Development of triple-functionalized calcium phosphate nanoparticles as an advanced drug delivery system for bone tissue repair.

Introduction: During tissue repair or regeneration, several bioactive molecules are released and interact with each other and act as complex additives or inhibitors for tissue reconstruction. In this study, the bone-healing effects of the combination treatment with tumor necrosis factor-α (TNF-α) inhibition, vascular endothelial growth factor A (VEGF-A) and bone morphogenetic protein-7 (BMP-7) release by gene silencing, and gene transfection with calcium phosphate nanoparticles (CaP) in the rat femoral head was histologically, morphologically, and biochemically evaluated. Methods: A triple-functionalized paste of CaP carrying plasmid DNA encoding for BMP-7 and for VEGF), and siRNA against TNF-α was developed and denoted as CaP3mix. To compare the effects of 3mixCaP, CaP with plasmid DNA encoding BMP-7, VEGF, or siRNA encoding TNF-α was prepared and denoted as CaP/PEI/pBMP-7/SiO2, CaP/PEI/pVEGF/SiO2, or CaP/PEI/siRNA-TNF-α/SiO2, respectively. The bone healing in bone defects in the rat femoral head was investigated after 10 and 21 days of implantation. Results: The levels of bone formation-related markers OCN, Runx2, and SP7 increased at the protein and gene levels in 3mixCaP after 10 days, and 3mixCaP significantly accelerated bone healing compared with the other treatments after 21 days of implantation. Conclusion: The triple-functionalized CaP paste loading plasmid DNA encoding BMP-7 and VEGF and siRNA encoding TNF-α is a promising bioactive material for bone tissue repair.

Functionalized calcium phosphate nanoparticles to direct osteoprotegerin to bone lesion sites in a medaka (Oryzias latipes) osteoporosis model.

Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity in vivo. For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

Uptake of Functional Ultrasmall Gold Nanoparticles in 3D Gut Cell Models.

Ultrasmall gold nanoparticles (2 nm) easily penetrate the membranes of intestinal murine epithelial cells (MODE-K) and colorectal cancer cells (CT-26). They are also taken up by 3D spheroids (400 µm) of these cell types and primary gut organoids (500 µm). In contrast, dissolved dyes are not taken up by any of these cells or 3D structures. The distribution of fluorescent ultrasmall gold nanoparticles inside cells, spheroids, and gut organoids is examined by confocal laser scanning microscopy. Nanoparticles conjugated with the cytostatic drug doxorubicin and a fluorescent dye exhibit significantly greater cytotoxicity toward CT-26 tumor spheroids than equally concentrated dissolved doxorubicin, probably because they enter the interior of a spheroid much more easily than dissolved doxorubicin. Comprehensive analyses show that the cellular uptake of ultrasmall gold nanoparticles occurs by different endocytosis pathways.

A New Water-Soluble Thermosensitive Star-Like Copolymer as a Promising Carrier of the Chemotherapeutic Drug Doxorubicin.

A new water-soluble thermosensitive star-like copolymer, dextran-graft-poly-N-iso-propilacrylamide (D-g-PNIPAM), was created and characterized by various techniques (size-exclusion chromatography, differential scanning calorimetry, Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) spectroscopy). The viability of cancer cell lines (human transformed cervix epithelial cells, HeLa) as a model for cancer cells was studied using MTT and Live/Dead assays after incubation with a D-g-PNIPAM copolymer as a carrier for the drug doxorubicin (Dox) as well as a D-g-PNIPAM + Dox mixture as a function of the concentration. FTIR spectroscopy clearly indicated the complex formation of Dox with the D-g-PNIPAM copolymer. The size distribution of particles in Hank's solution was determined by the DLS technique at different temperatures. The in vitro uptake of the studied D-g-PNIPAM + Dox nanoparticles into cancer cells was demonstrated by confocal laser scanning microscopy. It was found that D-g-PNIPAM + Dox nanoparticles in contrast to Dox alone showed higher toxicity toward cancer cells. All of the aforementioned facts indicate a possibility of further preclinical studies of the water-soluble D-g-PNIPAM particles' behavior in animal tumor models in vivo as promising carriers of anticancer agents.

Biological and Medical Applications of Calcium Phosphate Nanoparticles.

Calcium phosphate nanoparticles have a high biocompatibility and biodegradability due to their chemical similarity to human hard tissue, for example, bone and teeth. They can be used as efficient carriers for different kinds of biomolecules such as nucleic acids, proteins, peptides, antibodies, or drugs, which alone are not able to enter cells where their biological effect is required. They can be loaded with cargo molecules by incorporating them, unlike solid nanoparticles, and also by surface functionalization. This offers protection, for example, against nucleases, and the possibility for cell targeting. If such nanoparticles are functionalized with fluorescing dyes, they can be applied for imaging in vitro and in vivo. Synthesis, functionalization and cell uptake mechanisms of calcium phosphate nanoparticles are discussed together with applications in transfection, gene silencing, imaging, immunization, and bone substitution. Biodistribution data of calcium phosphate nanoparticles in vivo are reviewed.

Delivery of toll-like receptor 3 ligand poly(I:C) to the liver by calcium phosphate nanoparticles conjugated with an F4/80 antibody exerts an anti-hepatitis B virus effect in a mouse model.

Hepatitis B virus (HBV) is a global health issue, but currently available anti-HBV drugs have limited success. Previously, introduction of the Toll-like receptor (TLR)-3 ligand poly(I:C) to the liver via hydrodynamic injection (HI) was shown to effectively suppress HBV replication in a chronic HBV replication mouse model. However, this method cannot be applied in human beings. To improve the liver targeting of poly(I:C) via intravenous injection, calcium phosphate nanoparticles (CPNs) carrying poly(I:C) with or without antibodies were constructed, and their anti-HBV effects were investigated. We found that significantly more anti-F4/80-conjugated and IgG2α-conjugated nanoparticles were taken up in liver cells both in vivo and in vitro. In addition, these nanoparticles produced pronounced immunostimulatory effects in vitro in primary liver cells. Importantly, treatment with nanoparticles carrying poly(I:C) increased the production of intrahepatic cytokines and chemokines and enhanced T cell responses, significantly reducing HBsAg, HBeAg and HBV DNA levels in the mice. Compared to nonconjugated and isotype-antibody-conjugated nanoparticles, the anti-F4/80-conjugated nanoparticles demonstrated the strongest anti-HBV effects. In summary, nanoparticles carrying poly(I:C) conjugated with an F4/80 antibody promoted liver targeting, and they may represent a suitable alternative to HI for future anti-HBV treatment. STATEMENT OF SIGNIFICANCE: HBV chronically infects approximately 250 million individuals worldwide but current anti-HBV drugs have limited success. Introduction of toll-like receptor 3 ligand poly(I:C) into liver by hydrodynamic injection has been proven to promote HBV clearance in mouse model. However, this technique is not clinically suitable for human patients. We have constructed calcium phosphate nanoparticles carrying poly(I:C) with specific antibody targeting liver nonparenchymal cells. The uptake into relevant liver cells and the anti-HBV effects were studied. After intravenous injection into mice, the uptake rate of anti-F4/80-conjugated nanoparticels was enhanced in liver, and these nanoparticles exert effective anti-HBV effects in vivo. This may provide important insight into future HBV immunotherapy based on nanoparticle-mediated drug delivery.

Colloidal stability, cytotoxicity, and cellular uptake of HfO2 nanoparticles.

The colloidal stability, cytotoxicity, and cellular uptake of hafnium oxide (HfO2 ) nanoparticles (NPs) were investigated in vitro to assess safety and efficacy for use as a deliverable theranostic in nanomedicine. Monoclinic HfO2 NPs, ~60-90 nm in diameter and ellipsoidal in shape, were directly prepared without calcination by a hydrothermal synthesis at 83% yield. The as-prepared, bare HfO2 NPs exhibited colloidal stability in cell culture media for at least 10 days without significant agglomeration or settling. The viability (live/dead assay) of human epithelial cells (HeLa) and monocyte-derived macrophages (THP-1) did not fall below 95% of untreated cells after up to 24 h exposure to HfO2 NPs at concentrations up to 0.80 mg/ml. Similarly, the mitochondrial activity (MTT assay) of HeLa and THP-1 cells did not fall below 80% of untreated cells after up to 24 h exposure to HfO2 NPs at concentrations up to 0.40 mg/ml. Cellular uptake was confirmed and visualized in both HeLa and THP-1 cells by fluorescence microscopy of HfO2 NPs labeled with Cy5 and transmission electron microscopy (TEM) of bare HfO2 NPs. TEM micrographs provided direct observation of macropinocytosis and endosomal compartmentalization within 4 h of exposure. Thus, the HfO2 NPs in this study exhibited colloidal stability, cytocompatibility, and cellular uptake for potential use as a deliverable theranostic in nanomedicine.

The effect of short silica fibers (0.3 µm 3.2 µm) on macrophages.

Silica fibers with a dimension of 0.3 µm ∙ 3.2 µm2 nm were prepared by a modified Stöber synthesis as model particles. The particles were characterized by scanning electron microscopy, elemental analysis, thermogravimetry and X-ray powder diffraction. Their uptake by macrophages (THP-1 cells and NR8383 cells) was studied by confocal laser scanning microscopy and scanning electron microscopy. The uptake by cells was very high, but the silica fibers were not harmful to NR8383 cells in concentrations up to 100 µg mL-1. Only above 100 µg mL-1, significant cell toxic effects were observed, probably induced by a high dose of particles that had sedimented on the cells and led to the adverse effects. The chemotactic response as assessed by the particle-induced migration assay (PICMA) was weak in comparison to a control of agglomerated silica particles. The as-prepared fibers were fully X-ray amorphous but crystallized to ß-cristobalite after heating to 1000 °C and converted to α-cristobalite upon cooling to ambient temperature. The fibers had sintered to larger aggregates but retained their elongated primary shape. The particle cytotoxicity towards THP-1 cells was not significantly enhanced by the crystallization.

Structural analysis of an endoprosthesis designed with graded density lattice structures.

The most common causes of conducting a hip revision surgery after total hip replacement are aseptic loosening (aseptic instability) of the endoprosthesis, bone destruction as a result of contact with the endoprosthesis, and a periprosthetic fracture. These are the effects of load transfer to the bone tissue in arthroplasty resulting due to the difference in stiffness of the endoprosthesis and the bone. Titanium alloy is widely used in endoprostheses manufacturing because of its high biocompatibility, good wear properties, and corrosion resistance, but such endoprostheses are stiffer than the femur. These problems have raised interest in searching for the best materials and topology for a femoral implant. Nowadays additive technology is of great interest as it enables to create materials with graded density. These materials consist of multiple lattice structures with variable parameters and topology. By varying the parameters of lattice structures one can adjust the mechanical properties of the material as required. These materials find their application in hip endoprostheses manufacturing, allowing to adjust the parameters of the lattice structures, and deliver a product with femur-like mechanical properties. The porous structure also ensures bone tissue ingrowth into the prosthesis. The authors designed and simulated an endoprosthesis made of graded density lattice structures with femur-like mechanical properties. Using a numerical simulation software Ansys Mechanical authors determined the effect of the topology on the structural behavior of the femur and defined the endoprosthesis-femur combined performance under various load cases.

Gene transfection achieved by utilizing antibacterial calcium phosphate nanoparticles for enhanced regenerative therapy.

Protamine-coated multi-shell calcium phosphate (CaP) was developed as a non-viral vector for tissue regeneration therapy. CaP nanoparticles loaded with different amounts of plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) were used to treat MC3T3E1 cells, and the yield of the released BMP-2 or IGF-1 was measured using ELISA 3 days later. Collagen scaffolds containing CaP nanoparticles were implanted into rat cranial bone defects, and BMP-2 and IGF-1 yields, bone formation, and bone mineral density enhancement were evaluated 28 days after gene transfer. The antibacterial effects of CaP nanoparticles against Streptococcus mutans and Aggregatibacter actinomycetemcomitans increased with an increase in the protamine dose, while they were lower for Staphylococcus aureus and Porphyromonas gingivalis. In the combination treatment with BMP-2 and IGF-1, the concentration ratio of BMP-2 and IGF-1 is an important factor affecting bone formation activity. The calcification activity and OCN mRNA of MC3T3E1 cells subjected to a BMP-2:IGF-1 concentration ratio of 1:4 was higher at 14 days. During gene transfection treatment, BMP-2 and IGF-1 were released simultaneously after gene transfer; the loaded dose of the plasmid DNA encoding IGF-1 did not impact the BMP-2 or IGF-1 yield or new bone formation ratio in vitro and in vivo. In conclusion, two growth factor-releasing systems were developed using an antibacterial gene transfer vector, and the relationship between the loaded plasmid DNA dose and resultant growth factor yield was determined in vitro and in vivo.

Synthesis and characterization of PLGA/HAP scaffolds with DNA-functionalised calcium phosphate nanoparticles for bone tissue engineering.

Porous scaffolds of poly(lactide-co-glycolide) (PLGA; 85:15) and nano-hydroxyapatite (nHAP) were prepared by an emulsion-precipitation procedure from uniform PLGA-nHAP spheres (150-250 µm diameter). These spheres were then thermally sintered at 83 °C to porous scaffolds that can serve for bone tissue engineering or for bone substitution. The base materials PLGA and nHAP and the PLGA-nHAP scaffolds were extensively characterized by X-ray powder diffraction, infrared spectroscopy, thermogravimetry, differential scanning calorimetry, and scanning electron microscopy. The scaffold porosity was about 50 vol% as determined by relating mass and volume of the scaffolds, together with the computed density of the solid phase (PLGA-nHAP). The cultivation of HeLa cells demonstrated their high cytocompatibility. In combination with DNA-loaded calcium phosphate nanoparticles, they showed a good activity of gene transfection with enhanced green fluorescent protein (EGFP) as model protein. This is expected enhance bone growth around an implanted scaffold or inside a scaffold for tissue engineering.

Transport of ultrasmall gold nanoparticles (2 nm) across the blood-brain barrier in a six-cell brain spheroid model.

The blood-brain barrier (BBB) is an efficient barrier for molecules and drugs. Multicellular 3D spheroids display reproducible BBB features and functions. The spheroids used here were composed of six brain cell types: Astrocytes, pericytes, endothelial cells, microglia cells, oligodendrocytes, and neurons. They form an in vitro BBB that regulates the transport of compounds into the spheroid. The penetration of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm; hydrodynamic diameter 3-4 nm) across the BBB was studied as a function of time by confocal laser scanning microscopy, with the dissolved fluorescent dye (FAM-alkyne) as a control. The nanoparticles readily entered the interior of the spheroid, whereas the dissolved dye alone did not penetrate the BBB. We present a model that is based on a time-dependent opening of the BBB for nanoparticles, followed by a rapid diffusion into the center of the spheroid. After the spheroids underwent hypoxia (0.1% O2; 24 h), the BBB was more permeable, permitting the uptake of more nanoparticles and also of dissolved dye molecules. Together with our previous observations that such nanoparticles can easily enter cells and even the cell nucleus, these data provide evidence that ultrasmall nanoparticle can cross the blood brain barrier.

Ultrasmall gold nanoparticles (2 nm) can penetrate and enter cell nuclei in an in vitro 3D brain spheroid model.

The neurovascular unit (NVU) is a complex functional and anatomical structure composed of endothelial cells and their blood-brain barrier (BBB) forming tight junctions. It represents an efficient barrier for molecules and drugs. However, it also prevents a targeted transport for the treatment of cerebral diseases. The uptake of ultrasmall nanoparticles as potential drug delivery agents was studied in a three-dimensional co-culture cell model (3D spheroid) composed of primary human cells (astrocytes, pericytes, endothelial cells). Multicellular 3D spheroids show reproducible NVU features and functions. The spheroid core is composed mainly of astrocytes, covered with pericytes, while brain endothelial cells form the surface layer, establishing the NVU that regulates the transport of molecules. After 120 h cultivation, the cells self-assemble into a 350 µm spheroid as shown by confocal laser scanning microscopy. The passage of different types of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm) both into the spheroid and into three constituting cell types was studied by confocal laser scanning microscopy. Three kinds of covalently fluorophore-conjugated gold nanoparticles were used: One with fluorescein (FAM), one with Cy3, and one with the peptide CGGpTPAAK-5,6-FAM-NH2. In 2D cell co-culture experiments, it was found that all three kinds of nanoparticles readily entered all three cell types. FAM- and Cy3-labelled nanoparticles were able to enter the cell nucleus as well. The three dissolved dyes alone were not taken up by any cell type. A similar situation evolved with 3D spheroids: The three kinds of nanoparticles entered the spheroid, but the dissolved dyes did not. The presence of a functional blood-brain barrier was demonstrated by adding histamine to the spheroids. In that case, the blood-brain barrier opened, and dissolved dyes like a FITC-labelled antibody and FITC alone entered the spheroid. In summary, our results qualify ultrasmall gold nanoparticles as suitable carriers for imaging or drug delivery into brain cells (sometimes including the nucleus), brain cell spheroids, and probably also into the brain. STATEMENT OF SIGNIFICANCE: 3D brain spheroid model and its permeability by ultrasmall gold nanoparticles. We demonstrate that ultrasmall gold nanoparticles can easily penetrate the constituting cells and sometimes even enter the cell nucleus. They can also enter the interior of the blood-brain barrier model. In contrast, small molecules like fluorescing dyes are not able to do that. Thus, ultrasmall gold nanoparticles can serve as carriers of drugs or for imaging inside the brain.

Genetic immunization against hepatitis B virus with calcium phosphate nanoparticles in vitro and in vivo.

Calcium phosphate nanoparticles were loaded with plasmid DNA and toll-like receptor ligands (TLR), i.e. CpG or flagellin, to activate antigen-presenting cells (APCs) like dendritic cells (DCs). The functionalized nanoparticles were studied in vitro on HeLa, C2C12 and BHK-21 cell lines, focusing on the expression of two specific proteins. EGFP-DNA, encoding for enhanced green fluorescent protein (EGFP), was used as a model plasmid to optimize the transfection efficiency in vitro by fluorescence microscopy and flow cytometry. Calcium phosphate nanoparticles loaded with TLR ligands and plasmid DNA encoding for the hepatitis B virus surface antigen (pHBsAg) were evaluated by in vitro and in vivo immunization experiments to identify a possible candidate for a prophylactic hepatitis B virus (HBV) vaccine. The nanoparticles induced a strong expression of HBsAg in the three cell lines. In splenocytes, the expression of the co-stimulatory molecules CD80 and CD86 was enhanced. After intramuscular injection in mice, the nanoparticles induced the expression of HBsAg, the antigen-specific T cell response, and the antigen-specific antibody response (IgG1). STATEMENT OF SIGNIFICANCE: Hepatitis B is one of the most frequent viral infections worldwide. For preventive immunization, nanoparticles can be used which carry both an adjuvant (a stimulatory molecule) and DNA encoding for a viral antigen. After administration of such nanoparticles to cells, they are taken up by cells where the DNA is transcribed into the viral antigen (a protein). This viral antigen is inducing a virus-specific immune response. This was shown both by in vitro cell culture as well as by an extensive in vivo study in mice.

In vivo biodistribution of calcium phosphate nanoparticles after intravascular, intramuscular, intratumoral, and soft tissue administration in mice investigated by small animal PET/CT.

Calcium phosphate nanoparticles were covalently surface-functionalized with the ligand DOTA and loaded with the radioisotope 68Ga. The biodistribution of such 68Ga-labelled nanoparticles was followed in vivo in mice by positron emission tomography in combination with computer tomography (PET-CT). The biodistribution of 68Ga-labelled nanoparticles was compared for different application routes: intravenous, intramuscular, intratumoral, and into soft tissue. The particle distribution was measured in vivo by PET-CT after 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and ex vivo after 5 h. After intravenous injection (tail vein), the nanoparticles rapidly entered the lungs with later redistribution into liver and spleen. The nanoparticles remained mostly at the injection site following intramuscular, intratumoral, or soft tissue application, with less than 10 percent being mobilized into the blood stream. STATEMENT OF SIGNIFICANCE: The in vivo biodistribution of DOTA-terminated calcium phosphate nanoparticles was followed by PET/CT. To our knowledge, this is the first study of this kind. Four different application routes of clinical relevance were pursued: Intravascular, intramuscular, intratumoral, and into soft tissue. Given the high importance of calcium phosphate as biomaterial and for nanoparticular drug delivery and immunization, this is most important to assess the biofate of calcium phosphate nanoparticles for therapeutic application and also judge biodistribution of nanoscopic calcium phosphate ceramics, including debris from endoprostheses and related implants.

Effective Activation of Human Antigen-Presenting Cells and Cytotoxic CD8+ T Cells by a Calcium Phosphate-Based Nanoparticle Vaccine Delivery System.

The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses. Nanoparticles (NPs) are considered to be efficient tools for the initiation of potent immune responses. Calcium phosphate (CaP) NPs are a class of biodegradable nanocarriers that are able to deliver immune activating molecules across physiological barriers. Therefore, the aim of this study was to assess whether Toll-like receptor (TLR) ligand and viral antigen functionalized CaP NPs are capable of inducing efficient maturation of human antigen presenting cells (APC). To achieve this, we generated primary human dendritic cells (DCs) and stimulated them with CpG or poly(I:C) functionalized CaP NPs. DCs were profoundly stronger when activated upon NP stimulation compared to treatment with soluble TLR ligands. This is indicated by increased levels of costimulatory molecules and the secretion of proinflammatory cytokines. Consequently, coculture of NP-stimulated APCs with CD8+ T cells resulted in a significant expansion of virus-specific T cells. In summary, our data suggest that functionalized CaP NPs are a suitable tool for activating human virus-specific CD8+ T cells and may represent an excellent vaccine delivery system.

Reduction of inflammation in a chronic periodontitis model in rats by TNF-α gene silencing with a topically applied siRNA-loaded calcium phosphate paste.

We developed a calcium phosphate-based paste containing siRNA against TNF-α and investigated its anti-inflammatory and bone-healing effects in vitro and in vivo in a rat periodontitis model. The bioactive spherical CaP/PEI/siRNA/SiO2 nanoparticles had a core diameter of 40-90 nm and a positive charge (+23 mV) that facilitated cellular uptake. The TNF- α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. CaP/PEI/siRNA/SiO2 nanoparticles cancelled the suppression of alkaline phosphatase (ALP) activity in LPS-stimulated bone marrow-derived cells. In vivo, ALP mRNA was up-regulated, TNF-α mRNA was down-regulated, and the amount of released TNF-α was significantly reduced after topical application of the calcium phosphate-based paste containing siRNA-loaded nanoparticles. The number of TNF-α-positive cells in response to CaP/PEI/siRNA/SiO2 nanoparticle application was lower than that observed in the absence of siRNA. Elevated ALP activity and numerous TRAP-positive cells (osteoclasts) were observed in response to the application of all calcium phosphate pastes. These results demonstrate that local application of a paste consisting of siRNA-loaded calcium phosphate nanoparticles successfully induces TNF-α silencing in vitro and in vivo and removes the suppression of ALP activity stimulated by inflammation. STATEMENT OF SIGNIFICANCE: We developed a calcium phosphate-based paste containing nanoparticles loaded with siRNA against TNF-α. The nanoparticles had a core diameter of 40-90 nm and positive charge (+23 mV). The anti-inflammatory and osteoinductive effects of the paste were investigated in vitro and in vivo in a rat periodontitis model. In vitro, the TNF-α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. The ALP activity of bone marrow-derived cells was recovered. In vivo, TNF-α mRNA was down-regulated and the amount of released TNF-α was significantly reduced, whereas the ALP mRNA was up-regulated. Elevated ALP activity and TRAP-positive cells were observed by immunohistochemistry.

Calcium Phosphate Nanoparticle-Based Vaccines as a Platform for Improvement of HIV-1 Env Antibody Responses by Intrastructural Help.

Incorporation of immunodominant T-helper epitopes of licensed vaccines into virus-like particles (VLP) allows to harness T-helper cells induced by the licensed vaccines to provide intrastructural help (ISH) for B-cell responses against the surface proteins of the VLPs. To explore whether ISH could also improve antibody responses to calcium phosphate (CaP) nanoparticle vaccines we loaded the nanoparticle core with a universal T-helper epitope of Tetanus toxoid (p30) and functionalized the surface of CaP nanoparticles with stabilized trimers of the HIV-1 envelope (Env) resulting in Env-CaP-p30 nanoparticles. In contrast to soluble Env trimers, Env containing CaP nanoparticles induced activation of naïve Env-specific B-cells in vitro. Mice previously vaccinated against Tetanus raised stronger humoral immune responses against Env after immunization with Env-CaP-p30 than mice not vaccinated against Tetanus. The enhancing effect of ISH on anti-Env antibody levels was not attended with increased Env-specific IFN-γ CD4 T-cell responses that otherwise may potentially influence the susceptibility to HIV-1 infection. Thus, CaP nanoparticles functionalized with stabilized HIV-1 Env trimers and heterologous T-helper epitopes are able to recruit heterologous T-helper cells induced by a licensed vaccine and improve anti-Env antibody responses by intrastructural help.

In vitro study of the anticancer activity of various doxorubicin-containing dispersions.

Introduction: The aim of this research was to study the impact of various doxorubicin (Dox)-containing nanofluids, e.g. singlewalled carbon nanotube (SWCNT)+Dox, graphene oxide (GO)+Dox and DextranPNIPAM (copolymer)+Dox mixtures on HeLa cells (human transformed cervix epithelial cells, as a model for cancer cells) depending on their concentration. Methods: Structural analysis of GO+Dox complex was accomplished using Hartree-Fock level of theory in 6-31G** basis set in Gaussian. Dynamic light scattering (DLS), zeta-potential, scanning electron microscopy and confocal laser scanning microscopy were used. The cell viability was analyzed by the MTT assay. Results: The viability of HeLa cells was studied with the MTT assay after the incubation with various Dox-containing dispersions depending on their concentration. The size of the particles was determined by DLS. The morphology of the nanoparticles (NPs) was studied by scanning electron microscopy and their uptake into cells was visualized by confocal laser scanning microscopy. It was found that the Dextran-PNIPAM+Dox nanofluid in contrast to Dox alone showed higher toxicity towards HeLa cells up to 80% after 24 hours of incubation, whereas the SWCNT+Dox and GO+Dox nanofluids at the same concentrations protected cells from Dox. Conclusion: The importance of Dextran-PNIPAM copolymer as a universal platform for drug delivery was established, and the huge potential of Dextran-PNIPAM+Dox NPs as novel anticancer agents was noted. Based on the in vitro study of the SWCNT+Dox and GO+Dox nanofluids, it was concluded that SWCNT and GO NPs can be effective cytoprotectors against the highly toxic drugs.