Showing papers in "Journal of Biomedical Nanotechnology in 2015"

Nanocomposite Hydrogels and Their Applications in Drug Delivery and Tissue Engineering.

Abstract: Traditional hydrogels usually possess inferior mechanical properties as well as lacking multi-functionalities. Nano-sized particles/fillers, both inorganic and organic materials, have unique chemical, physical, and biological functions, and have been extensively studied as biomaterials or bio-functional materials. Nanocomposite hydrogels, which combine the advantages of both nano-fillers and hydrogel matrices, may result in improved mechanical and biological properties and find their potential applications in biomedical field. This paper reviews recent developments in the synthesis, preparation, and characterization of nanocomposite hydrogels; their biomedical applications, such as drug delivery matrices and tissue engineering scaffolding materials are also summarized.

Towards the Design of 3D Fiber-Deposited Poly(ε-caprolactone)/lron-Doped Hydroxyapatite Nanocomposite Magnetic Scaffolds for Bone Regeneration.

Abstract: In the past few years, researchers have focused on the design and development of three-dimensional (3D) advanced scaffolds, which offer significant advantages in terms of cell performance. The introduction of magnetic features into scaffold technology could offer innovative opportunities to control cell populations within 3D microenvironments, with the potential to enhance their use in tissue regeneration or in cell-based analysis. In the present study, 3D fully biodegradable and magnetic nanocomposite scaffolds for bone tissue engineering, consisting of a poly(e-caprolactone) (PCL) matrix reinforced with iron-doped hydroxyapatite (FeHA) nanoparticles, were designed and manufactured using a rapid prototyping technique. The performances of these novel 3D PCL/FeHA scaffolds were assessed through a combination of theoretical evaluation, experimental in vitro analyses and in vivo testing in a rabbit animal model. The results from mechanical com- pression tests were consistent with FEM simulations. The in vitro results showed that the cell growth in the magnetized scaffolds was 2.2-fold greater than that in non-magnetized ones. In vivo experiments further suggested that, after only 4 weeks, the PCL/FeHA scaffolds were completely filled with newly formed bone, proving a good level of histocompatibility. All of the results suggest that the introduction of magnetic features into biocompatible materials may confer significant advantages in terms of 3D cell assembly.

Recent Developments in Active Tumor Targeted Multifunctional Nanoparticles for Combination Chemotherapy in Cancer Treatment and Imaging

Abstract: Nanotechnology and combination therapy are two major fields that show great promise in the treatment of cancer. The delivery of drugs via nanoparticles helps to improve drug's therapeutic effectiveness while reducing adverse side effects associated wifh high dosage by improving their pharmacokinetics. Taking advantage of molecular markers over-expressing on tumor tissues compared to normal cells, an "active" molecular marker targeted approach would be-beneficial for cancer therapy. These actively targeted nanoparticles would increase drug concentration at the tumor site, improving efficacy while further reducing chemo-resistance. The multidisciplinary approach may help to improve the overall efficacy in cancer therapy. This review article summarizes recent developments of targeted multifunctional nanoparticles in the delivery, of various drugs for a combinational chemotherapy approach to cancer treatment and imaging.

Polymeric Nanocarriers for Non-Viral Gene Delivery.

Abstract: Gene therapy holds great promise for the treatment of acquired and inherited diseases Non-viral gene delivery systems are gaining recognition as alternatives to viral gene vectors due to their safety and low immunogenicity The effective delivery of nucleic acids requires overcoming many biological barriers that would otherwise hinder transfection efficiency Developing safe and efficient vectors that can overcome these obstacles is at the heart of current gene delivery research Polymeric nanocarriers, such as polyethylenimine (PEI), poly(L-lysine) (PLL), poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA), polyamidoamine (PAMAM), chitosan and poly(amino-co-ester)s (PAEs), play an important role in gene delivery In this review, we first describe the current understanding of the biological barriers to non-viral gene delivery and recent progress in addressing these barriers Several of the most widely used non-viral polymeric nanocarriers are discussed, and detailed strategies that have accumulated from nearly two decades of research aimed at overcoming biological barriers and improving gene transfer are highlighted

Application of C60 Fullerene-Doxorubicin Complex for Tumor Cell Treatment In Vitro and In Vivo.

Abstract: Development of nanocarriers for effective drug delivery to molecular targets in tumor cells is a real problem in modern pharmaceutical chemistry. In the present work we used pristine C60 fullerene as a platform for delivery of anticancer drug doxorubicin (Dox) to its biological targets. The formation of a complex of C60 fullerene with Dox (C60 + Dox) is described and physico-chemical characteristics of such complex are presented. It was found that Dox conjugation with C60 fullerene leads to 1.5-2-fold increase in Dox toxicity towards various human tumor cell lines, compared with such effect when the drug is used alone. Cytotoxic activity of C60 + Dox complex is accompanied by an increased level of cell produced hydrogen peroxide at early time point (3 h) after its addition to cultured cells. At the same time, cellular production of superoxide radicals does not change in comparison with the effect of Dox alone. Cytomorphological studies have demonstrated that C60 + Dox complexes kill tumor cells by apoptosis induction. The results of in vivo experiments using Lewis lung carcinoma in mice confirmed the enhancement of the Dox toxicity towards tumor cells after drug complexation with C60 fullerene. The effect of such complex towards tumor-bearing mice was even more pronounced than that in the in vitro experiment with targeting human tumor cells. The tumor volume decreased by 2.5 times compared with the control, and an average life span of treated animals increased by 63% compared with control. The obtained results suggest a great perspective of application of C60 + Dox complexes for chemotherapy of malignant tumors.

ZnO Nanorod-Based Non-Enzymatic Optical Glucose Biosensor.

Abstract: The highly sensitive, interference-free and non-enzymatic optical sensing of glucose has been made possible for the first time using the hydrothermally synthesized ZnO nanorods. The UV irradiation of glucose-treated ZnO nanorods decomposes glucose into hydrogen peroxide (H2O2) and gluconic acid by UV oxidation. The ZnO nanorods play the role of a catalyst similar to the oxidase used in the enzymatic glucose sensors. The photoluminescence (PL) intensity of the near-band edge emission of the ZnO nanorods linearly decreased with the increased concentration of H2O2. Therefore, the glucose concentration is monitored over the wide range of 0.5-30 mM, corresponding to 9-540 mg/dL. The concentration range of the linear region in the calibration curve is suitable for its clinical use as a glucose sensor, because the glucose concentration of human serum is typically in the range of 80-120 mg/dL. In addition, the optical glucose sensor made of the ZnO nanorods is free from interference by bovin serum albumin, ascorbic acid or uric acid, which are also present in human blood. The non-enzymatic ZnO-nanorod sensor has been demonstrated with human serum samples from both normal persons and diabetic patients. There is a good agreement between the glucose concentrations measured by the PL quenching and standard clinical methods.

Role of Mesoporous Wollastonite (Calcium Silicate) in Mesenchymal Stem Cell Proliferation and Osteoblast Differentiation: A Cellular and Molecular Study.

Abstract: Wollastonite (calcium silicate) has been widely used in bone tissue engineering, but its mechanism of action on the regulation of mesenchymal stem cell proliferation and differentiation to osteoblasts still remains unclear. The current study utilized an inexpensive source of rice straw ash to synthesize wollastonite with mesoporous architecture. Mesoporous-wollastonite (m-WS) particles were characterized by transmission electron microscopy (TEM), N2 adsorption-desorption isotherms, and Fourier transform infrared (FT-IR) spectroscopy. These particles were found to be biocompatible with mouse mesenchymal stem cells (C3H10T1/2) and significantly stimulated cell proliferation by promoting the entry of the cell population from the G0/G1 phase into the S and G2/M phases via the upregulated expression of the cyclin B1 and cyclin E genes. Under osteogenic conditions, m-WS particles promoted osteoblast differentiation as indicated by calcium deposits and upregulated mRNA expression of osteoblast differentiation marker genes determined by real-time RT-PCR, depicting the osteoconductive nature of these particles. Runx2, a bone-specific transcription factor responsible for the expression of osteoblast differentiation marker genes, was upregulated in C3H10T1/2 cells. The expression of Runx2 co-regulators like Sirt-1, a positive regulator, and HDAC-4, a negative regulator, were upregulated and downregulated, respectively, by m-WS particles in these cells. Thus, this study provides a detailed insight into the effect of m-WS particles on mesenchymal stem cells at the molecular and cellular levels for in vitro bone formation.

Metallic Nanomaterials for Bone Tissue Engineering.

Abstract: Conventional grafting techniques for bone regeneration are currently being replaced by tissue engineering approaches of using 3D biomimetic materials. Of these biomaterials, metals have the highest mechanical strength; moreover, they play a major role in accelerating bone formation and promoting bone regeneration. They act as cofactors for enzymes, serving as a structural component of bone forming enzymes and proteins, stimulating angiogenesis, increasing extra-cellular matrix synthesis, promoting bone formation, and inhibiting bone resorption. Metals have the inherent ability to promote osseointegration and osteoconductivity and possess antimicrobial activity. The current developments in bone tissue engineering focus on metal surface modifications by physical and chemical treatments to improve their bioactivity. Based on the recent literature available, this review aims at discussing the biological role of metals, namely Zn, Ti, Zr, B, Sr, Mg, Ag, and Cu along with their surface modifications for significantly enhanced bone regeneration.

Multicolor Nitrogen-Doped Carbon Dots for Live Cell Imaging.

Abstract: Doping carbon dots with nitrogen atoms considerably enhances their fluorescence properties. However, the mechanism by which the carbon dots are doped is not fully understood. We developed a facile bottom-up hydrothermal carbonization (HTC) process that uses glucose and glycine as precursors for the synthesis of photoluminescent nitrogen-doped carbon dots. The as-prepared nitrogen-doped carbon dots were mono-dispersed spherical particles with a diameter of -2.8 nm. The doped nitrogen atoms assumed pyridinic type and pyrrolic type configurations to participate in the nanocrystal structure of the carbon dots. It appeared that the nitrogen doping introduces a new internal structure. The aqueous solution of nitrogen-doped carbon dots showed excitation wavelength-dependent multicolor photoluminescence. Further, these nitrogen-doped carbon dots readily entered the cytoplasm of A549 cancer cells and showed no significant cytotoxicity. The internalized nitrogen-doped carbon dots were localized to the cell membrane and cytoplasm, particularly around the nucleus. Further, the as-prepared, biocompatible, nitrogen-doped carbon dots demonstrated the potential to be used as fluorescent probes for multicolor live cell labeling, tracking, and imaging.

Surface Functionalization of Chemically Reduced Graphene Oxide for Targeted Photodynamic Therapy.

Abstract: In this study, using chemically reduced graphene oxide (GO) as a model nanocarbon, we successfully developed a facile surface-functionalization strategy of nanocarbons to allow both biocompatibility and receptor targeted drug delivery. Polyvinylpyrrolidone (PVP) coating improves aqueous dispersibility and biocompatibility of GO, and provides anchoring sites for ACDCRGDCFCG peptide (RGD4C). Aromatic photosensitizer chlorin e6 (Ce6) can be effectively loaded into the rGO-PVP-RGD system via hydrophobic interactions and π-π stacking. The nanodelivery system can significantly increase the accumulation of Ce6 in tumor cells and lead to an improved photodynamic therapy (PDT) efficacy as compared to Ce6 alone. The facile surface functionalization strategy can be applied to other nanomaterials such as carbon nanotubes, and inorganic nanomaterials.

Fabrication of Curcumin Micellar Nanoparticles with Enhanced Anti-Cancer Activity.

Abstract: Although curcumin is effective in killing cancer cells, its low water solubility and inadequate bioavailability remain major limitations to its therapeutic application. Formulating curcumin micellar nanoparticles (Cur-NPs) encapsulated with a biodegradable polymer can significantly improve curcumin's solubility, stability, and bioavailability in vitro. In this study, differently sized Cur-NPs coated with polyvinyl alcohol (PVA) were engineered. The particle size, encapsulation efficiency, in vitro release, stability, cytotoxicity, and cellular uptake of these Cur-NPs were characterized in several cancer cell lines. The results showed that, relative to solubilized curcumin, Cur-NPs demonstrated higher cytotoxicity against cancer cells. To our knowledge, this study is the first to demonstrate that the extent of the anti-cancer potency and cellular uptake of Cur-NPs is directly correlated to particle size, where Cur-NPs with the smallest size (28 nm) were the most potent. Confocal microscopy revealed the subcellular localization of the smaller Cur-NPs (28 nm) to be in both the nucleus and cytoplasm, while the larger particles (200 nm) were only localized in the cytoplasm.

Unique Biological Degradation Behavior of Stöber Mesoporous Silica Nanoparticles from Their Interiors to Their Exteriors.

Abstract: The degradation behavior of mesoporous silica nanoparticles (MSNs) influences their biological applications. The present study was a systematic investigation of the biological degradation behavior of mesoporous silica synthesized by the Stober method. Different sized Stober mesoporous silica nanoparticles were prepared and immersed in simulated body fluid, and degradation curves were obtained by measuring the dissolved silicon content of the fluid. Structural changes during degradation were observed by transmission electron microscope (TEM). The Stober mesoporous silica nanoparticles tended to become hollow during the degradation process, and each particle was almost completely degradable from its interior to its exterior. Because of this unique degradation behavior, the morphology of the Stober mesoporous silica nanoparticles can be retained even after over 85% of the silica degraded. Thus, during degradation, the dispersibility of the silica particles was superior to that of MSNs prepared in aqueous phases. Furthermore, the degradation behavior, intracellular distribution, and structural transformation of Stober mesoporous silica nanoparticles in human embryo kidney 293T cells were investigated by measuring the silicon content in culture medium and analyzing TEM images. When these silica nanoparticles degraded in cells, their size and dispersibility remained unchanged, which would reduce the biological toxicity associated with the accumulation of silica aggregates in tissues. Overall, these results demonstrate that Stober mesoporous silica nanoparticles can degrade in biological medium from inside to outside and maintain their good dispersibility, which suggests that these nanoparticles have great potential for applications as degradable biomedical materials such as drug carriers.

Folate Receptor-Targeted Dendrimer-Methotrexate Conjugate for Inflammatory Arthritis.

Abstract: Generation 5 (G5) poly(amidoamide) (PAMAM) dendrimers are synthetic polymers that have been broadly applied as drug delivery carriers. Methotrexate (MTX), an anti-folate metabolite, has been successfully used as an anti-inflammatory drug to treat rheumatoid arthritis (RA) in the clinic. In this study, we examine the therapeutic efficacy of G5 PAMAM dendrimer methotrexate conjugates (G5-MTX) that also have folic acid (FA) conjugated to the G5-MTX (G5-FA-MTX) to target inflammation-activated folate receptors overexpressing macrophages. These cells are thought to play an important role in the development of RA. With G5 serving as a control, the in vitro binding affinities of G5-FA-MTX and G5-MTX to activated macrophages were assessed in RAW264.7, NR8383 and primary rat peritoneal macrophages. The results indicated that the binding of either conjugate to macrophages was concentration- and temperature-dependent and could be blocked by the presence of 6.25 mM free FA (p < 0.005). The preventive effects of G5-MTX and G5-FA-MTX conjugates on the development of arthritis were explored on an adjuvant-induced inflammatory arthritis model and had similar preventive effects in inflammatory arthritis at a MTX equivalent dose of 4.95 μmol/kg. These studies indicated that when multiples of MTX are conjugated on dendritic polymers, they specifically bind to folate receptor overexpressing macrophages and have comparable anti-inflammatory effects to folate targeted MTX conjugated polymers.

Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

Abstract: Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated that the developed DOX-PLGA/PEI/P-gp shRNA NBs is a potential, safe and efficient theranotic agent for cancer therapy and diagnostics.

Polymer-Block-Polypeptides and Polymer-Conjugated Hybrid Materials as Stimuli-Responsive Nanocarriers for Biomedical Applications.

Abstract: Stimuli-responsive nanocarriers are a class of soft materials that includes natural polymers, synthetic polymers, and polypeptides. Recently, modern synthesis tools such as atom transfer radical polymerization, reversible addition-fragmentation chain transfer polymerization, nitroxide-mediated radical polymerization, ring-opening polymerization of α-amino acid N-carboxyanhydrides, and various "click" chemistry strategies were simultaneously employed for the design and synthesis of nanosized drug delivery vehicles. Importantly, the research focused on the improvement of the nanocarrier targetability and the site-specific, triggered release of therapeutics with high drug loading efficiency and minimal drug leakage during the delivery to specific targets. In this context, nanocarriers responsive to common stimuli such as pH, temperature, redox potential, light, etc. have been widely used for the controlled delivery of therapeutics to pathological sites. Currently, different synthesis and self-assembly strategies improved the drug loading efficacy and targeted delivery of therapeutic agents to the desired site. In particular, polypeptide-containing hybrid materials have been developed for the controlled delivery of therapeutic agents. Therefore, stimuli-sensitive synthetic polypeptide-based materials have been extensively investigated in recent years. This review focuses on recent advances in the development of polymer-block-polypeptides and polymer-conjugated hybrid materials that have been designed and evaluated for various stimuli-responsive drug and gene delivery applications.

PEGylated Dendrimer-Doxorubicin Cojugates as pH-Sensitive Drug Delivery Systems: Synthesis and In Vitro Characterization.

Abstract: To achieve liver-specific delivery of antitumor drug doxorubicin (DOX), PEGylated dendrimer-DOX conjugates were designed and synthesized, whereas DOX was conjugated to dendrimers via hydrazone bonds and the dendrimers were functionalized with galactose moieties. The release rates of DOX from the conjugates at pH 5.0 were much faster than those at pH 7.4 due to the pH-sensitive cleavage of the hydrazone bonds. The conjugates were shown to effectively kill HepG2 cells in vitro. Compared to other conjugates, the PEGylated dendrimer-DOX one with multiple galactose moieties (Dendrimer-DOX-PEG-Gal) demonstrated HepG2 cells specificity, higher efficacy and good biosafety due to the lower IC50 value and higher cellular uptake confirmed by in vitro cytotoxicity assays, confocal laser scanning microscopy and flow cytometric studies. These results suggest that Dendrimer-DOX-PEG-Gal is an efficient and biocompatible candidate for the specific delivery of antitumor drug to HepG2 cells and could be used as liver cancer specific drug delivery system.

Mucoadhesive Amphiphilic Methacrylic Copolymer-Functionalized Poly(ε-caprolactone) Nanocapsules for Nose-to-Brain Delivery of Olanzapine.

Abstract: Nose-to-brain drug delivery has been proposed to overcome the low absorption of drugs in central nervous system due to the absence of brain-blood barrier in the olfactory nerve pathway. However, the presence of a mucus layer and quick clearance limit the use of this route. Herein, amphiphilic methacrylic copolymer-functionalized poly(e-caprolactone) nanocapsules were proposed as a mucoadhesive system to deliver olanzapine after intranasal administration. In vitro evaluations showed that these nanocapsules were able to interact with mucin (up to 17% of increment in particle size and 30% of reduction of particle concentration) and nasal mucosa (2-fold higher force for detaching), as well as to increase the retention of olanzapine (about 40%) on the nasal mucosa after continuous wash. The olanzapine-loaded amphiphilic methacrylic copolymer-functionalized PCL nanocapsules enhanced the amount of drug in the brain of rats (1.5-fold higher compared to the drug solution). In accordance with this finding, this formulation improved the prepulse inhibition impairment induced by apomorphine, which is considered as an operational measure of pre-attentive sensorimotor gating impairment present in schizophrenia. Besides, nanoencapsulated olanzapine did not affect the nasal mucosa integrity after repeated doses. These data evidenced that the designed nanocapsules are a promising mucoadhesive system for nose-to-brain delivery of drugs.

Monolayer Graphene-Directed Growth and Neuronal Differentiation of Mesenchymal Stem Cells.

Abstract: The development of an efficient platform for the growth and neuronal differentiation of stem cells is crucial for autologous cell therapy and tissue engineering to treat various neuronal disorders and neurodegenerative diseases. In this study, we describe the use of highly uniform graphene platforms that provide unique environments where unusual three-dimensional spheroids of human mesenchymal stem cells (hMSCs) are formed, which is advantageous for the differentiation of hMSCs into neurons. We suppose that graphene regulates the interactions at cell-substrate or cell-cell interfaces, consequently promoting the neurogenesis of hMSCs as well as the outgrowth of neurites, which was evidenced by the graphene-induced upregulation of early neurogenesis-related genes. We also demonstrated that the differentiated neurons from hMSCs on graphene are notably sensitive to external ion stimulation, and their neuronal properties can be maintained even after detaching and re-seeding onto a normal cell culture substrate, suggesting the enhanced maturity of resulting neuronal cells. Thus, we conclude that monolayer graphene is capable of regulating the growth and neural differentiation of hMSCs, which would provide new insight and strategy not only for autologous stem cell therapy but for tissue engineering and regenerative medicine based on graphene scaffolds.

Influence of Surface Charge and Polymer Coating on Internalization and Biodistribution of Polyethylene Glycol-Modified Iron Oxide Nanoparticles.

Abstract: The aim of this study was to investigate the influence of the surface charge and coating of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on their in vitro and in vivo behaviors. Neutral and negatively-charged PEG-based SPIONs were synthesized and compared to Resovist, a carboxydextran-based SPION currently used in clinics. Their cytotoxicity, cell internalization, and potential as contrast agents for magnetic resonance imaging were assessed. Neutral pegylated SPIONs were internalized less readily by the reticuloendothelial system and showed a lower uptake by the liver, compared to negatively-charged SPIONs (with carboxydextran and PEG). These results suggested that the charge of functionalized SPIONs was more relevant for their biological interactions than the nature of their coating.

Active Targeting of Nano-Photosensitizer Delivery Systems for Photodynamic Therapy of Cancer Stem Cells.

Abstract: Cancer stem cells are believed to be the basis for tumor initiation, development, metastasis and recurrence; are resistant to most traditional therapies (e.g., chemotherapy and radiotherapy); and have the ability to self-renew, proliferate and differentiate. Photodynamic therapy may be a promising novel treatment for drug-resistant cancer stem cells because of the selectivity of the photosensitizer. One of the most important issues to overcome in photodynamic therapy is the photosensitizer-targeted delivery to tumor cells, especially cancer stem cells. Nano-photosensitizers comprising molecular photosensitizers and water-dispersible nanocarriers with or without moieties possessing the ability for specific binding to cancer cells or cancer stem cells are a promising strategy for active targeted photosensitizer delivery and photodynamic therapy-targeted therapy of tumors. In this review, we highlight current and future prospects for potential strategies based on nanoscience and nanotechnology for nano-photosensitizer-targeted delivery in the photodynamic therapy treatment of cancer cells, especially cancer stem cells.

Enhancing methotrexate tolerance with folate tagged liposomes in arthritic mice

Abstract: Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor β present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor β was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor β. These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance.

Topical Skin Cancer Therapy Using Doxorubicin-Loaded Cationic Lipid Nanoparticles and lontophoresis.

Abstract: The topical administration of chemotherapeutics is a promising approach for the treatment of skin cancer; however, different pharmaceutical strategies are required to allow large amounts of drug to penetrate tumors. This work examined the potential of the anodic iontophoresis of doxorubicin-loaded cationic solid lipid nanoparticles (DOX-SLN) to increase the distribution and tumor penetration of DOX. A double-labeled cationic DOX-SLN composed of the lipids stearic acid and monoolein and a new BODIPY dye was prepared and characterized. The skin distribution and penetration of DOX were evaluated in vitro using confocal microscopy and vertical diffusion cells, respectively. The antitumor potential was evaluated in vivo through the anodic iontophoresis of DOX-SLN in squamous cell carcinoma induced in nude BALB/c mice. The encapsulation of DOX drastically altered the DOX partition coefficient and increased the distribution of DOX in the lipid matrix of the stratum corneum (SC). The association with iontophoresis created high-concentration drug reservoir zones in the follicles of the skin. Although the iontophoresis of a DOX solution increased the penetration of DOX in the viable epidermis by approximately 4-fold, the iontophoresis of cationic DOX-SLN increased the DOX penetration by approximately 50-fold. In vivo, the DOX-SLN iontophoretic treatment was effective in inhibiting tumor cell survival and tumor growth and was accompanied by an increase in keratinization and consequent cell death. These results indicate a strong and synergic effect of iontophoresis with DOX-SLN and provide a potential strategy for the treatment of skin cancer.

PAMAM Dendrimer/pDNA Functionalized-Magnetic Iron Oxide Nanoparticles for Gene Delivery.

Abstract: Herein, we report an easy and ingenious method to functionalize magnetic iron oxide nanoparticles (MNPs) with plasmid DNA (pDNA) to obtain nanohybrid systems suitable for nucleic acid therapy. The nanohybrids were prepared by combining complexes of dendrimers and pDNA (dendriplexes) and poly(styrene) sulfonate-coated MNPs through electrostatic interactions. The effects of the dendrimer generation (generations 2, 4 and 6) and the amine to phosphate group (N/P) ratio on the hydrodynamic diameter, zeta potential, cell viability, cellular internalization and transfection efficiency of the nanohybrids were systematically investigated at different transfection conditions (including incubation time, pDNA concentration, presence or absence of an external magnetic field, and presence or absence of fetal bovine serum). The results confirmed that the nanohybrids were able to transfect NIH 3T3 cells, and that the level of gene expression (the luciferase protein reporter gene was used) was strongly dependent on the dendrimer generation, the N/P ratio, and the pDNA concentration. The best system was based on dendriplex-coated MNPs formed by generation 6 dendrimers at an N/P ratio of 10 that, at optimized conditions, led to a gene expression level which was not significantly different from that obtained only using dendriplexes. In summary, a coherent set of results was reached indicating the potential of the developed nanohybrids as effective gene delivery nanomaterials.

Rapid and Sensitive Determination of HIV-1 Virus Based on Surface Enhanced Raman Spectroscopy.

Abstract: In this study, rapid and sensitive detection of human immunodeficiency virus (HIV)-1 was performed based on immunoreactions with an Au nanodot fabricated indium tin oxide (ITO) substrate using surface-enhanced Raman spectroscopy (SERS). Highly ordered Au nanodots (ca. 20 nm) were electrochemically fabricated over a large surface area (20 mm x 10 mm) of an ITO substrate using a simple deposition method with Triton X-100. On the Au nanodot surface, monoclonal antibody fragments against gp120 were selectively bound by gold-sulfur interactions. Various concentrations (35 fg/mL to 350 pg/mL) of HIV-1 virus-like particles (HIV-1 VLPs) were used for the measurements. The presence of HIV-1 VLPs was rapidly (within 5 s) and successfully determined by SERS due to specific immunoreactions on the Au nanodots without the use of labeling probes. The results showed the possibility of using SERS-related methods as a new immunoassay for the study of biomolecular interactions and detection of low viral loads. Moreover, based on its high sensitivity and chemical specificity, SERS could be used as a promising clinical tool for detecting infectious small biological components.

Genetic Variation of BCL2 (rs2279115), NEIL2 (rs804270), LTA (rs909253), PSCA (rs2294008) and PLCE1 (rs3765524, rs10509670) Genes and Their Correlation to Gastric Cancer Risk Based on Universal Tagged Arrays and Fe3O4 Magnetic Nanoparticles.

Abstract: With the help of Fe3O4 nagnetic nanoparticles as a solid carrier and an excellent tool for separation, six SNP loci (rs2279115 of BCL2 gene, rs804270 of NEIL2 gene, rs909253 of LTA gene, rs2294008 of PSCA gene, rs3765524 and rs10509670 of PLCE1 gene) were selected to evaluate their relation to gastric cancer risk. Using two kinds of functionalized magnetic nanoparticles and universal tagged arrays, the whole operation procedure including genome DNA extraction and SNP genotyping was performed. All genotypes and allele frequencies were calculated in the cases and controls respectively to analyze their association with gastric cancer risk. Totally 200 pathological samples and 134 normal control subjects were collected. The results demonstrated that four SNP loci (rs2279115, rs804270, rs909253 and rs3765524) showed a potential association with gastric cancer risk, and the other two (rs2294008, rs10509670) possessed no difference/association among cases and controls.

Arginine-Glycine-Aspartic Acid-Modified Lipid-Polymer Hybrid Nanoparticles for Docetaxel Delivery in Glioblastoma Multiforme.

Abstract: Hybrid nanoparticles consisting of lipids and the biodegradable polymer, poly (D,L-lactide-co-glycolide) (PLGA), were developed for the targeted delivery of the anticancer drug, docetaxel. Transmission electron microscopic observations confirmed the presence of a lipid coating over the polymeric core. Using coumarin-6 as a fluorescent probe, the uptake efficacy of RGD conjugated lipid coated nanoparticles (RGD-L-P) by C6 cells was increased significantly, compared with that of lipid-polymer hybrid nanoparticles (L-P; 2.5-fold higher) or PLGA-nanoparticles (PLGA-P; 1.76-fold higher). The superior tumor spheroid penetration of RGD-L-P indicated that RGD-L-P could target effectively and specifically to C6 cells overexpressing integrin α(v)β3. The anti-proliferative activity of docetaxel-loaded RGD-L-P against C6 cells was increased 2.69- and 4.13-fold compared with L-P and PLGA-P, respectively. Regarding biodistribution, the strongest brain-localized fluorescence signals were detected in glioblastoma multiforme (GBM)-bearing rats treated with 1,10-Dioctadecyl-3,3,30,30-tetramethylindotricarb-ocyanine iodide (DiR)-loaded RGD-L-P, compared to rats treated with DiR-loaded L-P or PLGA-P. The median survival time of GBM-bearing rats treated with docetaxel-loaded RGD-L-P was 57 days, a fold increase of 1.43, 1.78, 3.35, and 3.56 compared with animals given L-P (P < 0.05), PLGA-P (P < 0.05), Taxotere (P < 0.01) and saline (P < 0.01), respectively. Collectively, these results support RGD-L-P as a promising drug delivery system for the specific targeting and the treatment of GBM.

Local Administration of Gold Nanoparticles Prevents Pivotal Pathological Changes in Murine Models of Atopic Asthma.

Abstract: Although gold nanoparticles have been shown to exhibit a range of beneficial biological properties, including antiinflammatory and anti-oxidant effects, their putative impact on allergic asthma has not been addressed. In this study, we evaluated the potential of nasal-instilled gold nanoparticles to prevent allergen-induced asthma in distinct murine models of this disease. Swiss-Webster (outbred) and A/J (inbred) mice were sensitized with ovalbumin and then treated with intranasal injections of gold nanoparticles (6 and 60 μg/kg), 1 h before ovalbumin challenges. Lung function, leukocyte infiltration, mucus exacerbation, extracellular matrix deposition, cytokine generation and oxidative stress were evaluated 24 h after the last challenge. In both mice strains, gold nanoparticles clearly inhibited (70-100%) allergen-induced accumulation of inflammatory cells as well as the production of both pro-inflammatory cytokines and reactive oxygen species. In A/J mice, recognized as genetic asthma prone animals, instilled gold nanoparticles clearly prevented mucus production, peribronchiolar fibrosis and airway hyper-reactivity triggered by allergen provocation. In conclusion, these findings demonstrate that gold nanoparticles prevented pivotal features of asthma, including airway hyper-reactivity, inflammation and lung remodelling. Such protective effects are accounted for by reduction in lung tissue generation of pro-inflammatory cytokines and chemokines, in a mechanism probably related to down-regulation in the levels of oxidative stress.

Improving Anti-Tumor Activity of Curcumin by Polymeric Micelles in Thermosensitive Hydrogel System in Colorectal Peritoneal Carcinomatosis Model.

Abstract: In this work, we prepared an in situ gel-forming composite drug delivery system (DDS) to treat colorectal peritoneal carcinomatosis. The composite DDS was based on curcumin loaded polymeric micelles (Cur-M) and thermosensitive hydrogel. Cur-M had a particle size of 27.1 ± 1.3 nm with polydisperse index of 0.149 ± 0.017, and the drug loading and encapsulation efficiency of Cur-M were 14.82 ± 0.07 and 98.83 ± 0.45%, respectively. The prepared Cur-M in thermosensitive hydrogel system (Cur-H) was a free-flowing sol at ambient temperature, and converted into non-flowing gel at body temperature, serving as a drug depot. In vitro drug release behavior suggested that Cur-H and Cur-M could release Cur in an extent period, and Cur-H showed a slower cumulative release rate. In addition, compared with free Cur, Cur-M showed higher cytotoxicity and apoptotic induction efficiency. Furthermore, colorectal peritoneal carcinomatosis mouse model was used to evaluate the anti-tumor activity of Cur-H, and the results suggested that Cur-H could inhibit tumor growth and metastasis, and prolonged survival of tumor-bearing mice. Immunohistochemical and immunofluorescent staining of tumor tissues in each group were conducted. The results demonstrated that tumors in Cur-H group showed lower proliferation activity, more apoptotic cells, and fewer microvessels. Besides, pharmacokinetic studies of Cur-H and Cur-M by intraperitoneal administration were performed. Compared with Cur-M, Cur-H showed a higher AUC and longer t½. Thus, the above results suggested that Cur-H may have potential applications in colorectal peritoneal carcinomatosis.

Biodistribution, Clearance, and Toxicology of Polymeric Micelles Loaded with 0.9 or 5 nm Gold Nanoparticles.

Abstract: Long-circulating gold nanoparticles (AuNPs) have garnered a great deal of interest as both imaging and therapeutic agents. However, their protracted elimination and long-term persistence within many organ systems remains a concern for clinical translation. To improve the excretion of long-circulating nanoparticles, we prepared -80 nm biodegradable polymeric micelles with 0.9 nm or 5 nm AuNPs tightly packed within the hydrophobic core. These gold-loaded polymeric micelles (GPMs) were expected to allow for improved excretion of gold, compared with single large AuNPs, owing to the smaller size and larger surface-to-volume ratio of the individual AuNPs within the micelle. Following intravenous administration of GPMs, organs were harvested and examined for gold content using inductively coupled plasma optical emission spectrometry (ICP-OES) for up to 3 months post-injection. While both GPM formulations showed significant clearance of gold over time, micelles containing 0.9 nm AuNPs showed a 72% and 67% reduction in gold content in the liver and spleen, respectively, between 1 day and 3 months post-injection, compared with a 38% and 35% reduction in mice receiving 5 nm GPMs. Furthermore, feces and urine analysis revealed approximately 7.5 and 100 times more gold, respectively, in mice that received 0.9 nm GPMs one day after injection. These findings suggest that the excretion profile of inorganic nanomaterials may be improved if clusters of small inorganic materials are used in favor of single solid particles.

Synthesis and Characterization of Novel Polycarbonate Based Polyurethane/Polymer Wrapped Hydroxyapatite Nanocomposites: Mechanical Properties, Osteoconductivity and Biocompatibility.

Abstract: The present investigation reports the preparation of two types of 2D rod-like nano-hydroxyapatite (nHA) (unmodified and Polypropylene glycol (PPG) wrapped) of varying high-aspect ratios, by modified co-precipitation methods, without any templates. These nHA were successfully introduced into novel synthesized Thermoplastic Polyurethane (TPU) matrices based on polycarbonate soft segments, by both in-situ and ex-situ techniques. Physico-mechanical properties of the in-situ prepared TPU/nHA nanocomposites were found to be superior compared to the ex-situ counterparts, and pristine nHA reinforced TPU. Improved biocompatibility of the prepared nanocomposites was confirmed by MTT assays using osteoblast-like MG63 cells. Cell proliferation was evident over an extended period. Osteoconductivity of the nanocomposites was observed by successful formation of an apatite layer on the surface of the samples, after immersion into simulated body fluid (SBF). Prothrombin time (PT) and activated partial thromboplastin time (APTT), as calculated from coagulation assays, displayed an increase in the clotting time, particularly for the PPG-wrapped nHA nanocomposites, prepared through the in-situ technique. Only 0.3% of hemolysis was observed for the in-situ prepared nanocomposites, which establishes the antithrombotic property of the material. The key parameters for enhancing the technical properties and biocompatibility of the nanocomposites are: the interfacial adhesion parameter (B(σy)), the polymer-filler affinity, the aspect ratio of filler and non-covalent modifications, and the state of dispersion. Thus, the novel TPU/polymer wrapped nHA nanocomposites have great potential for biomedical applications, in particular for vascular prostheses, cardiovascular implants, scaffolds, and soft and hard tissues implants.