Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis.

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analysis of the Frequency Shift versus Force Gradient of a Dynamic AFM Quartz Tuning Fork Subject to Lennard-Jones Potential Force.

A self-sensing and self-actuating quartz tuning fork (QTF) can be used to obtain its frequency shift as function of the tip-sample distance. Once the function of the frequency shift versus force gradient is acquired, the combination of these two functions results in the relationship between the force gradient and the tip-sample distance. Integrating the force gradient once and twice elucidates the values of the interaction force and the interatomic potential, respectively. However, getting the frequency shift as a function of the force gradient requires a physical model which can describe the equations of motion properly. Most papers have adopted the single harmonic oscillator model, but encountered the problem of determining the spring constant. Their methods of finding the spring constant are very controversial in the research community and full of discrepancies. By circumventing the determination of the spring constant, we propose a method which models the prongs and proof mass as elastic bodies. Through the use of Hamilton's principle, we can obtain the equations of motion of the QTF, which is subject to Lennard-Jones potential force. Solving these equations of motion analytically, we get the relationship between the frequency shift and force gradient.

5-Methoxytryptophan-dependent inhibition of oral squamous cell carcinoma metastasis.

The metastatic status of oral cancer is highly associated with the overall survival rate of patients. Previous studies have revealed that the endogenous tryptophan metabolite 5-methoxytryptophan (5-MTP) can downregulate cyclooxygenase-2 expression; suppress tumor proliferation, migration, and invasion; and reduce the tumor size. To improve the understanding of the molecular mechanisms involved in the regulation of 5-MTP in the tumorigenesis of oral cancer, we conducted a comparative wound healing and transwell invasion assays. Our results revealed that 5-MTP reduce oral cancer cell migration and invasion ability. In addition, the results of an in vivo assay demonstrated that the growth of primary tumors was significantly inhibited by 5-MTP in OC3 oral cancer cells and in invasive OC3-I5 oral cancer cells. Moreover, enlarged spleens were observed in OC3-I5-implanted severe combined immunodeficiency mice although 5-MTP can inhibit spleen enlargement. Through comparative proteomics, we identified 32 differentially regulated protein spots by using 2D-DIGE/MALDI-TOF MS analyses. Some of the differentially regulated proteins such as amadillo-repeat-containing X-linked protein 1, phosphoglycerate kinase 1, tropomyosin alpha-1, and tropomyosin alpha-4 may be associated with the 5-MTP-dependent inhibition of oral cancer growth and metastasis. We conclude that 5-MTP plays a crucial role in inhibiting in vitro and in vivo cancer invasion and metastasis.

Redox-Sensitive Polymer/SPIO Nanocomplexes for Efficient Magnetofection and MR Imaging of Human Cancer Cells.

Magnetofection has received increasing attention for its great potential on gene therapy. To promote its clinical therapeutic applications, development of safe and effective magnetic nanocarriers is in high demand. Herein, we present a redox-sensitive polymer/metal nanocomplex system (PSPIO) for efficient magnetofection and magnet resonance imaging (MRI) on cancer cells. PSPIO was prepared by modifying SPIO with redox-sensitive polyethylenimine (SSPEI) via a ligand exchange process. PSPIO could efficiently condense plasmid DNA (pDNA) into nanoparticles, which exhibited several favorable properties for gene delivery, including protection of nucleic acids from enzymatic degradation, stable colloids in serum, and redox-responsive pDNA release. As a potential MR imaging agent, PSPIO displayed good magnetization (28.3 emu/g) and dose-dependent T2-weighted imaging contrast (R2 = 291.1 s(-1) mM(-1)) in vitro. The use of redox-sensitive SSPEI polymer contributed to much lower cytotoxicity of PSPIO compared to nondegradable bPEI25k. In vitro transfection efficiency of PSPIO was significantly enhanced under an external magnetic field. In the presence of serum, PSPIO exhibited higher transgene expression than SSPEI or bPEI25k polymer on mouse glioma (ALTS1C1) or human prostate cancer (PC3) cell lines. Taken together, it is demonstrated that PSPIO possess great potential for cancer gene therapy and molecular imaging.

Decoy receptor 3 suppresses B cell functions and has a negative correlation with disease activity in rheumatoid arthritis.

OBJECTIVES: The decoy receptor 3 (DcR3) is a member of the tumour necrosis factor (TNF) receptor superfamily and may regulate inflammation. The aim of this study was to investigate the role of DcR3 in B cell functions and its correlation to disease activity in patients with rheumatoid arthritis (RA). METHODS: The concentrations of DcR3 and TNF-α were measured by ELISA. B cell proliferation was assessed by quantification of 3H-thymidine uptake. Staphylococcus aureus Cowan (SAC) strain were used to stimulate B cell proliferation and TNF-α production. RESULTS: Compared to the osteoarthritis (OA) patients, the RA group had higher synovial DcR3 levels (3273.6±1623.2 vs. 1594.8±1190.0 pg/ml, p=0.003), which were negatively correlated with the serum erythrocyte sedimentation rate and Disease Activity Score using 28 joint counts (DAS28) scores (r=-0.560, p=0.002; r=-0.579, p<0.001, respectively). Although the RA B cells have more active characteristics, B cell proliferation induced by SAC was successfully suppressed by recombinant DcR3.Fc fusion protein with an average inhibition of 44.8%. Moreover, DcR3.Fc fusion protein was found to suppress SAC-induced TNF-α production by B cells in 8 RA patients (average inhibition 47.0%). CONCLUSIONS: The results of our study indicated that the inhibition of B cell functions by DcR3 may partially explain the negative correlation between DcR3 level and disease activity in RA patients. Our findings imply that DcR3 may be used as a biomarker for disease activity and a potential therapeutic agent in the treatment of RA.

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-ßR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

3D-Bioprinted GelMA Scaffold with ASCs and HUVECs for Engineering Vascularized Adipose Tissue.

The purpose of tissue engineering is to reconstruct parts of injured tissues and to resolve the shortage of organ donations. However, the main concern is the limited size of engineered tissue due to insufficient oxygen and nutrition distribution in large three-dimensional (3D) tissue constructs. To provide better support for cells inside the scaffolds, the vascularization of blood vessels within the scaffold could be a solution. This study compared the effects of different culturing systems using human adipose tissue-derived stem/stromal cells (ASCs), human umbilical vein endothelial cells (HUVECs), and coculture of ASCs and HUVECs in 3D-bioprinted gelatin methacrylate (GelMA) hydrogel constructs. The in vitro results showed that the number of live cells was highest in the coculture of ASCs and HUVECs in the GelMA hydrogel after culturing for 21 days. Additionally, the tubular structure was the most abundant in the GelMA hydrogel, containing both ASCs and HUVECs. In the in vivo test, blood vessels were present in both the HUVECs and the coculture of ASCs and HUVECs hydrogels implanted in mice. However, the blood vessel density was the highest in the HUVEC and ASC coculture groups. These findings indicate that the 3D-bioprinted GelMA hydrogel coculture system could be a promising biomaterial for large tissue engineering applications.

Isolation and characterizations of multidrug-resistant human cancer cells by a biodegradable nano-sensor.

Multidrug resistance (MDR) remains a significant challenge in cancer therapy, with inherent and acquired resistance distinct. While conventional drug selection processes enable the isolation of cancer cells with acquired multidrug resistance, identifying cancer cells with inherent drug resistance remains challenging. Herein, we proposed a molecular beacon (MB)-based strategy to identify and isolate the inherent MDR cancer cells. A lipid/PLGA core-shell nanoparticulate system (DNCP) was designed to deliver MB for intracellular MDR1 mRNA imaging. DNCP-MB - possess a surface potential of -8 mV and a size of 150 nm - demonstrated effective delivery of MB, remarkable selectivity towards the selected intracellular mRNA targets, and low cytotoxicity. Following DNCP transfection, fluorescence-activated cell sorting (FACS) was employed to differentiate MCF-7 cells into two distinct sub-populations: the Top 10 cells with a high level of MDR gene expression and the Bottom 10 cells with a low level of MDR gene expression, which represent inherent drug-resistant and non-drug-resistant cells, respectively. Intriguingly, we observed a positive correlation between elevated MDR1 mRNA expression and increased migration, enhanced proliferation rate, and tighter spheroid formation. Moreover, we conducted RNA sequencing analysis on the Top 10, Bottom 10, and MCF-7/ADR cells. The findings revealed a notable disparity in the gene ontology enrichment analysis of differentially expressed genes between the Top 10 and Bottom 10 cells when compared to the Bottom 10 and MCF-7/ADR cells. This novel approach provides a promising avenue for isolating inherent drug-resistant cells and holds significant potential in unraveling the mechanisms underlying inherent drug resistance.

Programmed antigen capture-harnessed dendritic cells by margination-hitchhiking lung delivery.

Adoptive T cells and immunotherapy suppress the most destructive metastatic tumors and prevent tumor recurrence by inducing T lymphocytes. However, the heterogeneity and immune privilege of invasive metastatic clusters often reduce immune cell infiltration and therapeutic efficacy. Here, the red blood cells (RBC)-hitchhiking mediated lung metastasis delivery of multi-grained iron oxide nanostructures (MIO) programming the antigen capture, dendritic cell harnessing, and T cell recruitment is developed. MIO is assembled to the surface of RBCs by osmotic shock-mediated fusion, and reversible interactions enable the transfer of MIO to pulmonary capillary endothelial cells by intravenous injection by squeezing RBCs at the pulmonary microvessels. RBC-hitchhiking delivery revealed that >65% of MIOs co-localized in tumors rather than normal tissues. In alternating magnetic field (AMF)-mediated magnetic lysis, MIO leads to the release of tumor-associated antigens, namely neoantigens and damage-associated molecular patterns. It also acted as an antigen capture agent-harnessed dendritic cells delivers these antigens to lymph nodes. By utilizing site-specific targeting, erythrocyte hitchhiker-mediated delivery of MIO to lung metastases improves survival and immune responses in mice with metastatic lung tumors.

Programmed T cells infiltration into lung metastases with harnessing dendritic cells in cancer immunotherapies by catalytic antigen-capture sponges.

T lymphocytes served as immune surveillance to suppress metastases by physically interacting with cancer cells. Whereas tumor immune privilege and heterogeneity protect immune attack, it limits immune cell infiltration into tumors, especially in invasive metastatic clusters. Here, a catalytic antigen-capture sponge (CAS) containing the catechol-functionalized copper-based metal organic framework (MOF) and chloroquine (CQ) for programming T cells infiltration is reported. The intravenously injected CAS accumulates at the tumor via the folic acid-mediated target and margination effect. In metastases, Fenton-like reaction induced by copper ions of CAS disrupts the intracellular redox potential, i.e., chemodynamic therapy (CDT), thereby reducing glutathione (GSH) levels. Furthermore, CQ helps inhibit autophagy by inducing lysosomal deacidification during CDT. This process leads to the breakdown of self-defense mechanisms, which exacerbates cytotoxicity. The therapies promote the liberation of tumor-associated antigens, such as neoantigens and damage-associated molecular patterns (DAMPs). Subsequently, the catechol groups present on CAS perform as antigen reservoirs and transport the autologous tumor-associated antigens to dendritic cells, resulting in prolonged immune activation. The CAS, which is capable of forming in-situ, serves as an antigen reservoir in CDT-mediated lung metastasis and leads to the accumulation of immune cells in metastatic clusters, thus hindering metastatic tumors.

Magnetic nanocomplexes for gene delivery applications.

Gene delivery is an indispensable technique for various biomedical applications such as gene therapy, stem cell engineering and gene editing. Recently, magnetic nanoparticles (MNPs) have received increasing attention for their use in promoting gene delivery efficiency. Under magnetic attraction, gene delivery efficiency using viral or nonviral gene carriers could be universally enhanced. Besides, magnetic nanoparticles could be utilized in magnetic resonance imaging or magnetic hyperthermia therapy, providing extra theranostic opportunities. In this review, recent research integrating MNPs with a viral or nonviral gene vector is summarized from both technical and application perspectives. Applications of MNPs in cutting-edge research technologies, such as biomimetic cell membrane nano-gene carriers, exosome-based gene delivery, cell-based drug delivery systems or CRISPR/Cas9 gene editing, are also discussed.

Arginine-tocopherol bioconjugated lipid vesicles for selective pTRAIL delivery and subsequent apoptosis induction in glioblastoma cells.

The incorporation of specific therapeutic gene into glioblastoma offers potent therapeutic strategy to treat the disease. Non-viral gene delivery vectors are of particular interest due to their tuneable transfection efficiency and easy scale-up. Herein, we demonstrate successful delivery of plasmid encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (pTRAIL) using arginine-conjugated tocopherol lipid (AT) nanovesicles into glioblastoma cell lines. Another cationic lipid, glycine-conjugated tocopherol lipid (GT) having glycine in the head group region is also synthesized as a control lipid. Both lipid-derived liposomes effectively condensed the pDNA and the corresponding biomacromolecular assemblies (lipoplexes) are efficiently transfected into different cell lines. AT-based liposomes exhibit higher transfection efficacy in various cell lines, particularly selective in glioma cell lines. At an optimized N/P ratio, both the liposomal formulations show low cytotoxicity. AT-based lipoplexes have superior cellular uptake in U87 than the control lipid GT. The expression of TRAIL protein regulated death receptor and apoptosis signaling pathway is assayed by western blot using transfection of AT-based/pTRAIL into U87 cell lines. Induction of apoptosis in U87 cells exposed to AT-based/pTRAIL plasmid is evaluated by MTT assay as well as Annexin V-propidium iodide dual-staining assay. All results indicate that the developed AT-based/pTRAIL system offers a potentially safe and efficient therapeutic strategy for glioblastoma gene therapy.

Heparin mimicking polymer promotes myogenic differentiation of muscle progenitor cells.

Heparin and heparan sulfate mediated basic fibroblast growth factor (bFGF) signaling plays an important role in skeletal muscle homeostasis by maintaining a balance between proliferation and differentiation of muscle progenitor cells. In this study we investigate the role of a synthetic mimic of heparin, poly(sodium-4-styrenesulfonate) (PSS), on myogenic differentiation of C2C12 cells. Exogenous supplementation of PSS increased the differentiation of C2C12 cells in a dose-dependent manner, while the formation of multinucleated myotubes exhibited a nonmonotonic dependence with the concentration of PSS. Our results further suggest that one possible mechanism by which PSS promotes myogenic differentiation is by downregulating the mitogen activated extracellular regulated signaling kinase (MAPK/ERK) pathway. The binding ability of PSS to bFGF was found to be comparable to heparin through molecular docking calculations and by native PAGE. Such synthetic heparin mimics could offer a cost-effective alternative to heparin and also reduce the risk associated with batch-to-batch variation and contamination of heparin.

Thermoprecipitation of Glutathione S-Transferase by Glutathione-Poly(N-isopropylacrylamide) Prepared by RAFT Polymerization.

Herein, we report an effective and rapid method to purify glutathione S-transferase (GST) using glutathione (GSH)-modified poly(N-isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide-pNIPAAm reached 95% within 30 min as determined by UV-Vis monitoring of the release of pyridine-2-thione. GST was successfully thermoprecipitated upon heating the GSH-pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large-scale purification of GST-tagged proteins.

Ultrasonic Transdermal Delivery System with Acid-Base Neutralization-Generated CO2 Microbubble Cavitation.

Transdermal delivery systems provide a convenient noninvasive approach for drug administration through the skin, and they have been widely developed for in-home health care. The stratum corneum of the skin surface limits drug penetration, but ultrasound (US)-stimulated microbubble (MB) cavitation can enhance skin permeability to promote transdermal drug penetration. However, the specific materials and complex fabrication of MBs influence the scope of application in transdermal delivery systems. Hence, we studied the mixture of citric acid and NaHCO3 agents to generate shell-free CO2-MBs by acid-base neutralization effect. The generation rate of CO2-MBs was 36.3 ± 10 MBs/s and the mean size was 110 ± 14 µm under US sonication (3.1 MHz, 0.5 W/cm2, 50% duty cycle, 1 min). The penetration of Evans blue and FITC-conjugated hyaluronic acid in rat abdominal skin by CO2-MB cavitation improved 2.4 ± 0.3 and 2.1 ± 0.1 fold, respectively. The penetration depth of Evans blue (27.1 ± 5.1 µm) reached the epidermal layer, providing the potential for inducing transcutaneous immunization. Therefore, we proposed a simple and self-operating ultrasonic transdermal delivery system with CO2-MB cavitation to improve drug penetration for in-home health care development.

A preliminary study of Parkinson's gene therapy via sono-magnetic sensing gene vector for conquering extra/intracellular barriers in mice.

BACKGROUND: Non-virus genetic treatment for Parkinson's disease (PD) via plasmid glial cell-line derived neurotrophic factor (pGDNF) has shown potential for repairing damaged dopaminergic neurons. However, development of this gene therapy is largely hampered by the insufficient transfection efficiency as a result of the cell membrane, lysosome, and cytoskeleton meshwork. METHODS: In this study, we propose the use of polyethylenimine (PEI)-superparamagnetic iron oxide-plasmid DNA (pDNA)-loaded microbubbles (PSp-MBs) in conjunction with focused ultrasound (FUS) and two-step magnetic navigation to provide cavitation, proton sponge effect and magnetic effects to increase the efficiency of gene delivery. RESULTS: The gene transfection rate in the proposed system was 2.2-fold higher than that of the commercial agent (TransIT®-LT1). The transfection rate could be boosted ∼11%, ∼10%, and 6% by cavitation-magnetic hybrid enhanced cell membrane permeabilization, proton sponge effect, and magnetic-assisted cytoskeleton-reorganization, respectively. In vivo data suggested that effective gene delivery with this system results in a 3.2-fold increase in recovery of dopaminergic neurons and a 3.9-fold improvement in the motor behavior when compared to untreated genetic PD mice. CONCLUSIONS: We proposed that this novel FUS-magnetic hybrid gene delivery platform could be integrated with a variety of therapeutic genes for treating neurodegenerative diseases in the future.

Stem Cell-Based Delivery of Gold/Chlorin e6 Nanocomplexes for Combined Photothermal and Photodynamic Therapy.

Combining photothermal and photodynamic modalities has shown encouraging therapeutic efficacy against various malignant cancers. Developing a delivery method for targeting and penetrating tumors is still a major focus for advancing this therapeutic approach. Herein, we report a novel strategy involving the utilization of stem cells as a live carrier to codeliver photothermal and photodynamic agents for cancer therapy. To this end, a novel gold nanorod (AuNR)-PEG-PEI (APP)/chlorin e6 (Ce6)-loaded adipose-derived stem cell (ADSC) system is proposed in which AuNRs and Ce6 act as the photothermal and photodynamic agents, respectively. To integrate with stem cells, the APP/Ce6 nanocomplexes exhibit advantages of low drug leakage, low cytotoxicity, efficient cellular uptake, and redox-responsive release. After loading of APP/Ce6 nanocomplexes, the ADSCs still maintained good tumor tropism and were capable of penetrating into the tumor spheroids. The photothermal effect induced by exposure to near-infrared light irradiation at 808 nm promoted the release of Ce6 from the stem cells into the surroundings and hence increased its availability to treat cancer cells. APP/Ce6-loaded ADSCs exerted effective dose-dependent in vitro anticancer activities via anticipated photothermal and photodynamic effects. In a murine CT26 colon cancer model, APP/Ce6 delivered by ADSCs resulted in superior tumor suppression compared to other delivery strategies. It was also noted that in vivo applications of APP/Ce6-loaded ADSCs did not induce noticeable detrimental effects on normal tissues/organs.

Synthesis of semitelechelic maleimide poly(PEGA) for protein conjugation by RAFT polymerization.

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.

Synthesis of a pyridyl disulfide end-functionalized glycopolymer for conjugation to biomolecules and patterning on gold surfaces.

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.

Differences in cytotoxicity of poly(PEGA)s synthesized by reversible addition-fragmentation chain transfer polymerization.

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.