Promoting dentinogenesis of DPSCs through inhibiting microRNA-218 by using magnetic nanocarrier delivery.

PURPOSE: The purposes of this study are to explore the roles of microRNA-218 (miR-218) delivered by a newly designed magnetic nanocarrier: GCC-Fe3O4 (GCC-Fe) in dentinogenesis potentials of human dental pulp stem cells (DPSCs). METHODS: Human DPSCs were obtained from impacted wisdom teeth of healthy donors under the permission of National Taiwan University Hospital institutional review board (NTUH IRB). Meanwhile, the transfection efficiency of GCC-Fe was evaluated. After transfecting miR-218 (GFm) and miR-218 inhibitor (GFmi) into DPSCs for 24 h, the dentinogenesis potentials of DPSCs were then evaluated with Alizarin Red S (ARS) staining with or without induction for 1, 4, and 9 days. Possible signaling pathway was further investigated by Western Blotting. RESULTS: We found that the magnetic GCC-Fe3O4 nanocarrier was serum endurable with about 90% transfection efficiency in DPSCs under normal culture condition. Results of ARS staining indicated that miR-218 was negatively regulating dentinogenesis potentials of DPSCs after induction. When the miR-218 inhibitor was delivered, calcium deposits in DPSCs were increased significantly. We also discovered that the effects of miR-218 were further regulated through the MAPK/ERK pathway. CONCLUSION: We identified that miR-218 had a negative regulation role in the dentinogenesis of DPSCs. By inhibiting miR-218, the mineralization potentials of DPSCs were promoted after induction. In addition, we also confirmed that the highly efficient magnetic GCC-Fe3O4 nanocarrier not only was suitable for gene manipulation in biomedical studies, but also ideal for future clinical applications due to its serum endurable property.

Developing a novel cholesterol-based nanocarrier with high transfection efficiency and serum compatibility for gene therapy.

BACKGROUND/PURPOSE: Primary cells are sensitive to culture conditions, which can be more difficult to get efficient transfection. The purpose of this study is to develop a serum-compatible cholesterol-based nanocarrier for delivering therapeutic nucleic acids into cells efficiently for future clinical gene therapy. METHODS: A novel cationic 3-ß-[N-(2-guanidinoethyl)carbamoyl]-cholesterol (GEC-Chol) was mixed with cholesterol and superparamagnetic iron oxide (SPIO) nanoparticles to form GCC-Fe3O4 nanocarrier. Transfection efficiency and cytotoxicity in serum and non-serum conditions were evaluated. Florescent-labeled oligonucleotides (ODNs) were transfected as indicators. Fluorescent microscopy, confocal microscopy, and flow cytometry analysis were used for evaluations. Besides, we also delivered functional antisense c-myc ODNs as surrogates for specific gene manipulation in vitro. RESULTS: Results indicated that GCC-Fe3O4 nanocarrier could have size down to less than 135 nm, which structure was highly stable and consistent over time. It also showed great transfection efficiency and low cytotoxicity in both serum and non-serum conditions. Our results demonstrated that GCC-Fe3O4 nanocarrier had exceeded 90% transfection efficiency, which was much better than common commercialized transfection reagents under same conditions. Such nanocarrier not only worked well in cell lines, but also ideal for gene delivery in primary cells. CONCLUSION: With high transfection efficiency and serum compatibility, this novel biocompatible cholesterol-based nanocarrier provides an ideal platform especially for RNAi-based gene manipulation. It also opens a wide range of biomedical applications for in vivo cell tracking and gene therapeutics for clinical usage.

Aspirin protects human coronary artery endothelial cells against atherogenic electronegative LDL via an epigenetic mechanism: a novel cytoprotective role of aspirin in acute myocardial infarction.

AIMS: L5 is the most negatively charged subfraction of human low-density lipoprotein (LDL) and is the only subfraction of LDL capable of inducing apoptosis in cultured vascular endothelial cells (ECs) by inhibiting fibroblast growth factor-2 (FGF2) transcription. We examined whether plasma L5 levels are elevated in patients with ST-segment elevation myocardial infarction (STEMI) and whether aspirin provides epigenetic protection of human coronary artery ECs (HCAECs) exposed to L5. METHODS AND RESULTS: Plasma L5 levels were compared between patients with STEMI (n = 10) and control subjects with chest pain syndrome but a normal coronary arteriogram (n = 5). L5 was isolated from the plasma of STEMI patients and control subjects, and apoptosis, FGF2 expression, and FGF2 promoter methylation were examined in HCAECs treated with L5 and aspirin. Plasma L5 levels were significantly higher in STEMI patients than in control subjects (P < 0.001). Treatment of HCAECs with L5 resulted in reduced survival and FGF2 expression and increased CpG methylation of the FGF2 promoter. Co-treatment of HCAECs with L5 and a physiologically relevant, low concentration of aspirin (0.2 mM) attenuated the adverse effects of L5 on HCAEC survival, FGF2 expression, and FGF2 promoter methylation. In contrast, high concentrations of aspirin (≥1.0 mM) accentuated the effects of L5. CONCLUSIONS: Our results show that L5 levels are significantly increased in STEMI patients. Furthermore, L5 impairs HCAEC function through CpG methylation of the FGF2 promoter, which is suppressed in the presence of low-concentration aspirin. Our results provide evidence of a novel mechanism of aspirin in the prevention of MI.

Photothermal cancer therapy via femtosecond-laser-excited FePt nanoparticles.

FePt nanoparticles (NPs) have recently been revealed to be significant multifunctional materials for the applications of biomedical imaging, drug delivery and magnetic hyperthermia due to their novel magnetic properties. In this study, a newly discovered photothermal effect activated by the near infrared (NIR) femtosecond laser for FePt NPs was demonstrated. The threshold laser energy to destroy cancer cells was found to be comparable to that of gold nanorods (Au NRs) previously reported. Through the thermal lens technique, it was concluded that the temperature of the FePt NPs can be heated up to a couple of hundreds degree C in picoseconds under laser irradiation due to the excellent photothermal transduction efficiency of FePt NPs. This finding boosts FePt NPs versatility in multifunctional targeted cancer therapy.

In vivo tumor targeting and imaging with anti-vascular endothelial growth factor antibody-conjugated dextran-coated iron oxide nanoparticles.

BACKGROUND: Active targeting by specific antibodies combined with nanoparticles is a promising technology for cancer imaging and detection by magnetic resonance imaging (MRI). The aim of the present study is to investigate whether the systemic delivery of antivascular endothelial growth factor antibodies conjugating to the surface of functionalized supermagnetic iron oxide nanoparticles (anti-VEGF-NPs) led to target-specific accumulation in the tumor. METHODS: The VEGF expression in human colon cancer and in Balb/c mice bearing colon cancers was examined by immunohistochemistry. The distribution of these anti-VEGF-NPs particles or NPs particles were evaluated by MRI at days 1, 2, or 9 after the injection into the jugular vein of Balb/c mice bearing colon cancers. Tumor and normal tissues (liver, spleen, lung, and kidney) were collected and were examined by Prussian blue staining to determine the presence and distribution of NPs in the tissue sections. RESULTS: VEGF is highly expressed in human and mouse colon cancer tissues. MRI showed significant changes in the T*(2) signal and T(2) relaxation in the anti-VEGF-NP- injected-mice, but not in mice injected with NP alone. Examination of paraffin sections of tumor tissues stained for the iron constituent of the NPs with Prussian blue revealed a strong blue reaction in the tumors of anti-VEGF-NP-treated mice, but only a weak reaction in mice injected with NPs. In both groups, at all time points, Prussian blue-stained liver and spleen sections showed only light staining, while stained cells were rarely detected in kidney and lung sections. Transmission electron microscopy showed that many more electron-dense particles were present in endothelial cells, tumor cells, and extracellular matrix in tumor tissues in mice injected with anti-VEGF-NPs than in NP-injected mice. CONCLUSION: These results demonstrated in vivo tumor targeting and efficient accumulation of anti-VEGF-NPs in tumor tissues after systemic delivery in a colon cancer model, showing that anti-VEGF-NPs have potential for use as a molecular-targeted tumor imaging agent in vivo.

PEGylated guanidinylated polyallylamine as gene-delivery carrier.

A novel cationic co-polymer was developed by grafting poly(ethylene glycol) (PEG) on guanidinylated polyallylamine (PAA) for gene delivery. Characterization of PEG-g-guanidinylated PAA/DNA complexes demonstrated that particle size increased and surface charge decreased with increasing the amount of PEG. The results of cytotoxicity assay proved that grafted PEG could effectively decrease the cytotoxicity of the complexes. In transfection efficiency assay, HeLa cells treated with PEG(2)-g-guanidinylated PAA (formed with 17.5 µmol guanidinylated PAA and 2 µmol PEG)/DNA (0.2 µg EGFP plasmid) complexes showed a very high level of EGFP expression. In conclusion, combination of guanidinylation and PEGylation could effectively decrease the cytotoxicity and significantly increase the transfection efficiency of PAA.

A variable gene delivery carrier--biotinylated chitosan/polyethyleneimine.

A variable gene delivery system has been developed based on conjugating chitosan to biotin through a functionalized poly(ethylene glycol) (PEG) spacer, which can be used to further bind different molecules on the outer layer of a polymer/DNA complex by streptavidin (SA)-biotin linkage. In this study, TAT-conjugated SA was used as the model molecule to prove the conjugation function of the prepared complex. In addition, low-molecular-weight poly(ethyleneimine) (PEI) was added into the polymer/DNA complex to increase the transfection efficiency. The results of the luciferase assay show that the transfection efficiency of the prepared complex was significantly correlated with the amount of PEI and was further enhanced when TAT was conjugated to the complex by SA-biotin linkage. Considered to have negligible cytotoxic effects, the variable gene delivery complex prepared in this study would be of considerable potential as carriers for in vitro applications.

Effects of focused ultrasound and microbubbles on the vascular permeability of nanoparticles delivered into mouse tumors.

Ultrasound sonication with microbubbles (MBs) was evaluated for enhancement of the release of nanoparticles from vasculature to tumor tissues. In this study, tumor-bearing Balb/c mice were insonicated with focused ultrasound (FUS) in the tumors after the injection of MBs (SonoVue) and then lipid-coated quantum dot (LQD) nanoparticles (130 +/- 25 nm) were injected through the tail vein. We studied the effects of the injected MB dose (0-300 microL/kg), sonication duration (0-300 s) and treatment-procedure sequence on the accumulation of nanoparticles in the tumors 24 h after the treatment and the time response of the accumulation (0.5-24 h). After the treatment, the mice were sacrificed and perfused and then the tumor tissues were harvested for quantifying the amount of nanoparticles using graphite furnace atomic absorption spectrometry (GF-AAS). The results showed that pulsed-FUS sonication with MBs can effectively enhance the vascular permeability for LQD nanoparticle delivery into the sonicated tumors. It indicates that this technique is promising for a better nanodrug delivery for tumor chemotherapy.

Quantitative and qualitative investigation into the impact of focused ultrasound with microbubbles on the triggered release of nanoparticles from vasculature in mouse tumors.

Ultrasound-mediated microbubble destruction may enhance the release of nanoparticles from vasculature to tumor tissues. In this study, we used four different sizes of lipid-coated CdSe quantum dot (LQD) nanoparticles ranging from 30 to 180 nm, 1.0-MHz pulsed focused ultrasound (FUS) with a peak acoustic pressure of 1.2-MPa, and an ultrasound contrast agent (UCA; SonoVue) at a dose of 30 microL/kg to investigate any enhancement of targeted delivery. Tumor-bearing male Balb/c mice were first injected with UCA intravenously, were then sonicated at the tumors with FUS, and were finally injected with 50 microL of the LQD solution after the sonication. The mice were sacrificed about 24h after the sonication, and then we quantitatively and qualitatively evaluated the deposition of LQDs in the tumors by using graphite furnace atomic absorption spectrometry (GF-AAS), photoluminescence spectrometry (PL), and harmonic generation microscopy (HGM). Further, immunoblotting analysis served to identify the biochemical markers reflecting the vascular rupture. The experimental results show that the amount of LQDs deposited in tumor tissues was greater in cases of FUS/UCA application, especially for smaller LQDs, being 4.47, 2.27, 0.99, and 0.82 (microg Cd)/(g tumor) for 30, 80, 130, and 180 nm of LQDs, respectively; compared to 1.12, 0.75, 0.26, and 0.34 (microg Cd)/(g tumor) in absence of FUS/UCA. The immunoblotting analysis further indicates that FUS-induced UCA oscillation/destruction results in rupture areas in blood vessels increasing the vascular permeability and thus justifying for the higher quantity of nanoparticles deposited in tumors.

Two-photon fluorescence correlation spectroscopy of lipid-encapsulated fluorescent nanodiamonds in living cells.

Dynamics of fluorescent diamond nanoparticles in HeLa cells has been studied with two-photon fluorescence correlation spectroscopy (FCS). Fluorescent nanodiamond (FND) is an excellent fluorescent probe for bioimaging application, but they are often trapped in endosomes after cellular uptake. The entrapment prohibits FCS from being performed in a time frame of 60 s. Herein, we show that the encapsulation of FNDs within a lipid layer enhances the diffusion of the particles in the cytoplasm by more than one order of magnitude, and particles as small as 40 nm can be probed individually with high image contrast by two-photon excited luminescence. The development of the technique together with single particle tracking through one-photon excitation allows probing of both short-term and long-term dynamics of single FNDs in living cells.

Direct backward third-harmonic generation in nanostructures.

Direct-backward third harmonic generation (DBTHG) has been regarded as negligible or even inexistent due to the large value of wave-vector mismatch. In the past, BTHG signals were often interpreted as back-reflected or back-scattered forward-THG (FTHG). In this paper, we theoretically and experimentally demonstrate that backward third harmonic waves can be directly generated, and that their magnitude can be comparable with FTHG in nanostructures. Experimental data of DBTHG from ZnO thin films, CdSe quantum dots and Fe(3)O(4) nanoparticles agree well with simulation results based on the Green's function. An integral equation was also derived for fast computation of DBTHG in nano films. Our investigation suggests that DBTHG can be a potentially powerful tool in nano-science research, especially when combined with FTHG measurements.

Formulation of novel lipid-coated magnetic nanoparticles as the probe for in vivo imaging.

BACKGROUND: Application of superparamagnetic iron oxide nanoparticles (SPIOs) as the contrast agent has improved the quality of magnetic resonance (MR) imaging. Low efficiency of loading the commercially available iron oxide nanoparticles into cells and the cytotoxicity of previously formulated complexes limit their usage as the image probe. Here, we formulated new cationic lipid nanoparticles containing SPIOs feasible for in vivo imaging. METHODS: Hydrophobic SPIOs were incorporated into cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and polyethylene-glycol-2000-1,2-distearyl-3-sn-phosphatidylethanolamine (PEG-DSPE) based micelles by self-assembly procedure to form lipid-coated SPIOs (L-SPIOs). Trace amount of Rhodamine-dioleoyl-phosphatidylethanolamine (Rhodamine-DOPE) was added as a fluorescent indicator. Particle size and zeta potential of L-SPIOs were determined by Dynamic Light Scattering (DLS) and Laser Doppler Velocimetry (LDV), respectively. HeLa, PC-3 and Neuro-2a cells were tested for loading efficiency and cytotoxicity of L-SPIOs using fluorescent microscopy, Prussian blue staining and flow cytometry. L-SPIO-loaded CT-26 cells were tested for in vivo MR imaging. RESULTS: The novel formulation generates L-SPIOs particle with the average size of 46 nm. We showed efficient cellular uptake of these L-SPIOs with cationic surface charge into HeLa, PC-3 and Neuro-2a cells. The L-SPIO-loaded cells exhibited similar growth potential as compared to unloaded cells, and could be sorted by a magnet stand over ten-day duration. Furthermore, when SPIO-loaded CT-26 tumor cells were injected into Balb/c mice, the growth status of these tumor cells could be monitored using optical and MR images. CONCLUSION: We have developed a novel cationic lipid-based nanoparticle of SPIOs with high loading efficiency, low cytotoxicity and long-term imaging signals. The results suggested these newly formulated non-toxic lipid-coated magnetic nanoparticles as a versatile image probe for cell tracking.

Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy.

We demonstrated that lipid-enclosed CdSe quantum dots (LEQDs) can function as versatile contrast agents in epi-detection third harmonic generation (THG) microscopy for biological applications in vivo. With epi-THG intensities 20 times stronger than corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption, such high brightness LEQDs were proved for the abilities of cell tracking and detection of specific molecular expression in live cancer cells. Using nude mice as an animal model, the distribution of LEQD-loaded tumor cells deep in subcutaneous tissues were imaged with high THG contrast. This is the first demonstration that THG contrast can be manipulated in vivo with nanoparticles. By linking LEQDs with anti-Her2 antibodies, the expression of Her2/neu receptors in live breast cancer cells could also be easily detected through THG. Compared with fluorescence modalities, the THG modality also provides the advantage of no photobleaching and photoblinkin g effects. Combined with a high penetration 1230 nm laser, these novel features make LEQDs excellent THG contrast agents for in vivo deep-tissue imaging in the future.

Surfection: a new platform for transfected cell arrays.

Efficient high-throughput expression of genes in mammalian cells can facilitate large-scale functional genomic studies. Towards this aim, we developed a simple yet powerful method to deliver genes into cells by cationic polymers on the surface of substrates. Transfection can be achieved by directly contacting nucleic acid-cell mixtures with the cationic substrates, e.g. polyethylenimine/collagen-coated wells. This single-step matrix-surface- mediated transfection method, termed 'surfection', can efficiently deliver multiple plasmids into cells and can successfully assay siRNA-mediated gene silencing. This technology represents the easiest method to transfer combinations of genes in large-scale arrays, and is a versatile tool for live-cell imaging and cell-based drug screening.

Enhanced gene delivery to HER-2-overexpressing breast cancer cells by modified immunolipoplexes conjugated with the anti-HER-2 antibody.

Cationic liposome-mediated gene delivery to tumors has met with only limited success due to the low transfection efficiency and lack of target specificity. We developed a gene delivery system for HER-2-overexpressing cells by adding modified anti-HER-2 Fab' fragments to liposome/DNA complexes (lipoplexes). The modified anti-HER-2-Fab' was conjugated to liposomes containing cationic lipids such as 1,2-dioleoyl-3-(trimethylammonium) propane and cholesterol (1:1 w/w) using a maleimido-polyethyleneglycol-3400-1,2-dioleoyl-3-sn-phosphatidylethanolamine linker. The specific modification constricted the sizes of these immunolipoplexes to a range of 0.3- 0.7 microm, and they remained stable for a longer duration of time compared to the lipoplex controls (0.8-3.2 microm at 4 h). In addition, a 10-fold increase in luciferase activity was achieved after transfecting human breast cancer SK-BR3 cells with immunolipoplexes as compared to the control lipoplexes. Flow cytometry analysis demonstrated that 80% of SK-BR3 cells expressed the green fluorescent protein (GFP) 48 h after being transfected with immunolipoplexes, while only 40% of those with control lipoplexes and 3% of those with naked DNA alone expressed GFP. Furthermore, the anti-HER-2 immunolipoplexes showed specific enhancement of transfection efficiency in HER-2-overexpressing SK-BR3 cells (a 6-fold increase in luciferase activity) but not in HER-2-negative MCF-7 breast cancer cells. The enhancement of gene delivery by anti-HER-2 immunoliposomes was not affected by the presence of serum. These results demonstrate the feasibility of improving target-specific gene delivery to HER-2-overexpressing cells by insertion of lipid-modified anti-HER-2-Fab' into the preformed liposomes.

Synergistic effect of polyethylenimine and cationic liposomes in nucleic acid delivery to human cancer cells.

Polyethylenimine (PEI) and other polycations are good vehicles for transferring genes into the cells. In earlier reports, poly-L-lysine and protamine have been shown to improve gene delivery with cationic liposomes. In this study, PEI, combined with different cationic liposomes, was studied to determine the optimal conditions for gene delivery. The reporter genes, luciferase and green fluorescent protein, were used to transfect human HeLa, HepG2 and hepatoma 2.2.15 cells with various combinations of PEIs (0.8 and 25 kDa), poly-L-lysine (15-30 kDa), protamine and cationic liposomes. The highest expression level was achieved by using the combination of PEI 25 kDa (0.65 microg/microg of DNA, nitrogen-to-DNA phosphate (N/P) ratio=4.5) with 10 nmol of DOTAP-cholesterol (DOTAP-Chol, 1:1 w/w). This DNA complex formulation dramatically increased the luciferase expression 10- to 100-fold, which was much higher than those of other polycations alone, cationic liposomes alone or the combination. In addition, PEI/DOTAP-Chol combination had little cytotoxicity than DOTAP-Chol or other cationic liposomes alone. The effect of oligonucleotide (ODN) delivery facilitated by PEI and cationic liposomes was also studied in the hepatoma cell lines. We demonstrated an antisense ODN of p53 delivered by PEI/DOTAP-Chol combination effectively inhibited the biosynthesis of p53 protein in HepG2 (68% inhibiton) and 2.2.15 cells (43% inhibition). Thus, the large PEI could synergistically increase the transfection efficiency when combined with the cationic liposomes.

Simple and efficient liposomal encapsulation of topotecan by ammonium sulfate gradient: stability, pharmacokinetic and therapeutic evaluation.

Topotecan (TPT), a topoisomerase I inhibitor, is presently undergoing clinical evaluation worldwide. Previous studies have shown that entrapping TPT within multi-lamellar vesicle liposome can stabilize the lactone moiety, which is structurally important for biological activity. However, low drug:lipid ratios due to the amphipathic character and small entrapment volume in the unilamellar vesicle limits the development of pharmaceutically acceptable liposomal formulation. With an aim to improve on this drawback, we herein describe a method that utilizes the ammonium sulfate gradient to entrap TPT into liposomes. By this method, the encapsulation efficiency was over 90% and a drug:lipid molar ratio as high as 1:5.4 was reached. In comparison with free drug, liposome-encapsulated TPT is more stable in physiological conditions and shows higher in vitro cytotoxicity. Because of increased blood circulation time, the initial plasma concentration and area under the plasma concentration of liposomal drugs were 14 and 40 times, respectively, of those of free drug. Furthermore, liposome encapsulation enhanced the antitumor activity of TPT in syngeneic murine C-26 and human HTB-9 xenograft models in vivo. At a dose of 5 mg/kg, the tumor growth delay of liposomal formulation was significantly than that of free TPT. Based on these results, we believe that this liposomal TPT formulation is worthy of further clinical study.

The roles of interleukin-1 and interleukin-1 receptor antagonist in antigen-specific immune responses.

Despite evidence that interleukin (IL)-1 promotes the proliferation of some T helper 2 (Th2) cell clones in vitro, the physiological role of IL-1 in the regulation of antigen-specific immune responses remains undefined. Using a liposome-DNA delivery system, we transiently expressed IL-1 receptor antagonist (IL-1Ra) to suppress IL-1 functions at the site of the antigen-specific primary immune response. Our data indicate, for the first time, that IL-1Ra downregulates antigen-specific IL-4 and IgE responses, with concomitant enhancement of interferon-gamma and IgG2a responses in vivo. In addition, IL-1 can promote Th2 development in an IL-4-independent manner in vitro. Thus, the balance between endogenous IL-1 and IL-1Ra during the primary immune response can be an important factor in determining the antigen-specific effector function of T cells.