility of click reactions to introduce a resilin-like polypeptide (RLP) that is capable of photoinitiated thiol-ene crosslinking. Lysine residues of the RLP were functionalized with norbornene acid as confirmed via 1H-NMR spectroscopy. The RLPNs were subsequently photocrosslinked with multi-arm PEG thiols in the presence of a photoinitiator to form elastic hybrid hydrogels. The crosslinking reaction and resulting RLP-PEG networks demonstrated cytocompatibility with human mesenchymal stem cells in both 2D cell-adhesion and 3D photoencapsulation studies.

Resilin‐Based Hybrid Hydrogels for Cardiovascular Tissue Engineering

The outstanding elastomeric properties of natural resilin, an insect protein, have motivated the engineering of resilin-like polypeptides (RLPs) as a potential material for cardiovascular tissue engineering. The RLPs, which incorporate biofunctional domains for cell-matrix interactions, are cross-linked into RLP-PEG hybrid hydrogels via a Michael-type addition of cysteine residues on the RLP with vinyl sulfones of an end-functionalized multi-arm star PEG. Oscillatory rheology indicated the useful mechanical properties of these materials. Assessments of cell viability via con-focal microscopy clearly show the successful encapsulation of human aortic adventitial fibroblasts in the three-dimensional matrices and the adoption of a spread morphology following 7 days of culture.

Nanogel surface coatings for improved single-molecule imaging substrates

Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG-BSA nanogel coatings will be highly useful for the SM analysis of proteins.

Phosphopantetheinyl Transferase-Catalyzed Formation of Bioactive Hydrogels for Tissue Engineering

Synthetic bioactive hydrogels have been widely recognized as key elements of emerging strategies to engineer tissues. However, the current shortage of highly specific and biocompatible methods to form and functionalize these materials hampers their wide pharmaceutical and medical use. In particular, enzymatic reactions are underexplored for the synthesis of bioactive hydrogels. Here, we present an approach by which phosphopantetheinyl transferase (PPTase), a small (16.2 kDa) enzyme that plays a key role in the biosynthesis of many natural products, was employed to catalyze covalent cross-linking of poly(ethylene glycol) (PEG)-based hydrogels. Gels were formed within minutes under physiological conditions by mixing two aqueous precursors containing multiarm PEG macromers end-functionalized with the PPTase substrate Coenzyme A (CoA) and a genetically engineered dimer of a carrier protein. The physicochemical properties of this new class of biomaterials were characterized. Bioactive hydrogels were produced by covalent incorporation of a CoA-functionalized cell adhesion peptide (RGDS), resulting in specific adhesion of primary fibroblasts on the hydrogel surfaces. 3D encapsulation of cells resulted in high cell viability (ca. 95%) and single cell migration over long distances within RGDS-modified gels.

Bioactive hydrogels made from step-growth derived PEG–peptide macromers

Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400 Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate–PEG–(peptide–PEG) m-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500 kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate–PEG–CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.

Synergistic Effect of Low Cytotoxic Linear Polyethylenimine and Multiarm Polyethylene Glycol: Study of Physicochemical Properties and In Vitro Gene Transfection

Novel star-shaped copolymers consisting of multiarm polyethylene glycol and low molecular weight linear polyethylenimines (MAPEG-LPEIs) with a high transfection efficiency and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. The cationic polymers were prepared by conjugating low molecular weight linear PEI (2.5 kDa) to six-arm PEG-NHS (10 kDa) in two different compositions. Two copolymers, MAPEG-LPEI(3) and MAPEG-LPEI(6) with molecular weights of 17.5 kDa and 25 kDa respectively, were synthesized. The MAPEG-LPEI(3)/pDNA and MAPEG-LPEI(6)/pDNA polyplexes are stably dispersed in aqueous media with a narrowly distributed size range of <200 nm as determined by dynamic light scattering. Furthermore, these polyplexes showed different surface charges depending upon the relative proportion of MAPEG and LPEI. Moreover, these polyplexes can protect pDNA from enzymatic degradation in serum containing media up to 24 h. These polyplexes were able to efficiently transfect luciferase-coded reporter gene into HeLa cancer cells and showed considerable gene transfection efficacy even in 50% serum-conditioned media in vitro. MAPEG-LPEI(6) exhibited higher transfection activity than that of MAPEG-LPEI(3) at the same weight ratios. Furthermore, MAPEG-LPEI/pDNA polyplexes were less toxic than LPEI/pDNA complexes as determined by MTT assay. These favorable results could be attributed to the combined effect of low molecular weight LPEI and multiarm PEG. The special structural features of the multiarm star-shaped central PEG core play an important role in achieving higher transfection efficiency as it imparts higher charge density to polyplexes and prevents the unwanted aggregation of the smaller polyplex particles. These two important factors contributed toward enhanced gene transfection. On the other hand, LPEI provides low cytotoxicity and effective complexation with pDNA in the designed architecture. Therefore it is possible to achieve enhanced gene transfection by using these two components, namely, pivotal multiarm PEG core and LPEI, in optimal ratio as observed in the case of MAPEG-LPEI(6).

Novel multiarm PEG‐based hydrogels for tissue engineering

Injectable scaffolds are promising substrates for regenerative medicine applications. In this study, multiarm amino-terminated poly(ethylene glycol) (PEG) hydrogels were crosslinked with genipin, a compound naturally derived from the gardenia fruit. Four- and eight-arm amino-terminated PEG hydrogels crosslinked with varying concentrations of genipin were characterized. Both surface and cross-sectional structures of PEG-based hydrogels were observed by scanning electron microscopy. In vitro gelation time, water uptake, swelling, and weight loss of PEG hydrogels in phosphate buffered saline at 37 degrees C were studied. The results showed that the eight-arm PEG demonstrated a much slower gelation time compared with the four-arm PEG, which may be due to the differing structures of the multiarm PEG hydrogels, which in turn affects the ability of genipin to react with the amine groups. Human adipose-derived stem cells were seeded onto the four- and eight-arm PEG hydrogels in vitro to assess the biological performance and applicability of the gels as cell carriers. The four-arm PEG hydrogel resulted in enhanced cell adhesion when compared with the eight-arm PEG hydrogel. Overall, these characteristics provide a potential opportunity for multiarm PEG hydrogels as injectable scaffolds in a variety of tissue engineering applications.

Immobilization of oriented protein molecules on poly(ethylene glycol)-coated Si(111).

A high-density poly(ethylene glycol) (PEG)-coated Si(111) surface is used for the immobilization of polyhistidine-tagged protein molecules. This process features a number of properties that are highly desirable for protein microarray technology: (i) minimal nonspecific protein adsorption; (ii) highly uniform surface functionality; (iii) controlled protein orientation; and (iv) highly specific immobilization reaction without the need of protein purification. The high-density PEG-coated silicon surface is obtained from the reaction of a multi-arm PEG (mPEG) molecule with a chlorine terminated Si(111) surface to give a mPEG film with thickness of 5.2 nm. Four out of the eight arms on each immobilized mPEG molecule are accessible for linking to the chelating iminodiacetic acid (IDA) groups for the binding of Cu(2+) ions. The resulting Cu(2+)-IDA-mPEG-Si(111) surface is shown to specifically bind 6x histidine-tagged protein molecules, including green fluorescent protein (GFP) and sulfotransferase (ST), but otherwise retains its inertness towards nonspecific protein adsorption. We demonstrate a particular advantage of this strategy: the possibility of protein immobilization without the need of prepurification. Surface concentrations of relevant chemical species are quantitatively characterized at each reaction step by X-ray photoelectron spectroscopy (XPS). This kind of quantitative analysis is essential in tuning surface concentration and chemical environment for optimal sensitivity in probe-target interaction.