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Differentiation of stem cells has shown to be strongly influenced through several cues provided by reciprocal interactions with the extracellular microenvironment, consisting of soluble bioactive agents and the extracellular matrix.While the dynamic extracellular matrix is difficult to mimic in its entirety, recent research has successfully mimicked individual matrix-centric cues using synthetic polymeric systems to influence differentiation of stem cells into tissue-specific lineages. Material properties that have been shown to direct this differentiation include chemical functionality, mechanical properties, as well as tissue-mimetic modifications such as mineralization. Another aspect of the extracellular microenvironment that has been mimicked in the controlled differentiation of stem cells is the presence of specific bioactive agents. Material-based delivery of these agents allows for the spatiotemporal variation in their presentation to stem cells, allowing for precise control over their terminally differentiated phenotype. Thus, the delivery of bioactive agents to cells via synthetic materials has also been an effective method to influence stem cell differentiation to various tissue-specific lineages. In this chapter, we discuss the use of synthetic materials to direct stem cell differentiation through both, capitulation of matrix-specific biochemical, mechanical and physical cues, as well as the controlled delivery of specific bioactive agents.

A pyridyl disulfide end-functionalized polymer with N-acetyl-d-glucosamine pendant side-chains was synthesized by atom transfer radical polymerization (ATRP). The glycopolymer was prepared from a pyridyl disulfide initiator catalyzed by a Cu(I)/Cu(II)/2,2'-bipyridine system in a mixture of methanol and water at 30 degrees C. The final polymer had a number-average molecular weight (M(n)) of 13.0 kDa determined by (1)H NMR spectroscopy and a narrow polydispersity index (1.12) determined by gel permeation chromatography (GPC). The pyridyl disulfide end-group was then utilized to conjugate the glycopolymer to a double-stranded short interfering RNA (siRNA). Characterization of the glycopolymer-siRNA by polyacrylamide gel electrophoresis (PAGE) showed 97% conjugation. The activated disulfide polymer was also patterned on gold via microcontact printing. The pyridyl disulfide allowed for ready immobilization of the glycopolymer into 200 microm sized features on the surface.

Maleimide end functionalized polymers for site-selective conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. A furan-protected maleimide chain transfer agent (CTA) was employed in the RAFT polymerization of poly(ethylene glycol) methyl ether acrylate (PEGA). Gel permeation chromatography (GPC) with laser light scattering detection indicated that 20,000 and 39,000 Da polyPEGA had been made with polydispersity indices of 1.25 and 1.36, respectively. The maleimide group on the polymer chain end was exposed by heating the poly(PEGA)s for 4 h. The deprotection efficiency was estimated to be 80 and 60% for poly(PEGA)(20 kDa) and poly(PEGA)(39 kDa), respectively. Maleimide-poly(PEGA)s were conjugated to V131C T4 lysozyme (T4L), and the resultant polymer-protein conjugates were characterized by size exclusion chromatography and gel electrophoresis.

A straightforward synthetic method to prepare pyridyl disulfide end functionalized poly(PEG acrylate) by RAFT polymerization for efficient and reversible conjugation to siRNA is described.

The frequency-domain optical coherence tomography (FD-OCT) has the advantages of high acquisition time and high system sensitivity, it makes to obtain cross-section image based on the broadband source or swept source. The phase shifting algorithm always applies to reduce the additional noise within FD-OCT and provide accurate signal process. In this paper, we present a new system structure of group delay based phase shifting by combining diffraction grating pairs in frequency domain OCT. For the parallel grating pairpsilas property, we separate the spectrum and stretch the optical pulse in time to produce group delay result utilizing diffraction grating pair. It makes the linearly group delay between the different frequencies and provides accurate phase shifting algorithm, promotes the system performance and accuracy.

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.

Treatments for renal cell carcinoma, while promising, are still limited by toxicity and cost. In the hopes of finding a novel compound or combination, we developed a plasmid containing the genes for interleukin-2 (IL-2) and soluble vascular endothelial growth factor receptor 2 (msFlk1). The plasmid, p2CMVIL2/msFlk1, demonstrated similar in vitro transgene expression of IL-2 or msFlk1 compared to their single-agent counterparts. Subcutaneous tumor growth was significantly inhibited in the p2CMVIL2/msFlk1 group when delivered locally by the non-viral water soluble polymer, WSLP and exhibited a 50% increase in survival over glucose and single-agent controls. In vivo experimentation demonstrated that WSLP/msFlk1 decreased microvessel density in pCMVmsFlk1 and p2CMVIL2/msFlk1 treated groups. Furthermore, tumor-infiltrating lymphocytes expressing CD45RO and CD68 were increased within the tumor microenvironment upon p2CMVIL2/msFlk1 treatment. To determine the effects of p2CMVIL2/msFlk1 in an experimental RENCA lung metastases model, therapeutic DNA was delivered systemically following complexation with the angiogenic endothelial-targeting polymer PEI-g-PEG-RGD. The p2CMVIL2/msFlk1 treatment significantly reduced metastases by 56% over single-agent therapy and increased survival proportions by 50% over all groups. Our work clearly demonstrates that non-viral delivery of p2CMVIL2/msFlk1 can inhibit RENCA growth in a synergistic manner and may represent a new treatment for renal carcinoma.

Feng-Yun J Huang,1 Te-Wei Lee,2 Chih-Hsien Chang,2 Liang-Cheng Chen,2 Wei-Hsin Hsu,2 Chien-Wen Chang,1 Jem-Mau Lo1 1Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan; 2Institute of Nuclear Energy Research, Longtan, Taiwan Purpose: In this study, the 188Re-labeled PEGylated nanoliposome (188Re-liposome) was prepared and evaluated as a therapeutic agent for glioma.Materials and methods: The reporter cell line, F98luc was prepared via Lentivector expression kit system and used to set up the orthotopic glioma-bearing rat model for non-invasive bioluminescent imaging. The maximum tolerated dose applicable in Fischer344 rats was explored via body weight monitoring of the rats after single intravenous injection of 188Re-liposome with varying dosages before the treatment study. The OLINDA/EXM 1.1 software was utilized for estimating the radiation dosimetry. To assess the therapeutic efficacy, tumor-bearing rats were intravenously administered 188Re-liposome or normal saline followed by monitoring of the tumor growth and animal survival time. In addition, the histopathological examinations of tumors were conducted on the 188Re-liposome-treated rats.Results: By using bioluminescent imaging, the well-established reporter cell line (F98luc) showed a high relationship between cell number and its bioluminescent intensity (R2=0.99) in vitro; furthermore, it could also provide clear tumor imaging for monitoring tumor growth in vivo. The maximum tolerated dose of 188Re-liposome in Fischer344 rats was estimated to be 333 MBq. According to the dosimetry results, higher equivalent doses were observed in spleen and kidneys while very less were in normal brain, red marrow, and thyroid. For therapeutic efficacy study, the progression of tumor growth in terms of tumor volume and/or tumor weight was significantly slower for the 188Re-liposome-treated group than the control group (P

We examined the potential application of a non-viral gene carrier, water soluble lipopolymer (WSLP) for delivering siRNA targeting vascular endothelial growth factor (VEGF) in vitro and in vivo. WSLP was complexed with siRNA designed to inhibit human VEGF expression or scrambled siRNA as a control. WSLP readily formed nano-sized complexes ( approximately 100 nm) with siRNA and protected siRNAs from enzymatic degradation in serum conditioned media. WSLP/siRNA complexes were transfected in PC-3 cells derived from human prostate adenocarcinomas and, then the siRNA delivery efficiency of the complexes was evaluated by VEGF production inhibition assay. VEGF production was efficiently inhibited by the WSLP/siRNA complexes, while complexes of WSLP with scrambled siRNA did not show this inhibitory effect. WSLP/siRNA complexes reduced the VEGF production by 40% when compared to unmodified branched polyethylenimine (bPEI, MW=1800). Moreover, WSLP/siRNA complexes reduced tumor volume by 55% at 21 days, and by 65% at 28 days when compared to controls. These results indicate that WSLP has potential as a siRNA delivering agent and can be applied for anti-angiogenic tumor therapy.

Naked plasmid DNA (pDNA)-based gene therapy has low delivery efficiency, and consequently, low therapeutic effect. We present a biodegradable nonionic triblock copolymer, PEG(13)-PLGA(10)-PEG(13), to enhance gene delivery efficiency in skeletal muscle. Effects of PEG(13)-PLGA(10)-PEG(13) on physicochemical properties of pDNA were evaluated by atomic force microscopy (AFM) imaging, gel electrophoresis and zeta-potential analysis. AFM imaging suggested a slightly compacted structure of pDNA when it was mixed with the polymer, while zeta-potential measurement indicated an increased surface potential of negatively charged pDNA. PEG(13)-PLGA(10)-PEG(13) showed a relatively lower toxicity compared to Pluronic P85 in a skeletal muscle cell line. The luciferase expression of pDNA delivered in 0.25% polymer solution was up to three orders of magnitude more than branched polyethylenimine (bPEI(25 k))/pDNA and three times more than that of naked pDNA five days after intramuscular administration. This in vivo gene delivery enhancement was also observed displaying a two-fold higher expression of human vascular endothelial growth factor (VEGF). Based on fluorescence labeled pDNA distribution, it is speculated that the greater diffusivity of PEG(13)-PLGA(10)-PEG(13)/pDNA compared to bPEI(25 k)/pDNA accounts for better transfection efficiency in vivo. To summarize, combining PEG(13)-PLGA(10)-PEG(13) with pDNA possesses the potential to improve gene delivery efficiency in skeletal muscle.

Frequency-domain optical coherence tomography (FD-OCT) has the advantages of an improved sensitivity and a reduced acquisition time since it enables cross-sectional images of the sample of interest to be obtained using a broadband source or a swept source. The phase-shifting method can always be applied to reduce the additional noise within FD-OCT and provide accurate signal processing. We present a phase-shifting method in which a parallel diffraction grating pair is inserted into the reference arm of the OCT system. The gratings separate the spectrum of the illuminating light and stretch the optical pulse in time thereby producing a linear group delay between the adjacent wavelengths in the superluminescent diode light source. The simulation results show that the proposed group-delay-based phase-shifting method improves the precision of the sample parameters extracted using the FD-OCT procedure compared to those obtained using traditional techniques such as the 0/4 phase-shifting method.

Toxicity of poly(ethylene glycol acrylate) (poly(PEGA)) synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using different chain transfer agents (CTA)s is described.