Importance of Polyacrylamide Hydrogel Diverse Chains and Cross-Linking Density for Cell Proliferation, Aging, and Death.

Diverse chains and cross-linking density of polymers are important for cell proliferation, aging, and death. In this study, by controlling the component ratio of N,N'-methylenebisacrylamide (Bis)/acrylamide (Acr), we prepared polyacrylamide (PAM) hydrogels with three different polymer structures using ultraviolet irradiation. Moreover, we quantified their Flory's cross-linking densities, gel concentrations, and mechanical properties and evaluated their influence to HL-7702 liver cell behavior and metabolism. Results showed that PAM hydrogel at a ratio of Bis/Acr = 1:50 (Acr-50) owned the highest cross-linking density (0.04), which provided abundant binding sites for cell adhesion and allowed for rapid cell proliferation. On the basis of the binding sites, cells had strong traction interaction from fibrillate adhesion with the polymers, allowed easy cell migration, and induced the living cell aggregations with a diameter of 800 µm. Cells in aggregation exhibited healthy cell phenotypes and functions, and also the mitosis of the cells in aggregation is exactly the same with the cells in tissue. It is concluded that cell proliferation, aging, and death can be controlled by adjusting the cross-linking density and diverse chains of matrix hydrogels. This work will be helpful to design new functional soft biomaterials for tissue regeneration in the future.

A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids.

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.

Preparation of Porous Core-Shell Poly L-Lactic Acid/Polyethylene Glycol Superfine Fibres Containing Drug.

In this paper, poly L-lactic acid (PLLA) blended with polyethylene glycol (PEG) was dissolved in methylene dichloride solution as the shell solution, and rapamycin (RAPA), was encapsulated inside the core of PLLA micro/nano fibres as a model drug. The effects of the blending ratio of PLLA to PEG, the concentration of the electrospinning solution, the voltage, the flow rate, and the encapsulation efficiency were studied. Uniform and porous RAPA-Loading PLLA fibres were obtained when the ratio of PLLA to PEG was 7/3, the concentration of PLLA was 3%, the applied voltage was 7.5 kV, and the pump speed was V(core) = 0.1 mL/h, V(shell) = 1 mL/h, repectively. The average diameter of PLLA fibres increased with the gradual increase in PLLA concentration. FTIR results showed that RAPA was successfully encapsulated into the core-co-shell PLLA fibres. Meanwhile, the RAPA-loading of coaxial electrospun PLLA fibres was significantly higher than that of the blending electrospun fibres. It was also found that the porous core-shell PLLA/PEG blending superfine fibres could regulate the appearance of pore on the surface of superfine fibres by adjusting the electrospinning parameters. The porous PLLA/PEG blending fibres can be used as drug carriers and, to improve the single way of drug release depending on the degradation of shell material to meet different need. It will be a remarkable breakthrough in the area for sustained and controlled release drug delivery system.

Methoxy poly(ethylene glycol) conjugated doxorubicin micelles for effective killing of cancer cells.

Methoxy poly(ethylene glycol) conjugated doxorubicin (mPEG-DOX) micelles are prepared for delivering drug effectively. The core of the unimolecular micelle is a DOX (doxorubicin) which is an anti-cancer chemotherapy drug, while the outer hydrophilic shell is composed of poly(ethylene glycol) (PEG) segments. Dynamic light scattering (DLS) analysis shows that the unimolecular micelles are uniform with a mean hydrodynamic diameter around 250 nm. The mPEG-DOX micelles can be internalized by the cancer cells and exhibit good cell uptake by the fluorescence microscopy. Obvious cytotoxicity is also observed when the concentration (count on DOX) is over 1 µg/mL. These findings indicate that these unique unimolecular micelles may offer a very promising approach for targeted cancer therapy.

Advances in applications of dendritic compounds.

Dendritic compounds (so-called dendrimers) with nano-scale represent unique symmetric and spherical structures. They are usually prepared through two fundamental methods based on molecular level which involved the convergent method and divergent method, although the synthetic methods have a tendency towards the diversity and functionalization. The outershell of dendrimers with rich peripheral reactive sites are easily modified by small functional molecules. Moreover, the higher generation dendrimers also possess more exposed functional groups on the surface prior to the previous one and they are much easily customized for much more applications. Consequently, based on previous researches, this review summarized wide applications of dendritic compounds in many fields which were investigated in detail, including gene vector, drug carrier, catalysis, sensor industry, photoelectric material, etc. Dendrimers provide promising tools for the cellular delivery of molecular cargos ranging in size from small molecules and peptides to proteins and DNA. More importantly, it is necessary to explore new synthetic methods and undiscovered applications of new dendrimers.

Preparation of SiO2/(PMMA/Fe3O4) nanoparticles using linolenic acid as crosslink agent for nucleic acid detection using chemiluminescent method.

Usually, magnetic nanoparticles (MNPs) are prepared based on the famous Stöber process in which divinylbenzene (DVA) is often used as a crosslink agent to synthesize SiO2/(PMMA/Fe3O4) nanoparticles. Compared with DVA, linolenic acid (LNA) is innoxious and can polymerize more easily for it has three unsaturated double bonds. In this paper, LNA was used as a new crosslink agent instead of DVA to synthesize the SiO2/(PMMA/Fe3O4) nanoparticles. The results showed that the core-shell structure could be observed obviously. The sizes of nanoparticles with core-shell structure range from 200 to 500 nm. The DNA probes which was immobilized on the surface of MNPs were used to capture the biotin modified complementary sequence of the probe, and the formed complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The specificity and sensitivity of this approach were investigated in this paper.

A mini-review of embedded 3D printing: supporting media and strategies.

Embedded 3D printing is an additive manufacturing method based on a material extrusion strategy. Its distinctive feature is that the printing process is carried out in a supporting medium, and the printed ink filaments can be embedded in the supporting medium. It makes the printing process almost undisturbed by gravity, and no additional support needs to be planned before the object is printed. In recent years, embedded 3D printing has been used in sensors, software robots, tissue engineering, dosage forms, and organ models, showing strong potential. This article summarizes embedded 3D printing's latest progress on supporting media and strategies in a short space.

Effect of single central base pair mismatch on the conformation and stability of surface immobilized DNA: molecular dynamics studies.

We combine molecular dynamics simulations and relative free energy calculation to investigate how single central base mismatch influence the conformation and stability of surface immobilized DNA. For comparison purpose, molecular dynamics simulations have been performed on a central mismatched duplex and the corresponding perfectly matched duplex, respectively. Simulations have been conducted with the generalized Born model. The relative free energy differences of the two systems suggest the single base-pair mismatch may lower the stability of DNA as expectation. The result obtained from the structural analysis shows the average conformations of the two DNA duplexes resemble the B-form, which suggests the central base pair mismatch has little effect on the overall conformation of DNA. Furthermore, our result shows the fluctuation of the perfectly matched duplex is obviously larger than that of the central base pair mismatched duplex. This study gives us a new atomic-level insight into DNA probe-target interaction on the surface. The results presented here will also aid in the design of DNA microarrays for single nucleotide polymorphisms screening.

Sulfosalicylic acid/Fe3+ based nanoscale coordination polymers for effective cancer therapy by the Fenton reaction: an inspiration for understanding the role of aspirin in the prevention of cancer.

Fenton reaction-based reactive oxygen species (ROS) generation provides a new idea for the design of ROS-mediated anticancer agents. Finding ways to increase iron uptake and to elevate the level of H2O2 in cells simultaneously is thus crucial to this strategy. Meanwhile, salicylic acid (SA) or its analogue, as the major metabolite of aspirin, has been reported to be closely associated with an intracellular redox-active product. In this work, a PEG-modified nanoscale coordination polymer (PFNC) via the self-assembly of 5-sulfosalicylic acid (SSA) with Fe3+ ions has been designed for the first time. The results show that the SSA dissociated from the PFNC can lead to the decrease of GSH and the accumulation of H2O2 in cancer cells, and thus elevate cellular ROS via the Fenton reaction. Owing to such intracellular oxidative stress, PFNC-induced ferroptotic cell death was further confirmed. In vitro cytotoxicity studies show that PFNCs display higher cytotoxicity on cancer cells than on normal cells. In vivo experiments further demonstrate that PFNCs not only possess high tumor accumulation, but also significantly inhibit the tumor growth without obvious damage toward the major organs. Based on the results, we expect that this work will provide an inspiration for understanding the role of SA, even aspirin, in the prevention of cancer.

Synthesis and characterization of SiO2/(PMMA/Fe3O4) magnetic nanocomposites.

Magnetic silica nanocomposites (magnetic nanoparticles core coated by silica shell) have the wide promising applications in the biomedical field and usually been prepared based on the famous Stöber process. However, the flocculation of Fe3O4 nanoparticles easily occurs during the silica coating, which limits the amount of magnetic silica particles produced in the Stöber process. In this paper, PMMA/Fe3O4 nanoparticles were used in the Stöber process instead of the "nude" Fe3O4 nanoparticles. And coating Fe3O4 with PMMA polymer beforehand can prevent magnetic nanoparticles from the aggregation that usually comes from the increasing of ionic strength during the hydrolyzation of tetraethoxysilane (TEOS) by the steric hindrance. The results show that the critical concentration of magnetic nanoparticles can increase from 12 mg/L for "nude" Fe3O4 nanoparticles to 3 g/L for PMMA/Fe3O4 nanoparticles during the Stöber process. And before the deposition of silica shell, the surface of PMMA/FeO4 nanoparticles had to be further modified by hydrolyzing them in CH3OH/NH3 x H2O mixture solution, which provides the carboxyl groups on their surface to react further with the silanol groups of silicic acid.

Real-time investigation of interactions between nanoparticles and cell membrane model.

Understanding the internal cellular processes of micron/nanoparticles will be important for particles toxicity studies or drug-delivery systems designing. The details and mechanisms of the cellular up-take process of micron/nano particles can hardly be described in real-time. In this study, cellular internalization of micron/nanoparticles was investigated by fluorescence spectroscopy, flow cytometry and sum frequency generation vibrational spectroscopy (SFG). Model cell membranes such as substrate supported lipid bilayers and lipid vesicles were used in this research. Especially, SFG was used to examine the behavior of each leaflet of the lipid bilayer while interacting with micron/nanoparticles. Experiments of SFG show direct evidences that micron/nanoparticles attachment lead to the lipid orientation. Vesicle dye-leakage model were used to study long-term interactions on model membrane. Results from this study provide in-depth insight into the molecular interactions between micron/nanoparticles and cell membranes, which will help to understand the particles toxicity and will be useful for the designing of micron/nanoparticles for applications.

Polyethylenimine-Mediated CCR5 Gene Knockout Using Transcription Activator-Like Effector Nucleases.

CCR5 acts as one of the key coreceptors for human immunodeficiency virus infection, which leads to acquired immune deficiency syndrome. CCR5 gene knockout comes up as an alternative method for treatment of the disease. Transcription activator-like effector nuclease is a powerful gene editing tool characterized by the ease of design, high rates of cleavage activity, and the accessibility of all ranges of transcription activator-like effector nucleases. In this study, transcription activator-like effector nuclease plasmids specifically targeting to knock out CCR5 gene were designed, constructed and then transfected to Hela and HEK293T cell lines with the assistance of the well-established gene delivery carrier polyethylenimine. The transfection efficiency of polyethylenimine reached 50-60% at the optimized N/P ratio of 6/1. The genomic CCR5 sequencing results suggested an estimated knockout efficiency of around 50% under the optimized condition. Thus, CCR5 gene knockout was achieved in cell lines by the polyethylenimine-mediated transfection of transcription activator-like effector nuclease plasmids of interest. More efforts such as the improvement of transfection efficiency of gene delivery carrier could be made to achieve higher gene knockout efficiency of transcription activator-like effector nucleases.

Long Blood Residence and Large Tumor Uptake of Ruthenium Sulfide Nanoclusters for Highly Efficient Cancer Photothermal Therapy.

Transition metal sulfide (TMS) holds great potential in cancer photothermal therapy (PTT) because of the high absorbance in the near-infrared (NIR) region. The short blood circulation time and limited tumor accumulation of TMS-based photothermal agents, however, limit their applications. Herein, we design a novel TMS-based PTT agent, ruthenium sulfide-based nanoclusters (NCs), to overcome the current limitations. We firstly develop a simple method to prepare oleic acid coated ruthenium sulfide nanodots (OA-RuS1.7 NDs) and assemble them into water-soluble NCs via sequentially coating with denatured bovine serum albumin (dBSA) and poly(ethylene glycol) (PEG). The obtained PEG-dBSA-RuS1.7 NCs possess excellent photothermal conversion ability. More significantly, they exhibit enhanced blood circulation time and tumor-targeting efficiency in vivo compared with other TMS-based PTT nanoagents, which may be attributed to their appropriate hydrodynamic diameter (~70 nm) and an ideal charge (~0 mV). These characteristics help the PEG-dBSA-RuS1.7 NCs to escape the removal by the reticuloendothelial system (RES) and kidney. All these advantages enable the PEG-dBSA-RuS1.7 NCs to selectively concentrate in tumor sites and effectively ablate the cancer cells upon NIR irradiation.

Low-fouling electrospun PLLA films modified with zwitterionic poly(sulfobetaine methacrylate)-catechol conjugates.

UNLABELLED: In this work, we modified a hydrophobic electrospun poly (l-lactic) acid (PLLA) film with poly (sulfobetaine methacrylate) (pSBMA)-catechol conjugates of different molecular weights to improve the biocompatibility of the film. These conjugates were synthesized via atom transfer radical polymerization. They consist of an ultra-low fouling pSBMA zwitterionic polymer with a surface-adhesive catechol moiety. X-ray photoelectron spectroscopy, contact angle and scanning electron microscopy experiments were performed to characterize films before and after modification with pSBMA-catechol conjugates. Enzyme-linked immunosorbent and fluorescently-labeled bovine serum albumin were used to study the interactions of proteins with these films. Results showed that low molecular weight zwitterionic pSBMA-catechol conjugates greatly discouraged protein adsorption as shown by use of single protein solutions on PLLA films when the modification was performed in ethanolic Tris-HCl solution. This work offers a convenient and effective method to modify electrospun PLLA films for biomedical applications. STATEMENT OF SIGNIFICANCE: In this work, we report a convenient and effective method to modify electrospun PLLA films using pSBMA-catechol conjugates via "graft-to" for biomedical applications. After pSBMA modification, the PLLA surface becomes hydrophilic with low contact angle and protein adsorption. Results showed that lower molecular weight zwitterionic pSBMA-catechol conjugate led to lower contact angles and better nonfouling properties on PLLA films when the coating was performed in a solution containing ethanol.

Light-Responsive Biodegradable Nanomedicine Overcomes Multidrug Resistance via NO-Enhanced Chemosensitization.

Multidrug resistance (MDR) is responsible for the relatively low effectiveness of chemotherapeutics. Herein, a nitric oxide (NO) gas-enhanced chemosensitization strategy is proposed to overcome MDR by construction of a biodegradable nanomedicine formula based on BNN6/DOX coloaded monomethoxy(polyethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA). On one hand, the nanomedicine features high biocompatibility due to the high density of PEG and biodegradable PLGA. On the other hand, the nanoformula exhibits excellent stability under physiological conditions but exhibits stimuli-responsive decomposition of BNN6 for NO gas release upon ultraviolet-visible irradiation. More importantly, after NO release is triggered, gas molecules are generated that break the nanoparticle shell and lead to the release of doxorubicin. Furthermore, NO was demonstrated to reverse the MDR of tumor cells and enhance the chemosensitization for doxorubicin therapy.

Synthesis and hybridization studies of DNA on functionalized polypropylene surfaces.

Polypropylene (PP) slice was treated with plasma in a mixture of nitrogen and hydrogen (1:2 V/V). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) demonstrated the successful grafting of amino groups. The as-treated slices were successively applied to the in-situ synthesis of oligonucleotides and an average coupling yield of more than 98% was achieved. The hybridization signals were recorded with fluorescent analysis system. The complementary and mismatched sequences were distinguished clearly, and the ratio of fluorescence intensity values (arbitrary units) for perfect match:single base mismatch:two bases mismatch:three bases mismatch is 108.6: 62.9: 22.4: 5.5. The results implied the plasmamodified PP surface was extremely stable, performed well in DNA hybridization assays, and could service as a good substrate for high-density oligonucleotide array synthesis, especially in a multistep molecular stamping method to fabricate DNA microarrays in-situ on a large scale.

Polyacrylamide gel film immobilized molecular beacon array for single nucleotide mismatch detection.

We reported polyacrylamide gel immobilized molecular beacon array for single nucleotide mismatch detection in this paper. Molecular beacons are oligonucleotide probes fluorescing upon hybridization to their complementary DNA/RNA targets with excellent sensitivity and high selectivity. The specially designed molecular beacon for immobilization contains a 15 base loop sequence with a 5 base pair stem, a polyT (20 bases) spacer, a 5'-end amino group for immobilization, a fluorescein in the middle of the sequence as the fluorophore, and a 3'-end DABCYL as the quencher. Between the 5'-end amino group and the stem, the polyT is used to minimize disability caused by 5'-end immobilization. The molecular beacon microarray was fabricated by a pin-based spotting robot and the hybridization was investigated by confocal microscope. A real-time hybridization process at room temperature was registered every minute for 20 min after the target solution was pumped into the hybridization cell. The result indicates that a polyacrylamide film coated glass slide provides an ideal solution-like environment for molecular beacon probes. The potential applications of this kind of molecular beacon array are mutation detection, disease mechanisms, disease diagnostics, etc. in a parallel, cost saving, and label-free detection way.

Delivery of PUMA Apoptosis Gene Using Polyethyleneimine-SMCC-TAT/DNA Nanoparticles: Biophysical Characterization and In Vitro Transfection Into Malignant Melanoma Cells.

A synthesized PEI-based gene delivery system, wherein PEI was crosslinked with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) conjugating trans-activating transcriptional activator (TAT), yielding PEI-SMCC-TAT (PST), a novel non-viral vector for apoptosis-related gene PUMA (p53 up regulated modulator of apoptosis), was designed and evaluated. Sulfo-SMCC is a commonly used heterobifunctional crosslinker and is soluble in water, making the crosslinking easier without organic reagent like DMSO or chloroform. The PST/pDNA nanoparticles were 171.9 nm at the optimal N/P ratio (50:1). DNA complexes of all the PST conjugation had much lower toxicity and exhibited enhancement in transfection efficiency in comparison with single PEI vector. The results also showed that the transfection efficiency of PST/pEGFP nanoparticles into malignant melanoma A375 cell increased, and PST carrying PUMA gene induced the apoptosis of A375 cells. It was suggested that PST could be a promising melanoma tumor-targeting nanovector, and have a good potential in clinical application.

Colorimetric detection of polynucleotides on polypropylene slices.

The gold-label-silver-stain method (GLSS) for DNA hybridization detection has been receiving increased interest as a colorimetric detective method, demonstrating the advantages of non-radioactivity, non-quenching effect of fluorescence and simplicity for analytical equipment. A colorimetric detection based on the GLSS method was applied to DNA arrays in situ synthesized on polypropylene (PP) slices. In this paper a simple plasma treatment was employed to graft amino (-NH2) on the polypropylene slice surfaces, where DNA probes were immobilized via in situ synthesis. Hybridization was accomplished by a sandwich hybridization format. With the amplification of Silver Enhancer Solution, the hybridization signals were recorded with a scanner. A target DNA concentration as low as 100 fM was detected. Complementary and mismatched sequences were clearly distinguished, and the ratio of the background-subtracted gray scale values for a perfect match, single-base mismatch, 2-base mismatch and 3-base mismatch is 22:16:9:4. The sensitivity of the in situ synthesis system was 3 orders of magnitude higher than that of the spotting system, and the signals of the former were about 2-times stronger than that of the latter under the same target DNA concentration.

Current progress in gene delivery technology based on chemical methods and nano-carriers.

Gene transfer methods are promising in the field of gene therapy. Current methods for gene transfer include three major groups: viral, physical and chemical methods. This review mainly summarizes development of several types of chemical methods for gene transfer in vitro and in vivo by means of nano-carriers like; calcium phosphates, lipids, and cationic polymers including chitosan, polyethylenimine, polyamidoamine dendrimers, and poly(lactide-co-glycolide). This review also briefly introduces applications of these chemical methods for gene delivery.