Assembly of Rolled-Up Collagen Constructs on Porous Alumina Textiles.

Developing new techniques to prepare free-standing tubular scaffolds has always been a challenge in the field of regenerative medicine. Here, we report a new and simple way to prepare free-standing collagen constructs with rolled-up architecture by self-assembling nanofibers on porous alumina (Al2O3) textiles modified with different silanes, carbon or gold. Following self-assembly and cross-linking with glutaraldehyde, collagen nanofibers spontaneously rolled up on the modified Al2O3 textiles and detached. The resulting collagen constructs had an inner diameter of approximately 2 to 4 mm in a rolled-up state and could be easily detached from the underlying textiles. Mechanical testing of wet collagen scaffolds following detachment yielded mean values of 3.5 ± 1.9 MPa for the tensile strength, 41.0 ± 20.8 MPa for the Young's modulus and 8.1 ± 3.7% for the elongation at break. No roll-up was observed on Al2O3 textiles without any modification, where collagen did not assemble into fibers, either. Blends of collagen and chitosan were also found to roll into fibrous constructs on silanized Al2O3 textiles, while fibrinogen nanofibers or blends of collagen and elastin did not yield such structures. Based on these differences, we hypothesize that textile surface charge and protein charge, in combination with the porous architecture of protein nanofibers and differences in mechanical strain, are key factors in inducing a scaffold roll-up. Further studies are required to develop the observed roll-up effect into a reproducible biofabrication process that can enable the controlled production of free-standing collagen-based tubes for soft tissue engineering.

Rapid fabrication and screening of tailored functional 3D biomaterials: Validation in bone tissue repair - Part II.

Regenerative medicine strategies place increasingly sophisticated demands on 3D biomaterials to promote tissue formation at sites where tissue would otherwise not form. Ideally, the discovery/fabrication of the 3D scaffolds needs to be high-throughput and uniform to ensure quick and in-depth analysis in order to pinpoint appropriate chemical and mechanical properties of a biomaterial. Herein we present a versatile technique to screen new potential biocompatible acrylate-based 3D scaffolds with the ultimate aim of application in tissue repair. As part of this process, we identified an acrylate-based 3D porous scaffold that promoted cell proliferation followed by accelerated tissue formation, pre-requisites for tissue repair. Scaffolds were fabricated by a facile freeze-casting and an in-situ photo-polymerization route, embracing a high-throughput synthesis, screening and characterization protocol. The current studies demonstrate the dependence of cellular growth and vascularization on the porosity and intrinsic chemical nature of the scaffolds, with tuneable 3D scaffolds generated with large, interconnected pores suitable for cellular growth applied to skeletal reparation. Our studies showed increased cell proliferation, collagen and ALP expression, while chorioallantoic membrane assays indicated biocompatibility and demonstrated the angiogenic nature of the scaffolds. VEGRF2 expression in vivo observed throughout the 3D scaffolds in the absence of growth factor supplementation demonstrates a potential for angiogenesis. This novel platform provides an innovative approach to 3D scanning of synthetic biomaterials for tissue regeneration.

Effect of Collagen Nanofibers and Silanization on the Interaction of HaCaT Keratinocytes and 3T3 Fibroblasts with Alumina Nanopores.

During wound healing, a complex cascade of cellular and molecular events occurs, which is governed by topographical and biochemical cues. Therefore, optimal tissue repair requires scaffold materials with versatile structural and biochemical features. Nanoporous anodic aluminum oxide (AAO) membranes exhibit good biocompatibility along with customizable nanotopography and antimicrobial properties, which has brought them into the focus of wound treatment. However, despite their good permeability, such bioinert ceramic nanopores cannot actively promote cell growth as they lack biochemical cues to support specific ligand-receptor interactions. Therefore, we modified AAO nanopores with the biochemical features of collagen nanofibers or amino groups provided by silanization with (3-aminopropyl)triethoxysilane (APTES) to design a permeable scaffold material that can additionally promote cell adhesion. Viability assays revealed that the metabolic activity of both 3T3 fibroblasts and HaCaT keratinocytes on bare and silanized AAO pores was comparable to glass controls until 72 h. Interestingly, both cell types showed a reduced proliferation on AAO with collagen nanofibers. Nevertheless, scanning electron and fluorescence microscopy revealed that 3T3 fibroblasts exhibited a well-spread morphology with filopodia attached to the nanoporous surface of the underlying AAO membranes or nanofibrous collagen networks, thus indicating a close interaction with the composites. Keratinocytes, although growing in clusters on bare and APTES-modified AAO, also adhered well on collagen-modified AAO membranes. When in contact with Escherichia coli suspensions for 20 h, the AAO membranes successfully prevented bacteria penetration irrespective of the biochemical functionalization. In summary, both functionalization strategies have high potential to specifically control molecular signaling and cell migration to further develop alumina nanopores for wound healing.

Quercetin-Loaded Luminescent Hydroxyapatite Nanoparticles for Theranostic Application in Monolayer and Spheroid Cultures of Cervical Cancer Cell Line In Vitro.

Nanoscale materials have been explored as better alternatives to conventional therapeutic agents in cancer theranostics in the recent period due to efficacy in overcoming biological, biomedical, and biophysical barriers. Analysis on the ability of copper nanocluster (CuNC)-doped hydroxyapatite nanoparticles (Cu-HXNPs) as suitable nanocarriers for anticell proliferative application was carried out. Having high adsorption capacity, the Cu-HXNPs could be loaded with the anticancer drug quercetin, which is a polyphenolic flavonoid compound, and were used as nanocarriers to be applied on HeLa (cancer cells) and HEK-293 (normal cells). The drug release profile was found to be pH-dependent, where maximum release of quercetin from quercetin-loaded Cu-HXNPs was observed in acidic pH as compared to physiological pH. The Cu-HXNPs could release quercetin, which could effectively decline proliferation of cancer cells via generation of reactive oxygen species. Moreover, the released quercetin significantly altered the cell cycle pattern and triggered the cells to undergo apoptosis. Additionally, the efficacy of Cu-HXNPs as a nanocarrier to release quercetin on 3D spheroids of HeLa had been checked, which demonstrated significant reduction in the viability of 3D spheroids. The luminescent CuNCs used for doping HXNPs endowed the nanocarrier with the imaging property, which was an excellent feature in confirming their uptake by the cells. Thus, the study suggested Cu-HXNPs to be a beneficial nanocarrier for both bioimaging and therapeutic purpose in the field of cancer theranostics.

Rapid fabrication and screening of tailored functional 3D biomaterials.

Three dimensional synthetic polymer scaffolds have remarkable chemical and mechanical tunability in addition to biocompatibility. However, the chemical and physical space is vast in view of the number of variables that can be altered e.g. chemical composition, porosity, pore size and mechanical properties to name but a few. Here, we report the development of an array of 3D polymer scaffolds, whereby the physical and chemical properties of the polymer substrates were controlled, characterized in parallel (e.g. micro-CT scanning of 24 samples) and biological properties screened. This approach allowed the screening of 48 different polymer scaffolds constructed in situ by means of freeze-casting and photo-polymerisation with the tunable composition and 3D architecture of the polymer scaffolds facilitating the identification of optimal 3D biomaterials. As a proof of concept, the array approach was used to identify 3D polymers that were capable of supporting cell growth while controlling their behaviour. Sitting alongside classical polymer microarray technology, this novel platform reduces the gap between the identification of a biomaterial in 2D and its subsequent 3D application.

Smartphone controlled interactive portable device for theranostics in vitro.

In this work, a smartphone controlled interactive theranostic device has been developed to perform in vitro photodynamic therapy (PDT) and diagnostic assays for treatment assessment on a single platform. Further, silver nanorod (Ag NR) was identified as a photosensitizer and its effect was studied in three different cell lines. PDT was achieved with Ag NRs using low irradiation (1.4 mW/cm2 at 632 nm) from light emitting diodes (LEDs) in the device. Specifically, PDT in conjugation with widely used chemotherapeutic drug doxorubicin (Dox) proved effective in killing of HeLa cancer cells and multicellular tumor spheroids at a minimum dose of Ag (2.5 µg/mL). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) assays performed with the device indicated the therapeutic success of the delivered PDT. The device is portable and can be adapted for different wavelength irradiations and radiation doses. Additionally, wireless operation using a custom designed smartphone application makes it convenient to use in complex environments without much of human intervention.

Copper Nanocluster-Doped Luminescent Hydroxyapatite Nanoparticles for Antibacterial and Antibiofilm Applications.

Novel strategies in the field of nanotechnology for the development of suitable multifunctional drug delivery vehicles have been pursued with promising upshots. Luminescent copper nanocluster-doped hydroxyapatite nanoparticles (HAP NPs) were synthesized and applied for the delivery of antibacterial drug kanamycin. The negatively charged doped HAP NPs could electrostatically interact with the positively charged kanamycin. The kanamycin-loaded doped HAP NPs showed pronounced activity in the case of Gram-negative bacteria compared to that in Gram-positive bacteria. Upon interaction with the bacteria, kanamycin could probably generate harmful agents such as hydroxyl radical that leads to bacterial cell damage. After being incorporated with copper nanoclusters (Cu NCs), the doped HAP NPs were applied for the bioimaging of bacterial cells. The biocompatibility of doped HAP NPs was also studied in HeLa cells. As compared to copper nanoclusters, the doped HAP NPs showed excellent biocompatibility even at higher concentrations of copper. The kanamycin-loaded doped HAP NPs were further applied toward Pseudomonas aeruginosa biofilm eradication. Thus, the as-synthesized copper nanocluster-doped HAP NPs were applied as nanocarriers for antibiotic drug delivery, bioimaging, and antibiofilm applications.

Gold-Nanocluster-Embedded Mucin Nanoparticles for Photodynamic Therapy and Bioimaging.

Effective delivery of a photosensitizer with the ability to trace its eventual progress forms an important aspect in photodynamic therapy (PDT). Further, the delivery mechanism might require possessing the ability to traverse through the complex mucus barrier that offers retention of therapeutic molecules. In this work, gold nanocluster (Au NC)-embedded mucin nanoparticles were synthesized by a rapid green synthetic procedure for application as nanocarriers and to achieve image-guided PDT. The mucin-based nanocarrier exhibited excellent biocompatibility toward normal cells (HEK 293T). The photosensitizer methylene blue (MB) was loaded onto these Au NC-mucin nanoparticles (NPs). HeLa cancer cells were treated with MB-loaded Au NC-mucin nanoparticles under irradiation of 640 nm light. The cell viability assay revealed that the viability of HeLa cells was reduced to 50% after treatment with MB-loaded Au NC-mucin NPs under 640 nm irradiation. The luminescence exhibited by Au NCs in the nanocarrier was applied for tracking the delivery of MB inside the HeLa cells using confocal microscopy. The flow cytometry assays elucidated the mechanism of cell death.

Boron Doped Carbon Dots with Unusually High Photoluminescence Quantum Yield for Ratiometric Intracellular pH Sensing.

Herein we report that boron doping in carbon dots results in increased photoluminescence (PL) quantum yield, which could be used for ratiometric intracellular pH sensing in cancer cell lines. Using a mixture of citric acid monohydrate, thiourea, and boric acid, microwave-assisted synthesis of boron doped blue emitting carbon dots (B-Cdots) with an average size of 3.5±1.0 nm was achieved. For B-Cdots, the maximum quantum yield (QY) was observed to be 25.8 % (11.1 % (w/w) H3 BO3 input concentration), whereas, the same was calculated to be 16.9 % and 11.4 % for Cdots (synthesized from citric acid monohydrate and thiourea only) and P-Cdots (phosphorus doped carbon dots; synthesized using citric acid monohydrate, thiourea and phosphoric acid) (11.1 % (w/w) H3 PO4 input concentration), respectively. The observed luminescence efficiencies as obtained from steady state and time-resolved photoluminescence measurements suggest an alternative emission mechanism due to boron/phosphorus doping in carbon dots. We furthermore demonstrated facile composite formation using B-Cdots and another carbon dots with orange emission in presence of polyvinyl alcohol (PVA), resulting in white light emission (0.31, 0.32; λex 380 nm). The white light emitting composite enabled ratiometric pH sensing in the aqueous medium and showed favorable uptake properties by cancerous cells for intracellular pH sensing as well.

Acoustic Propulsion of Vitamin C Loaded Teabots for Targeted Oxidative Stress and Amyloid Therapeutics.

We report the fabrication of ascorbic acid (AA) template nanomotors using buds of Camelia sinensis, undergoing fuel-free propulsion. The motors, namely, Teabots, display propulsion by converting the sound energy from the acoustic field into a mechanical one. The mesh-like structures of the anionic Teabots facilitate superior adsorption of ascorbic acid (AA-Teabots) undergoing a controlled release. The motors show antioxidant properties at the physiological pH range by scavenging intracellular reactive oxygen species. Interestingly, the percentage release of ascorbic acid is significantly higher under the influence of ultrasound exposure, as compared to the normal pH-dependent release. The motors were also efficient in the degradation of artificially synthesized toxic amyloid fibrils. The acoustic delivery of AA-Teabots could protect HEK-293 cells from oxidative injuries alongside preventing protein-aggregation derived diseases. Soon, such acoustic powered biocompatible AA-Teabots are envisioned to provide an attractive approach in proficient delivery and controlled release of therapeutic payloads at targeted zones.

Single Platform for Gene and Protein Expression Analyses Using Luminescent Gold Nanoclusters.

A single platform for gene and protein expression studies is proposed to pursue rapid diagnostics. A common method to synthesize gold (Au) nanoclusters on both DNA and protein template was developed using a benchtop device. The method of synthesis is rapid and versatile and can be applied to different classes of DNA/protein. Employing luminescent Au nanoclusters as the signal-generating agents, the device enables carrying out reverse transcriptase polymerase chain reaction and array-based analyses of multiple genes/proteins simultaneously using switchable holders and custom-designed software. The device and methods were applied to evaluate gene profiling related to apoptosis in HeLa cancer cells and further to analyze the protein expressions of glutathione-S-transferase (GST) and GST-tagged human granulocyte macrophage colony-stimulating factor (GST-hGMCSF) recombinant proteins purified from bacterial strains of BL21(DE3) Escherichia coli (E. coli). The device with user-friendly methods for diagnosis using the luminescence of Au nanoclusters offers potential use in disease diagnostics with a vision to extend health care facilities especially to remote geographical locations.

Fluorescence Resonance Energy Transfer-Based Wash-Free Bacterial Imaging and Antibacterial Application Using a Cationic Conjugated Polyelectrolyte.

The increase in bacterial infection and antibiotic resistance has posed a severe threat to the human health. This threat has warranted an imperative demand for the development of a new and effective bactericidal material to eradicate the antibiotic-resistant pathogenic bacteria. In this work, we report the wash-free imaging of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) bacteria using cationic conjugated polyelectrolyte[9,9-bis(6'-methylimidazoliumbromide)hexyl-fluorene- co-4,7-(2,1,3-benzothiadiazole)] (PFBT-MI) based on aggregation-induced fluorescence resonance energy transfer (FRET). Cationic imidazolium group strapped on the polymer side chain not only increases its solubility in water but also helps in binding with the negatively charged bacterial membrane via electrostatic interactions to turn on its bright yellow emission. The change in the fluorescence color of conjugated polyelectrolyte in the presence of bacteria could be visualized very easily via naked eyes under a UV lamp (365 nm). Furthermore, the antibacterial activity of PFBT-MI against both S. aureus and E. coli was observed because of the amphiphilic nature of the conjugated polyelectrolyte which in turn is due to the presence of ionic functionality and conjugated polymer backbone that can intercalate very proficiently into the bacterial membrane, which disrupts the membrane integrity and thus results in toxicity. Morphologically, the membrane damage was perceived via field emission scanning electron microscopy (FESEM) images, which clearly indicated the disruption of cell membrane upon exposure to PFBT-MI. The PFBT-MI acts as an effective antibacterial agent, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of (30 µM or 23.7 µg/mL) and (60 µM or 47.7 µg/mL) for S. aureus and for E. coli (60 µM or 47.7 µg/mL) and (100 µM or 79 µg/mL), respectively. Moreover, PFBT-MI shows less cytotoxicity against mammalian cells at concentration greater than MIC.

Phenylboronic Acid Templated Gold Nanoclusters for Mucin Detection Using a Smartphone-Based Device and Targeted Cancer Cell Theranostics.

A phenylboronic acid templated gold nanocluster probe was developed to detect biomarker mucin by a noninvasive fluorescence-based method using a point-of-care smartphone-based fluorescence detection device. The gold nanocluster probe is able to detect mucin specifically. The same probe was applied for in vitro targeted bioimaging of HeLa and Hep G2 cancer cells, and it demonstrated specific therapeutic effects toward cancer cells as well as multicellular tumor spheroids imparting theranostic properties. The module is found to be more effective toward HeLa cells, and a pathway of cell death was established using flow-cytometry-based assays.

Phytaspase-loaded, Mn-doped ZnS quantum dots when embedded into chitosan nanoparticles leads to improved chemotherapy of HeLa cells using in cisplatin.

OBJECTIVES: To investigate the potential of recombinant phytaspase loaded manganese (Mn) doped zinc sulphide (ZnS) quantum dots embedded chitosan nanoparticles for augmenting cisplatin induced chemotherapy of HeLa cells. RESULTS: The recombinant phytaspase was cloned into bacterial expression vector PGEX-4T-2. The expressed and purified recombinant plant phytaspase protein from Escherichia coli BL21 was immobilized onto the cationic nanocomposite. Confocal microscopy elucidated the delivery of these luminescent nanocomposites inside cervical cancer HeLa cells. A 50% reduction in the viability of HeLa cells was achieved only in the case of phytaspase-nanocomposites-cisplatin combination at a dose of phytaspase (42 nM), nanocomposites (56.3 µg/ml) and cisplatin (0.44 µg/ml). CONCLUSION: Luminescent cationic nanocomposites were developed for intracellular delivery of recombinant phytaspase, which due to its caspase-like activity assisted in substantiating the chemotherapeutic activity of apoptosis inducing drug-cisplatin.

Biomimetically crystallized protease resistant zinc phosphate decorated with gold atomic clusters for bioimaging.

A luminescent probe synthesised via biomimetic crystallization of zinc phosphate in the presence of protein fragment stabilised gold (Au) nanoclusters is reported. The engineered probe - with Au nanoclusters assembled on the crystal - was protease resistant and offered efficient bioimaging and uptake studies.

Magnetic Field Guided Chemotaxis of iMushbots for Targeted Anticancer Therapeutics.

We report controlled migrations of an intelligent and biocompatible "iMushbot" composed of Agaricus bisporus, mushroom microcapsules coated with magnetite nanoparticles. The otherwise randomly moving microbot could meticulously direct itself toward and away from the acid- and alkali-rich regions with the help of acid, acidic catalase, and alkali stimuli, emulating the chemotaxis of microorganisms. Although the catalytic decomposition of peroxide-fuel in alkali engendered the directed alkali taxis toward higher pH region, decomposition of peroxide fuel by the acidic catalase activity led to directed acid taxis toward the lower pH region. The presence of magnetite nanoparticles not only helped in improving the "activity" of the motor through the heterogeneous catalytic decomposition of the peroxide fuel but also provided a remote magnetic control on the chemotaxis. The mesoporous iMushbots having negative ζ-potential could easily be loaded with the cationic anticancer drugs, which were magnetically guided toward the cancerous cells to cause apoptosis. The iMushbots exhibited higher degree of drug retaining capacity inside alkaline pH and showed facile drug release preferentially in the lower pH environments. The experiments show the potential of the iMushbots in retaining and transporting drugs in alkaline medium such as human blood and releasing them in acidic medium such as the cancerous tissues for cell apoptosis.

Bimetallic silver nanoparticle-gold nanocluster embedded composite nanoparticles for cancer theranostics.

A bimetallic silver nanoparticle based gold nanocluster module has been developed for theranostic cellular application by a rapid and simple galvanic exchange method. The use of chitosan as a template has helped to convert the bimetallic system into composite nanoparticles, which can be easily delivered into cancer cells. The synthesized composite nanoparticles offer the advantage of combinatorial properties of the metals present in two different nanoscale levels enabling both killing and bioimaging of cancer cells. Detailed molecular events of cell death were illustrated for this combined module to decipher apoptotic mediated cell death.

Silver Nanocluster Embedded Composite Nanoparticles for Targeted Prodrug Delivery in Cancer Theranostics.

Cancer therapy with theranostic nanoparticles having the dual properties of concurrent delivery of therapeutics and its tracking offers a huge prospect to overcome the limitations of conventional therapy. Delivery of the nontoxic prodrug, which converts into the toxic drug due to cellular stimuli, offers a great deal of scope in cancer therapy. The paracetamol dimer (PD) generally considered as nontoxic is encapsulated with fluorescent silver nanocluster (Ag NC) embedded composite nanoparticles where it acts as a prodrug. This is possibly converted to a toxic metabolite due to elevated reactive oxygen species (ROS), leading to apoptosis mediated cell death. Conjugation of folic acid with these composite NPs offers the credibility of distinguishing between two different cancer cell lines such as HeLa, which overexpresses folic acid receptors, and A549, which down-regulates its expression, probed by the fluorescence intensity of Ag NCs. Importantly, Ag NCs along with PD synergistically induce prodrug mediated targeted cell death at a much reduced concentration of silver. Thus, theranostic nanocarriers have been developed offering the dual property of therapy and imaging based on the differential uptake.

Cationic BSA Templated Au-Ag Bimetallic Nanoclusters As a Theranostic Gene Delivery Vector for HeLa Cancer Cells.

A cationic BSA nanoparticle system incorporating bimetallic Au-Ag luminescent nanoclusters (NCs) was developed for the delivery of therapeutic suicide gene in HeLa cancer cells. Combinatorial therapy is achieved with cationic BSA Au-Ag NCs composite NPs loaded with pDNA, which binds efficiently with pDNA due to its positive nature. While successful delivery of a suicide gene (CD-UPRT) into cells by pDNA loaded composite NPs initiates a therapeutic response cascade by converting prodrug 5-FC to 5-FU, Au-Ag NCs results in ROS (reactive oxygen species) triggered apoptosis mediated cell death. Further, luminescence of Au-Ag NCs serves to track the gene delivery into cells. The detailed mechanism of uptake and manner of cell death have been demonstrated to comprehend the combinatorial therapeutic efficacy of the composite system.