Nontoxic, Biodegradable Hyperbranched Poly(ß-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(ß-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Synthesis of novel cholesterol-based cationic lipids for gene delivery.

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.

Bioactive glass-ionomer cement with potential therapeutic function to dentin capping mineralization.

We have developed a novel bioactive resin-modified glass-ionomer cement system with therapeutic function to dentin capping mineralization. In the system, the newly synthesized star-shape poly(acrylic acid) was formulated with water, Fuji II LC filler, and bioactive glass S53P4 to form resin-modified glass-ionomer cement. Compressive strength (CS) was used as a screening tool for evaluation. The commercial glass-ionomer cement Fuji II LC was used as a control. All the specimens were conditioned in simulated body fluid (SBF) at 37 degrees C prior to testing. The effect of aging in SBF on CS and microhardness of the cements was investigated. Scanning electron microscopy was used to examine the in vitro dentin surface changes caused by the incorporation of bioactive glass. The results show that the system not only provided strengths comparable to original commercial Fuji II LC cement but also allowed the cement to help mineralize the dentin in the presence of SBF. It appears that this bioactive glass-ionomer cement system has direct therapeutic impact on dental restorations that require root surface fillings.

Synthesis and preparation of novel 4-arm star-shape poly(carboxylic acid)s for improved light-cured glass-ionomer cements.

OBJECTIVE: The objective of this study was to synthesize and characterize novel 4-arm star-shape poly(acrylic acid)s (poly(AA)s) via atom-transfer radical polymerization (ATRP) technique, tether in situ light-curable methacrylate functionalities onto the poly(AA) backbone, use these star-shape poly(AA)s to formulate the light-cured glass-ionomer cements (LCGICs), and evaluate the mechanical strengths of the formed cements. MATERIALS AND METHODS: The 4-arm poly(AA)s were synthesized using ATRP and tethered with either 2-isocyanatoethyl methacrylate (IEM) or glycidyl methacrylate (GM). The polymers were formulated with 2-hydroxyethyl methacrylte (HEMA) or methacryloyl beta-alanine (MBA), water, initiators, and Fuji II LC filler. Compressive strength (CS) was used as a tool to evaluate the formed cements. The specimens were conditioned in distilled water at 37 degrees C for 24h prior to testing. RESULTS: The 4-arm poly(AA) showed a lower viscosity as compared to its linear counterpart. Both IEM-tethered and GM-tethered 4-arm poly(AA) constructed LCGICs showed significantly high mechanical strengths. Both types of co-monomer and grafting agent dramatically affected the mechanical strengths. The MBA-containing poly(AA) cements exhibited much higher CS than the HEMA-containing cements. The IEM-tethered poly(AA) cements showed much higher CS and DTS than the GM-tethered cements. CONCLUSIONS: This study developed a novel light-curable 4-arm star-shape poly(AA) system. The system was 13% in CS, 178% in DTS and 123% in FS, compared to Fuji II LC.

A novel comonomer-free light-cured glass-ionomer cement for reduced cytotoxicity and enhanced mechanical strength.

OBJECTIVE: The objective of this study was to develop a novel comonomer-free light-cured glass-ionomer system based on the 4-arm star-shape poly(acrylic acid). The mechanical strengths and in vitro cytotoxicity of the formed system were evaluated and compared with those of several representative commercial glass-ionomer cements. MATERIALS AND METHODS: The 4-arm poly(acrylic acid) was synthesized using ATRP and tethered with glycidyl methacrylate (GM). The GM-tethered polymer was formulated with water, photo-initiators, and Fuji II LC filler. Fuji II, Fuji II LC and Vitremer were used for comparison. Compressive strength (CS) and MTT assay were used as tools to evaluate the mechanical strengths and in vitro cytotoxicity of the cements, respectively. RESULTS: The experimental cement exhibited significantly high compressive, diametral tensile and flexural strengths as compared to commercial glass-ionomer cements, Fuji II, Fuji II LC and Vitremer. The effects of polymer/water (P/W) ratio, GM-grafting ratio, glass powder/polymer liquid (P/L) ratio and aging in water on strengths were significant. Similar to conventional glass-ionomer cement Fuji II, the eluates from the experimental cement showed little in vitro cytotoxicity to Balb/c mouse fibroblast cells, as compared to Fuji II LC and Vitremer that contain HEMA as a comonomer. CONCLUSIONS: It appears that this novel comonomer-free light-cured glass-ionomer cement will be a promising dental restorative because it demonstrated significantly improved mechanical strengths and almost no in vitro cytotoxicity as compared to current commercial light-cured glass-ionomer cements.

Novel injectable and in situ curable glycolide/lactide based biodegradable polymer resins and composites.

Novel in situ polymerizable liquid three-arm biodegradable oligomeric polyesters based upon glycolic acid (GA), L-lactic acid (LLA), and their copolymers are synthesized and characterized. Injectable and in situ curable polymer neat resins and their composites formulated with bioabsorbable beta-tricalcium phosphate are prepared at room temperature using photo- and redox-initiation systems, respectively. The cured neat resins show the initial compressive yield strength (YCS, MPa), modulus (M, MPa), ultimate compressive strength (UCS, MPa), and toughness (T, kN mm), ranging from 4.0 to 20.1, 201.5 to 730.2, 82.7 to 310.5, and 1.02 to 3.93. The cured composites show the initial YCS, M, UCS and T, ranging from 27.7 to 56.4, 1440 to 4870, 81.6 to 158.9, and 0.94 to 1.97. Increasing GA/LLA ratio increases all the initial compressive strengths of both neat resins and composites. Increasing filler content increases YCS and M but decreases UCS and T. A diametral tensile strength test shows the same trend as a compressive strength test. There seems to be an optimal flexural strength for the composite at the filler content around 43%. An increasing molar ratio increases curing time but decreases the degree of conversion (DC). An increasing filler content increases curing time but decreases exotherm and DC. During the course of degradation, all the materials show a burst degradation behavior within 24 h, followed by an increase in CS. The poly(glycolic acid) neat resin completely loses its strength at around Day 45. The composites completely lose their strengths at different time intervals, depending on their molar ratio and filler content. The degradation rate is found to be molar ratio and filler-content dependent.

Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.

Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.

Novel amino acid-constructed polyalkenoates for dental glass-ionomer restoratives.

Several methacrylate or acrylate derivatives of natural amino acids were synthesized and characterized. Based upon these derivatives, novel amino acid-constructed polyalkenoic acids were prepared and used to formulate glass-ionomer cements (GICs) with Fuji II glass filler. The effects of type of derivatives, molar ratio, molecular weight, and powder/liquid ratio were investigated. The results show that amino acid-constructed polyalkenoic acids can be formed only from amino acid methacrylate derivatives or by copolymerization of methacrylate with acrylate derivatives. Strong hydrogen bond interactions failed the polymer formation from acrylate derivatives. The cement composed of poly(methacryloyl glutamic acid-co-acryloyl beta-alanine) with the molar ratio of 8:2 demonstrated the best mechanical strengths along with a workable viscosity. By using the powder/liquid ratio of 3.0/1, the experimental cement exhibited a significantly higher FS (27.7 MPa), and nearly the same CS (198.5 MPa) and DTS (11.8 MPa), as compared to Fuji II (18.9 for FS, 189.1 for CS, and 11.4 MPa for DTS). During aging, the cement showed a significant increase in strength over 24 h, followed by a slow increase over 6 months.

Prevention of tissue injury by ribbon antisense to TGF-beta1 in the kidney.

Transforming growth factor-beta1 (TGF-beta1) is an important mediator of glomerulosclerosis and tubulointerstitial fibrosis in renal diseases. We designed ribbon-type antisense oligos of TGF-beta1, TGF-beta1 RiAS, and combined them with a short peptide of the nuclear localization signal to form a transfection complex of DNA/peptide/liposomes (DPL) for enhanced cellular uptake. When H4IIE cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. We then examined the ratio of the kidney weight per body weight in rats. Whereas the weight ratio was 0.47% for the normal kidney, the ratio was 0.99% on day 5 after unilateral ureteric obstruction (UUO). The ratios were 0.95% with PBS injection, 1.07% with scrambled RiAS, and 0.68% with TGF-beta1 RiAS. When examined for TGF-beta1 expression in the tissue, the level of TGF-beta1 mRNA was also significantly reduced following treatment with TGF-beta1 RiAS. Further, physical changes such as diminished dilation, atrophy, as well as apoptosis caused by UUO were also found to be markedly reduced by TGF-beta1 RiAS. The results show that ribbon antisense to TGF-beta1 when combined with efficient uptake can effectively block TGF-beta1 expression and preserve tissue integrity in kidneys with UUO.

Novel amino acid modified zinc polycarboxylates for improved dental cements.

OBJECTIVE: The objective of this study was to develop a novel amino acid modified zinc/calcium polycarboxylate cement system, formulate the cements, and evaluate their mechanical strengths. MATERIALS AND METHODS: Acrylate and methacrylate derivatives of four amino acids were synthesized and characterized using FT-IR and 1HNMR spectroscopy. The derivatives were formulated with polymer having pendent methacrylate group, water, and synthesized novel filler. Compressive (CS) as well as flexural strengths (FS) and viscosities of the resin liquids were used as tools to evaluate the formulations and formed cements. The specimens for CS and FS tests were conditioned in distilled water at 37 degrees C for 24 h and 7 d, respectively, prior to testing. RESULTS: The measured CS and FS of the cements was in the decreasing order of methacryloyl beta-alanine > acryloyl glutamic acid = acryloyl beta-alanine > methacryloyl glutamic acid > 2-hydroxyethyl methacrylate. Methacryloyl beta-alanine was selected for further formulations due to its relatively low solution viscosity and high CS as well as FS. Effects of polymer content, P/L ratio, tartaric acid and initiator concentration were significant. During aging, the cement showed a constant increase in CS for up to 1 month and then kept constant for up to 3 months. CONCLUSIONS: This study developed a novel amino acid modified zinc/calcium polycarboxylate cement system. This system was 85% higher in CS, 98% higher in DTS and 183% higher in FS, compared to Durelon.

Water sorption and dynamic mechanical properties of dentin adhesives with a urethane-based multifunctional methacrylate monomer.

OBJECTIVES: Our previous study showed the synthesis and characterization of a novel urethane-linked trimethacrylate monomer for use as a co-monomer in dentin adhesives. The objective of this work was to further investigate the performance of dentin adhesives containing a new monomer, with particular emphasis on the water sorption and viscoelastic behavior of the crosslinked networks. MATERIALS AND METHODS: Dentin adhesives contained 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) phenyl]-propane (BisGMA), 2-hydroxyethyl methacrylate (HEMA), and a new multifunctional methacrylate with urethane-linked groups-1,1,1-tri-[4-(methacryloxyethylaminocarbonyloxy)-phenyl]ethane (MPE) and were photo-polymerized in the presence or absence of water. Adhesives were characterized with regard to degree of conversion (DC), viscosity, water sorption/solubility, and dynamic mechanical analysis (DMA) and compared with BisGMA/HEMA controls. RESULTS: The experimental adhesives exhibited DC and solubility comparable to that of the control, regardless of the presence or absence of water. All the experimental adhesives tested showed less water sorption, lower tandelta peak heights, and higher rubbery modulus than the control. SIGNIFICANCE: Dentin adhesives containing a new multifunctional methacrylate showed better dynamic thermomechanical properties and water sorption relative to controls, without compromising DC and solubility. Thus, MPE, when included as a component of methacrylate dentin adhesives, may provide enhanced durability in the moist environment of the mouth.

Enzyme-catalyzed hydrolysis of dentin adhesives containing a new urethane-based trimethacrylate monomer.

A new trimethacrylate monomer with urethane-linked groups, 1,1,1-tri-[4-(methacryloxyethylamino-carbonyloxy)-phenyl]ethane (MPE), was synthesized, characterized, and used as a comonomer in dentin adhesives. Dentin adhesives containing 2-hydroxyethyl methacrylate (HEMA, 45% w/w) and 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (BisGMA, 30% w/w) in addition to MPE (25% w/w) were formulated with H(2)O at 0 (MPE0), 8 (MPE8), and 16 wt % water (MPE16) to simulate the wet demineralized dentin matrix and compared with controls [HEMA /BisGMA, 45/55 w/w, at 0 (C0), 8 (C8), and 16 wt % water (C16)]. The new adhesive showed a degree of double bond conversion and mechanical properties comparable with control, with good penetration into the dentin surface and a uniform adhesive/dentin interface. On exposure to porcine liver esterase, the net cumulative methacrylic acid (MAA) released from the new adhesives was dramatically (p < 0.05) decreased relative to the control, suggesting that the new monomer improves esterase resistance.

Dynamic mechanical analysis and esterase degradation of dentin adhesives containing a branched methacrylate.

A study of the dynamic mechanical properties and the enzymatic degradation of new dentin adhesives containing a multifunctional methacrylate are described. Adhesives contained 2-hydroxyethyl methacrylate, 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) phenyl]-propane, and a new multifunctional methacrylate with a branched side chain-trimethylolpropane mono allyl ether dimethacrylate (TMPEDMA). Adhesives were photopolymerized in the presence of 0, 8, and 16 wt % water to simulate wet bonding conditions in the mouth and compared with control adhesives. The degree of conversion as a function of irradiation time was comparable for experimental and control adhesives. In dynamic mechanical analysis, broad tan delta peaks were obtained for all samples, indicating that the polymerized networks are heterogeneous; comparison of the full-width-at-half-maximum values obtained from the tan delta curves indicated increased heterogeneity for samples cured in the presence of water and/or containing TMPEDMA. The experimental adhesive showed higher T(g) and higher rubbery modulus indicating increased crosslink density when compared with the control. The improvement in esterase resistance afforded by adhesives containing the TMPEDMA is greater when this material is photopolymerized in the presence of water, suggesting better performance in the moist environment of the mouth. The improved esterase resistance of the new adhesive could be explained in terms of the densely crosslinked network structure and/or the steric hindrance of branched alkyl side chains.

Enzymatic biodegradation of HEMA/bisGMA adhesives formulated with different water content.

Dentin adhesives may undergo phase separation when bonding to wet demineralized dentin. We hypothesized that adhesives exhibiting phase separation will experience enhanced biodegradation of methacrylate ester groups. The objective of this project was to study the effect of enzyme-exposure on the release of methacrylic acid (MAA) and 2-hydroxyethyl methacrylate (HEMA) from adhesives formulated under conditions simulating wet bonding. HEMA/bisGMA(2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane), 45/55 w/w ratio, was formulated with different water content: 0 Wt % (A00), 8 wt % (A08), and 16 wt % (A16). After a three day prewash, adhesive discs were incubated with/without porcine liver esterase (PLE) in phosphate buffer (PB, pH 7.4) at 37 degrees C for 8 days. Supernatants were collected daily and analyzed for MAA and HEMA by HPLC. For all formulations, daily MAA release in the presence of PLE was increased compared to MAA release in PB. HEMA release in the presence of PLE was not detected while HEMA release was consistently measured in PB. A08 and A16 released significantly larger amounts of HEMA compared to A00. Analysis of the cumulative release of analytes showed that the leachables in PLE was significantly increased (p < 0.05) as compared with that released in PB indicating that MAA release was not only formed from unreacted monomers but from pendant groups in the polymer network. However, the levels of analytes HEMA in PB or MAA in PLE were increased in A08 and A16 as compared with A00, which suggests that there could be a greater loss of material in HEMA/bisGMA adhesives that experience phase separation under wet bonding conditions.

Preparation and Properties of Novel Dentin Adhesives with Esterase Resistance.

A new methacrylate monomer, trimethylolpropane mono allyl ether dimethacrylate (TMPEDMA), was synthesized and evaluated. This branched methacrylate was designed to increase esterase-resistance when incorporated into conventional HEMA (2-hydroxyethyl methacrylate)/BisGMA (2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane) dental adhesives. The new adhesives, HEMA/BisGMA/TMPEDMA in a 45/30/25 (w/w) ratio were formulated with H(2)O at 0 (A0T) and 8 wt % water (A8T) and compared with control adhesives (HEMA/BisGMA, 45/55 (w/w), at 0 (A0) and 8 wt % (A8) water). Camphoroquinone (CQ), 2-(dimethylamino) ethyl methacrylate and diphenyliodonium hexafluorophosphate were used as photoinitiators. The new adhesives showed a degree of conversion comparable with the control and improved modulus and glass transition temperature (T(g)). Exposure of photopolymerized discs to porcine liver esterase for up to eight days showed that the net cumulative methacrylic acid (MAA) release in adhesives formulated with the new monomer and 8% water (A8T: 182 µg/mL) was dramatically (P < 0.05) decreased in comparison to the control (A8: 361.6 µg/mL). The results demonstrate that adhesives made with the new monomer and cured in water to simulate wet bonding are more resistant to esterase than conventional HEMA/BisGMA adhesive.

A novel light-cured glass-ionomer system for improved dental restoratives.

A novel light-cured glass-ionomer cement (LCGIC) system based on the 4-arm star-shape poly(acrylic acid) (AA) tethered with glycidyl methacrylate has been developed. The 4-arm poly(AA) polymer was synthesized using atom-transfer radical polymerization. The purified polymer was used to formulate with water and Fuji II LC filler to form LCGICs. Compressive strength (CS) was used as a screening tool for evaluation. The effects of grafting ratio, polymer/water (P/W) ratio, filler powder/polymer liquid (P/L) ratio and aging on strengths were investigated. All the specimens were conditioned in distilled water at 37 degrees C for 24 h prior to testing. The results show that the 4-arm poly(AA) polymer exhibited a lower viscosity as compared to its linear counterpart that was synthesized via conventional free-radical polymerization. This novel LCGIC system was 13% in CS, 86% in diametral tensile strength (DTS) and 123% in flexural strength higher but 93.6% in shrinkage lower than Fuji II LC. Increasing P/W ratio significantly increased both CS and DTS. Upon increasing grafting ratio, the CS was increased from 35% to 50% but not from 50% to 70%. Likewise, when P/L ratio was increased, the CS was increased from 2.2 to 2.7 but not from 2.7 to 3.0. During aging, the ultimate CS (MPa) was significantly increased from 209.2 at 1 h to 329.7 at 1 week. It appears that this novel LCGIC system will be a better dental restorative because it demonstrated improved mechanical strengths as well as little shrinkage and may eliminate cytotoxicity in current LCGICs caused by leached HEMA.

Preparation, formulation and evaluation of novel photo-cured glass ionomers based on co-polymers of (meth)acrylated amino acids.

A novel photo-cured amino-acid-constructed glass-ionomer cement system has been developed. Glutamic acid- and beta-alanine-based methacrylate and acrylate derivatives were synthesized, characterized and used to construct the polyalkenoic acids and formulated with water and Fuji II glass filler to form self-cured cement. Compressive strength (CS) of the cement and viscosity of the liquid were used as tools for evaluation. The effects of molar ratio and molecular weight (MW) were studied. The optimized co-polymer was further modified with glycidyl methacrylate (GM) and formulated with water, acrylic acid and Fuji II LC filler to form photo-cured cement. The effects of MW, GM tethering ratio, polymer liquid ratio and glass filler powder/polymer liquid (P/L) ratio were investigated. CS, flexural strength (FS) and viscosity were used as screening tools to find the optimal formulation. All the specimens were conditioned in distilled water at 37 degrees C for either 24 h or 7 days prior to testing. The results show that amino-acid-constructed polyalkenoic acids can be formed only from amino acid methacrylate derivatives or by co-polymerization of methacrylate with acrylate derivatives. Among the numerous co-polymers synthesized, poly(methacryloyl glutamic acid-co-acryloyl beta-alanine) or poly(MGA-co-ABA) with the molar ratio of 8:2 and MW of 19.5 kg/mol contributed the highest mechanical strengths and lower working viscosity to the cement. For photo-cured system, the effects of GM tethering ratio, polymer content and P/L ratio were significant. It is found that an appropriate ratio balance between these parameters is very important. The effect of molecular weight was not significant. The self-cured experimental cement was 32% higher in FS than Fuji II and the same in CS and DTS as Fuji II. The photo-cured experimental cement was 19%, 47% and 176% higher in CS, DTS and FS than Fuji II LC.