Incorporating nanoconfined chitin-fibrils in poly (ε-caprolactone) membrane scaffolds improves mechanical and chemical properties for biomedical application.

Engineered composite scaffolds composed of natural and synthetic polymers exhibit cooperation at the molecular level that closely mimics tissue extracellular matrix's (ECM) physical and chemical characteristics. However, due to the lack of smooth intermix capability of natural and synthetic materials in the solution phase, bio-inspired composite material development has been quite challenged. In this research, we introduced new bio-inspired material blending techniques to fabricate nanofibrous composite scaffolds of chitin nanofibrils (CNF), a natural hydrophilic biomaterial and poly (ɛ-caprolactone) (PCL), a synthetic hydrophobic-biopolymer. CNF was first prepared by acid hydrolysis technique and dispersed in trifluoroethanol (TFE); and second, PCL was dissolved in TFE and mixed with the chitin solution in different ratios. Electrospinning and spin-coating technology were used to form nanofibrous mesh and films, respectively. Physicochemical properties, such as mechanical strength, and cellular compatibility, and structural parameters, such as morphology, and crystallinity, were determined. Toward the potential use of this composite materials as a support membrane in blood-brain barrier application (BBB), human umbilical vein endothelial cells (HUVECs) were cultured, and transendothelial electrical resistance (TEER) was measured. Experimental results of the composite materials with PCL/CNF ratios from 100/00 to 25/75 showed good uniformity in fiber morphology and suitable mechanical properties. They retained the excellent ECM-like properties that mimic synthetic-bio-interface that has potential application in biomedical fields, particularly tissue engineering and BBB applications.

CXC chemokine receptor 4 (CXCR4) targeted gold nanoparticles potently enhance radiotherapy outcomes in breast cancer.

CXC chemokine receptor 4 (CXCR4) is overexpressed on most breast cancer cell surfaces including triple negative breast cancer (TNBC) which lacks traditional receptor overexpression. We targeted gold nanoparticles (GNPs) to this receptor via conjugation to anti-CXCR4 antibody (cGNPs). Irradiation of cells treated with cGNPs compared to PEGylated GNPs (pGNPs) resulted in more prominent radiosensitization of MDA-MB-231 cells with abundant CXCR4 overexpression than HTB-123 cells with moderate and MCF-7 cells with minimal CXCR4 overexpression. Overexpression of CXCR4 facilitated improved cellular internalization of cGNPs and irradiation of internalized cGNPs resulted in more unrepaired DNA double strand breaks and increased the production of oxygen free radicals compared to irradiation with non-internalized pGNPs. In a murine TNBC xenograft model, CXCR4 targeting potently increased tumor regrowth delay following radiation compared to radiation in the presence of pGNPs or vehicle alone. CXCR4 targeted GNPs enhance the efficacy of TNBC radiotherapy by increasing oxidative stress and DNA damage.

Electrodynamic assisted self-assembled fibrous hydrogel microcapsules: a novel 3D in vitro platform for assessment of nanoparticle toxicity.

Nanoparticle (NP) toxicity assessment is a critical step in assessing the health impacts of NP exposure to both consumers and occupational workers. In vitro assessment models comprising cells cultured in a two-dimensional tissue culture plate (2D-TCP) are an efficient and cost-effective choice for estimating the safety risks of NPs. However, in vitro culture of cells in 2D-TCPs distorts cell-integrin and cell-cell interactions and is not able to replicate an in vivo phenotype. Three-dimensional (3D) in vitro platforms provide a unique alternative to bridge the gap between traditional 2D in vitro and in vivo models. In this study, novel microcapsules of alginate hydrogel incorporated with natural polymeric nanofibers (chitin nanofibrils) and synthetic polymeric nanofibers poly(lactide-co-glycolide) are designed as a 3D in vitro platform. This study demonstrates for the first time that electrodynamic assisted self-assembled fibrous 3D hydrogel (3D-SAF hydrogel) microcapsules with a size in the range of 300-500 µm in diameter with a Young's modulus of 12.7-42 kPa can be obtained by varying the amount of nanofibers in the hydrogel precursor solutions. The 3D-SAF microcapsules were found to mimic the in vivo cellular microenvironment for cells to grow, as evaluated using A549 cells. Higher cellular spreading and prolonged proliferation of A549 cells were observed in 3D-SAF microcapsules compared to control microcapsules without the nanofibers. The 3D-SAF microcapsule integrated well plate was used to assess the toxicity of model NPs, e.g., Al2O3 and ZnO. The toxicity levels of the model NPs were found to be dependent on the chemistry of the NPs and their physical agglomeration in the test media. Our results demonstrate that 3D-SAF microcapsules with an in vivo mimicking microenvironment can be developed as a physiologically relevant platform for high-throughput toxicity screening of NPs or pharmaceutical drugs.

Nanonet-nano fiber electrospun mesh of PCL-chitosan for controlled and extended release of diclofenac sodium.

Electrospun nanofiber (EN) technology has been used in the past to generate electrostatically charged multilayer-nanofibers. This platform offers versatile applications including in tissue engineering, drug delivery, wound dressings, and high-efficiency particulate air filters. In this study, we synthesized for the first time nanonet-nanofiber electrospun meshes (NNEMs) of polycaprolactone (PCL)-chitosan (CH) using EN technology. The fabricated NNEMs were utilized for high payload delivery and controlled release of a water-soluble drug. Diclofenac Sodium (DS), a hydrophilic anti-inflammatory drug, was selected as a model drug because of its high aqueous solubility and poor compatibility with insoluble polymers. Various compositions of DS drug-loaded NNEMs (DS-NNEMs) were synthesized. The physicochemical properties such as structure, morphology, and aqueous stability and the chemical properties of DS-NNEMs were evaluated. High drug entrapment efficiency and concentration-dependent drug release patterns were investigated for up to 14 days. Furthermore, the biocompatibility of the DS-NNEMs was tested with NIH 3T3 cells. The physicochemical characterization results showed that the DS drug is a key contributing factor in the generation of nanonet-nanofiber networks during electrospinning. DS-NNEMs also enhanced 3T3 cell adhesion, viability, and proliferation in the nanonet-nano fiber network through the controlled release of DS. The presented EN technology-based biodegradable NNEM material is not only limited for the controlled release of hydrophilic anti-inflammatory drugs, but also can be a suitable platform for loading and release of antiviral drugs.

Nano-fibre Integrated Microcapsules: A Nano-in-Micro Platform for 3D Cell Culture.

Nano-in-micro (NIM) system is a promising approach to enhance the performance of devices for a wide range of applications in disease treatment and tissue regeneration. In this study, polymeric nanofibre-integrated alginate (PNA) hydrogel microcapsules were designed using NIM technology. Various ratios of cryo-ground poly (lactide-co-glycolide) (PLGA) nanofibres (CPN) were incorporated into PNA hydrogel microcapsule. Electrostatic encapsulation method was used to incorporate living cells into the PNA microcapsules (~500 µm diameter). Human liver carcinoma cells, HepG2, were encapsulated into the microcapsules and their physio-chemical properties were studied. Morphology, stability, and chemical composition of the PNA microcapsules were analysed by light microscopy, fluorescent microscopy, scanning electron microscopy (SEM), Fourier-Transform Infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The incorporation of CPN caused no significant changes in the morphology, size, and chemical structure of PNA microcapsules in cell culture media. Among four PNA microcapsule products (PNA-0, PNA-10, PNA-30, and PNA-50 with size 489 ± 31 µm, 480 ± 40 µm, 473 ± 51 µm and 464 ± 35 µm, respectively), PNA-10 showed overall suitability for HepG2 growth with high cellular metabolic activity, indicating that the 3D PNA-10 microcapsule could be suitable to maintain better vitality and liver-specific metabolic functions. Overall, this novel design of PNA microcapsule and the one-step method of cell encapsulation can be a versatile 3D NIM system for spontaneous generation of organoids with in vivo like tissue architectures, and the system can be useful for numerous biomedical applications, especially for liver tissue engineering, cell preservation, and drug toxicity study.

pH-Responsive PLGA Nanoparticle for Controlled Payload Delivery of Diclofenac Sodium.

Poly(lactic-co-glycolic acid) (PLGA) based nanoparticles have gained increasing attention in delivery applications due to their capability for controlled drug release characteristics, biocompatibility, and tunable mechanical, as well as degradation, properties. However, thorough study is always required while evaluating potential toxicity of the particles from dose dumping, inconsistent release and drug-polymer interactions. In this research, we developed PLGA nanoparticles modified by chitosan (CS), a cationic and pH responsive polysaccharide that bears repetitive amine groups in its backbone. We used a model drug, diclofenac sodium (DS), a nonsteroidal anti-inflammatory drug (NSAID), to study the drug loading and release characteristics. PLGA nanoparticles were synthesized by double-emulsion solvent evaporation technique. The nanoparticles were evaluated based on their particle size, surface charge, entrapment efficacy, and effect of pH in drug release profile. About 390-420 nm of average diameters and uniform morphology of the particles were confirmed by scanning electron microscope (SEM) imaging and dynamic light scattering (DLS) measurement. Chitosan coating over PLGA surface was confirmed by FTIR and DLS. Drug entrapment efficacy was up to 52%. Chitosan coated PLGA showed a pH responsive drug release in in vitro. The release was about 45% more at pH 5.5 than at pH 7.4. The results of our study indicated the development of chitosan coating over PLGA nanoparticle for pH dependent controlled release DS drug for therapeutic applications.

Targeted gold nanoparticles enhance sensitization of prostate tumors to megavoltage radiation therapy in vivo.

We report potent radiosensitization of prostate cancers in vitro and in vivo using goserelin-conjugated gold nanorods. Progressive receptor-mediated internalization of conjugated nanorods over time increases the radiation interaction cross-section of cells and contributes to the effects observed in vitro. The low concentrations of gold required, the long interval between injection of nanoparticles and radiation, and the use of megavoltage radiation to generate radiosensitization in vivo foretell the possibility of eventual clinical translation of this approach. FROM THE CLINICAL EDITOR: The ability of gold nanoparticles (AuNPs) to enhance the effect of physical radiation dose on tumor cells is known. This radiosensitization effect is thought to result from an increased number of photoelectric absorption events and the increased number of electrons present in gold. The authors here sought to further increase the amount and specificity of gold accumulation in prostatic cancer cells by conjugating gold nanorods to goserelin, a synthetic luteinizing hormone releasing hormone (LHRH) analogue that would bind to the LHRH receptor overexpressed in prostate cancers. It was shown that tumour cells were more sensitive to megavoltage radiation therapy. It is hoped that there would be eventual clinical translation of this approach.

Convergence of nanotechnology with radiation therapy-insights and implications for clinical translation.

Improvements in accuracy and efficacy in treating tumors with radiation therapy (RT) over the years have been fueled by parallel technological and conceptual advances in imaging and image-guidance techniques, radiation treatment machines, computational methods, and the understanding of the biology of tumor response to RT. Recent advances in our understanding of the hallmarks of cancer and the emergence of strategies to combat these traits of cancer have resulted in an expanding repertoire of targeted therapeutics, many of which can be exploited for enhancing the efficacy of RT. Complementing this advent of new treatment options is the evolution of our knowledge of the interaction between nanoscale materials and human tissues (nanomedicine). As with the changes in RT paradigms when the field has encountered newer and maturing disciplines, the incorporation of nanotechnology innovations into radiation oncology has the potential to refine or redefine its principles and revolutionize its practice. This review provides a summary of the principles, applications, challenges and outlook for the use of metallic nanoparticles in RT.

Engineered phage-based therapeutic materials inhibit Chlamydia trachomatis intracellular infection.

Developing materials that are effective against sexually transmitted pathogens such as Chlamydia trachomatis (Ct) and HIV-1 is challenging both in terms of material selection and improving bio-membrane and cellular permeability at desired mucosal sites. Here, we engineered the prokaryotic bacterial virus (M13 phage) carrying two functional peptides, integrin binding peptide (RGD) and a segment of the polymorphic membrane protein D (PmpD) from Ct, as a phage-based material that can ameliorate Ct infection. Ct is a globally prevalent human pathogen for which there are no effective vaccines or microbicides. We show that engineered phage stably express both RGD motifs and Ct peptides and traffic intracellularly and into the lumen of the inclusion in which the organism resides within the host cell. Engineered phage were able to significantly reduce Ct infection in both HeLa and primary endocervical cells compared with Ct infection alone. Polyclonal antibodies raised against PmpD and co-incubated with constructs prior to infection did not alter the course of infection, indicating that PmpD is responsible for the observed decrease in Ct infection. Our results suggest that phage-based design approaches to vector delivery that overcome mucosal cellular barriers may be effective in preventing Ct and other sexually transmitted pathogens.

Enhanced gene and siRNA delivery by polycation-modified mesoporous silica nanoparticles loaded with chloroquine.

PURPOSE: To prepare mesoporous silica-based delivery systems capable of simultaneous delivery of drugs and nucleic acids. METHODS: The surface of mesoporous silica nanoparticles (MSN) was modified with poly(ethylene glycol) (PEG) and poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA) or poly(2-(diethylamino)ethylmethacrylate) (PDEAEMA). The particles were then loaded with a lysosomotropic agent chloroquine (CQ) and complexed with plasmid DNA or siRNA. The ability of the synthesized particles to deliver combinations of CQ and nucleic acids was evaluated using luciferase plasmid DNA and siRNA targeting luciferase and GAPDH. RESULTS: The results show a slow partial MSN dissolution to form hollow silica nanoparticles in aqueous solution. The biological studies show that polycation-modified MSN are able to simultaneously deliver CQ with DNA and siRNA. The co-delivery of CQ and the nucleic acids leads to a significantly increased transfection and silencing activity of the complexes compared with MSN not loaded with CQ. CONCLUSION: PEGylated MSN modified with polycations are promising delivery vectors for combination drug/nucleic acid therapies.

N-hexanoyl chitosan-stabilized magnetic nanoparticles: enhancement of adenoviral-mediated gene expression both in vitro and in vivo.

We developed hexanoyl chloride-modified chitosan (Nac-6) stabilized iron oxide nanoparticles (Nac-6-IOPs) as magnetic nanoparticles for viral gene (Ad/LacZ) delivery via magnetofection. This vector, Nac-6-IOPs/Ad/LacZ, binds to K562 cells in the presence of external magnetic fields and results in enhanced expression of the transgene in those cells that do not exhibit the coxsackie-adenovirus receptor (CAR). Our results demonstrate that Nac-6-IOPs/Ad/LacZ is able to transduce K562 cells specifically with reduced infection of CAR- cells. The dramatic enhancement in intracellular trafficking of the adenovirus without genetically modified vesicles can lead to enhanced nuclear transfer, especially in CAR- cells. In vivo magnetofection results also clearly demonstrated that the present Nac-6-IOPs could be applied in other cell lines. Whether cells or organs, in the presence of magnetic fields Nac-6-IOPs/Ad/LacZ has high transduction efficiency. The newly formulated Nac-6-IOPs, introduced by magnetofection, provide a high-throughput gene screening both in vitro and in vivo.

N-hexanoyl chitosan stabilized magnetic nanoparticles: Implication for cellular labeling and magnetic resonance imaging.

This project involved the synthesis of N-hexanoyl chitosan or simply modified chitosan (MC) stabilized iron oxide nanoparticles (MC-IOPs) and the biological evaluation of MC-IOPs. IOPs containing MC were prepared using conventional methods, and the extent of cell uptake was evaluated using mouse macrophages cell line (RAW cells). MC-IOPs were found to rapidly associate with the RAW cells, and saturation was typically reached within the 24 h of incubation at 37 degrees C. Nearly 8.53 +/- 0.31 pg iron/cell were bound or internalized at saturation. From these results, we conclude that MC-IOPs effectively deliver into RAW cells in vitro and we also hope MC-IOPs can be used for MRI enhancing agents in biomedical fields.

Laboratory formulated magnetic nanoparticles for enhancement of viral gene expression in suspension cell line.

One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology including viral and non-viral vectors have been made, an ideal vector system has not yet been constructed. Due to the growing concerns over the toxicity and immunogenicity of viral DNA delivery systems, DNA delivery via improve viral routes has become more desirable and advantageous. The ideal improve viral DNA delivery system should be a synthetic materials plus viral vectors. The materials should also be biocompatible, efficient, and modular so that it is tunable to various applications in both research and clinical settings. The successful steps towards this improve viral DNA delivery system is demonstrated: a magnetofection system mediated by modified cationic chitosan-coated iron oxide nanoparticles. Dense colloidal cationic iron oxide nanoparticles serve as an uptake-enhancing component by physical concentration at the cell surface in presence of external magnetic fields; enhanced viral gene expression (3-100-fold) due to the particles is seen as compared to virus vector alone with little virus dose.

Novel production method and in-vitro cell compatibility of porous Ti-6Al-4V alloy disk for hard tissue engineering.

Porous metals are attractive due to its unique physical, mechanical, and new bone tissue ingrowth properties. In the present study, the production of highly porous Ti-6Al-4V parts by powder metallurgical technology and subsequently it's uses in in vitro bone tissue engineering is described. A space-holder method using carbamide with different particle size to produce parts with porosities between 35 and 70% were applied. The compressive strength and Young's modulus of porous Ti-6Al-4V were determined. Results indicated that stress and Young's modulus decrease with increasing porosity and pore size. The porous parts are characterized by scanning electron microscopy. Furthermore, study was to investigate the effects of three different porosities of porous Ti-6Al-4V (35, 50, and 70%) on proliferation, differentiation, and cell-matrix interaction of mouse osteoblast-like cells, MC-3T3. Results showed that the cell proliferation was significantly (p < 0.05) higher on 70% porous Ti-6Al-4V. However, synthesis of different types of extra cellular matrix proteins was also more abundant on 70% porous Ti-6Al-4V than 35 and 50% porous Ti-6Al-4V disk except some specific proteins. An increase in alkaline phosphate activity was significantly (p < 0.05) higher on 70 and 50% porous Ti-6Al-4V disk after 12 days of MC-3T3 cells incubation. Above all, results indicated that porosity (nearly 70%) of porous Ti-6Al-4V topography affects proliferation and differentiation of osteoblast-like MC-3T3 cells. The results showed that this novel process is a promise to fabricate porous biomaterials for bone implants.

Stabilization of gold nanoparticles by hydrophobically-modified polycations.

Surface-modified gold nanoparticles have pronounced benefits in the biomedical field due to their significant interaction with delivery materials. In the present study we used hydrophobically-modified polycations (i.e., N-acylated chitosan) to stabilize gold nanoparticles. Aliphatic hydrophobic groups, having carbon chains of different lengths, were first grafted onto the backbone of chitosan by N-acylation with fatty-acid chlorides in order to increase its hydrophobicity. Gold nanoparticles stabilized with native chitosan and N-acylated chitosan were prepared by the graft-onto approach. Chemical modification and its quantification were studied by Fourier-transform infrared (FT-IR) spectroscopy. Further, the stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-Vis, FT-IR, TEM, TGA and DLS. Spectral studies of gold nanoparticles show the backbone and the side chain functional groups of chitosan were not cleaved during the conjugation process. TEM observations revealed that the modified chitosan gold nanoparticles were well dispersed and spherical in shape with average size around 10-12 nm in triply-distilled water at pH 7.4, whereas the native chitosan gold nanoparticles appeared as clusters with 9.9 nm as average diameter and were dispersed only in dilute HCl. The size of modified chitosan gold nanoparticles varied depending on the length of grafting molecules.

Novel block copolymer (PPDO/PLLA-b-PEG): enhancement of DNA uptake and cell transfection.

The cationic lipid mediated uptake of plasmid DNA by cells in monolayer culture was significantly enhanced with an aqueous solution of the block copolymer poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol) (PPDO/PLLA-b-PEG). Plasmid uptake studies with DNA encoding the beta-galactosidase gene and cytotoxicity evaluations were performed on MCF-7, NIH 3T3 and CT-26 cell lines. Transfection yields and time courses for maximum release of FITC labeled DNA in MCF-7 cells were observed and quantified by beta-galactosidase assay and spectrofluorometry, respectively. The reported results suggest that the studied block copolymer might be useful for the enhancement of polycation mediated transfection and could find application in gene therapy.

Hydrophilic nanofibrous structure of polylactide; fabrication and cell affinity.

Microstructure and architecture of the scaffolds along with the surface chemistry exert profound effect on biological activity (cell distribution, proliferation, and differentiation). For the biological activity, scaffolds in tissue engineering have been widely designed. The objective of this study was to develop hydrophilic nanofibrous structure of polylactides (PLLA) polymer in the form of nonwoven mat by electrospinning technique, and further evaluate the fibroblast NIH3T3 cell proliferation, morphology, and cell-matrix interaction. Hydrophilicity of the PLLA fibers was improved by adding small fraction of low molecular weight polyethylene glycol (PEG) into the electrospinning solution. Four different ratio types (100/0, 80/20, 70/30, and 50/50) of PLLA/PEG electrospun matrices were fabricated, and the pore characteristics, tensile properties, contact angle, and hydrolytic degradation were observed. Furthermore, scanning electron microscope (SEM) and fluorescence actin staining images were used for micro-observation of cell-matrix interaction and cell morphology. It was found that the electrospun mat of PLLA/PEG (80/20), composed of fibers with diameters in the range 540-850 nm, majority of pore diameter less than 100 microm, tensile strength 8 MPa, elongation 150%, porosity more than 90%, and improved hydrophilicity with slow hydrolytic degradation, is favorable for biological activity of NIH3T3 fibroblast cell. Based on these results, the correct composition of PLLA and PEG in the porous electrospun matrix (i.e., PLLA/PEG (80/20)) will be a better candidate rather than other compositions of PLLA/PEG as well as hydrophobic PLLA for application in tissue engineering.

Deposition of gold nanoparticles on electrospun MgTiO3 ceramic nanofibers.

A simple method to deposit spherical gold nanoparticles on the surface of MgTiO3 ceramic nanofibers is presented. Electrospun MgTiO3/poly(vinyl acetate) (PVAc) hybrid nanofibers were calcined at 650 degrees C to obtain phase pure ceramic MgTiO3 nanofibers with 100-150 nm diameters. These ceramic nanofibers were immersed in an aqueous solution of HAuCl4 containing poly(vinyl alcohol) (PVA) as capping agent followed by photoreduction at 365 nm to get a novel Au-MgTiO3 nanocomposite. The formation of gold nanoparticles upon irradiation was confirmed by the appearance of a surface plasmon band (SPB) at 590 nm in the UV-visible absorption spectra. The surface morphology and elemental compositions were analyzed by the scanning electron microscope (SEM) equipped with energy dispersive X-ray (EDX), and transmission electron microscope (TEM). X-ray diffraction (XRD) and selected area diffraction (SAED) pattern in TEM revealed the crystallization of gold by exhibiting strong diffractions correspond to Au(111) and Au(200) crystalline planes in addition to the MgTiO3 diffraction.