Modified Titanium Surface with Nano Amorphous Calcium Phosphate@Chitosan Oligolactate as Ion Loading Platform with Multifunctional Properties for Potential Biomedical Application.

Titanium (Ti) is widely used in medical and dental implants. Calcium phosphate (CPs) coatings enhance Ti implants' osteoinductive properties, and additives further improve these coatings. Recently, a nano amorphous calcium phosphate (nACP) coating decorated with chitosan oligolactate (ChOL) and selenium (Se) showed immunomodulatory effects. This study investigates the surface morphology, composition, bioactivity, mechanical properties, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates. Amorphous calcium phosphate (ACP) was synthesized, and the nACP@ChOL-Se hybrid coating was deposited on Ti substrates using in situ anaphoretic deposition. Physico-chemical characterization was used to analyze the surface of the coating (scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy). The distribution of Se within the coating was examined with energy-dispersive X-ray spectroscopy (EDS). Bioactivity was evaluated in simulated body fluid (SBF), and adhesion was tested using a scratch test method. In vitro testing determined the release mechanism of Se. SEM images illustrated the surface morphology, while AFM provided a detailed analysis of surface roughness. XRD analysis revealed structural and phase composition, and EDS confirmed Se distribution within the coating. The coating exhibited bioactivity in SBF and showed good adhesion according to the scratch test. In vitro testing uncovered the release mechanism of Se from the coating. This study successfully characterized the surface morphology, composition, bioactivity, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates, offering insights for developing immunomodulatory coatings for medical and dental applications.

Immunomodulatory Effects Mediated by Nano Amorphous Calcium Phosphate/Chitosan Oligosaccharide Lactate Coatings Decorated with Selenium on Titanium Implants.

The aim of this work is in situ anodization/anaphoretic deposition of a nano amorphous calcium phosphate (ACP)/chitosan oligosaccharide lactate (ChOL) multifunctional hybrid coating decorated with selenium (Se) on a titanium substrate and in vivo investigation of its immunomodulatory and anti-inflammatory effect. Investigating phenomena at the implant-tissue interface of interest for controlled inflammation and immunomodulation was also the aim of the research. In our earlier research, we designed coatings based on ACP and ChOL on titanium with anticorrosive, antibacterial and biocompatible properties, while in the presented results we show that selenium addition makes this coating an immunomodulator. The immunomodulatory effect of the novel hybrid coating is characterized by the examination of the functional aspects in the tissue around the implant (in vivo): proinflammatory cytokines' gene expression, M1 (iNOS) and M2 (Arg1) macrophages, fibrous capsule formation (TGF-ß) and vascularization (VEGF). The EDS, FTIR and XRD analyses prove the formation of a ACP/ChOL/Se multifunctional hybrid coating on Ti and the presence of Se. A higher M2/M1 macrophage ratio in the ACP/ChOL/Se-coated implants compared to pure titanium implants (a higher level of Arg1 expression) is noted at all time points examined (after 7, 14 and 28 days). Lower inflammation measured by gene expression of proinflammatory cytokines IL-1ß and TNF, lower expression of TGF-ß in the surrounding tissue and higher IL-6 expression (solely at day 7 post-implantation) is noted in presence of the ACP/ChOL/Se-coated implants.

Hormone receptor binding, selectivity and cytotoxicity of steroid D-homo lactone loaded chitosan nanoparticles for the treatment of breast and prostate cancer cells.

Chemically modified steroids have a long history as anti-neoplastic drugs. Incorporation of a lactone moiety in the steroid nucleus, as in previously obtained 3ß-acetoxy-17-oxa-17a-homoandrost-5-en-16-one (A) and 3ß-hidroxy-17-oxa-17a-homoandrost-5-en-16-one (B), often results in enhanced anticancer properties. In this work, chitosan-based (Ch) nanoparticles were created and loaded with potent anticancer steroidal compounds, A and B. Changes to hormone receptor binding and cytotoxicity were then measured. In agreement with our previous results for A and B, A- and B-loaded Ch displayed cytotoxic properties against cancer cell lines. Both A-Ch and B-Ch showed activity toward estrogen negative breast cancer (MDA-MB-231) and androgen negative prostate cancer cell lines (PC-3). Greater selectivity toward cancer cells versus healthy lung fibroblast (MRC-5) was observed for B-Ch particles. Cell viability and cytotoxicity measurements after a recovery period indicate more robust recovery of healthy cells versus malignant cells. Compounds A and B or their Ch-encapsulated forms were shown to have negligible affinity for the ligand binding domain of estrogen receptor ß or the androgen receptor in a fluorescent yeast screen, suggesting a lack of estrogenicity and androgenicity. Steroid-loaded chitosan nanoparticles display strong cytotoxicity towards MDA-MB-231 and PC-3 with a lack of hormone activity, indicating their safety and efficacy.

Anodizing/Anaphoretic Electrodeposition of Nano-Calcium Phosphate/Chitosan Lactate Multifunctional Coatings on Titanium with Advanced Corrosion Resistance, Bioactivity, and Antibacterial Properties.

The aim of this work was to investigate corrosion resistivity, bioactivity, and antibacterial activity of novel nano-amorphous calcium phosphate (ACP) potentially multifunctional composite coatings with and without chitosan oligosaccharide lactate (ChOL), ACP + ChOL/TiO2 and ACP/TiO2 ACP + ChOL/TiO2, respectively, on the titanium substrate. The coatings were obtained by new single-step in situ anodization of the substrate to generate TiO2 and the anaphoretic electrodeposition process of ACP and ChOL. The obtained coatings were around 300 ± 15 µm thick and consisted of two phases, namely, TiO2 and hybrid composite phases. Both ACP/TiO2 and ACP + ChOL/TiO2 have improved corrosion stability, whereas the ACP + ChOL/TiO2 coating showed better corrosion stability. It was shown that at the very start of the deposition process, the formation of the ChOL/TiO2 layer takes place predominantly, which is followed by the inclusion of ChOL into ACP with simultaneous growth of TiO2. This deposition mechanism resulted in the formation of strongly bonded uniform stable coating with high corrosion resistance. In vitro bioactivity was investigated by immersion of the samples in simulated body fluid (SBF). There is in-bone-like apatite formation on both ACP/TiO2 and ACP + ChOL/TiO2 surfaces upon immersion into SBF, which was proven by X-ray diffraction and Fourier transform infrared spectroscopy. While ACP/TiO2 shows no antibacterial activity, ACP + ChOL/TiO2 samples exhibited three- to fourfold decreases in the number of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, after 420 min. The probable mechanism is binding ChOL with the bacterial cell wall, inhibiting its growth, altering the permeability of the cell membrane, and leading to bacterial death.

Rare-earth (Gd3+,Yb3+/Tm3+, Eu3+) co-doped hydroxyapatite as magnetic, up-conversion and down-conversion materials for multimodal imaging.

Taking advantage of the flexibility of the apatite structure, nano- and micro-particles of hydroxyapatite (HAp) were doped with different combinations of rare earth ions (RE3+ = Gd, Eu, Yb, Tm) to achieve a synergy among their magnetic and optical properties and to enable their application in preventive medicine, particularly diagnostics based on multimodal imaging. All powders were synthesized through hydrothermal processing at T ≤ 200 °C. An X-ray powder diffraction analysis showed that all powders crystallized in P63/m space group of the hexagonal crystal structure. The refined unit-cell parameters reflected a decrease in the unit cell volume as a result of the partial substitution of Ca2+ with smaller RE3+ ions at both cation positions. The FTIR analysis additionally suggested that a synergy may exist solely in the triply doped system, where the lattice symmetry and vibration modes become more coherent than in the singly or doubly doped systems. HAp:RE3+ optical characterization revealed a change in the energy band gap and the appearance of a weak blue luminescence (λex = 370 nm) due to an increased concentration of defects. The "up"- and the "down"-conversion spectra of HAp:Gd/Yb/Tm and HAp:Gd/Eu powders showed characteristic transitions of Tm3+ and Eu3+, respectively. Furthermore, in contrast to diamagnetic HAp, all HAp:RE3+ powders exhibited paramagnetic behavior. Cell viability tests of HAp:Gd/Yb/Tm and HAp:Gd/Eu powders in human dental pulp stem cell cultures indicated their good biocompatibility.

Effects of hydroxyapatite@poly-lactide-co-glycolide nanoparticles combined with Pb and Cd on liver and kidney parenchyma after the reconstruction of mandibular bone defects.

Reconstruction of bone defects with the use of biomaterials based on hydroxyapatite (HAp) has been a popular approach in medicine and dentistry. Most often the process of new bone formation is analyzed with the focus only on the region of the reconstructed defect. The effects of the therapy on distant organs have been rarely reported in the literature, especially not in synergy with the exposure to other bioactive chemicals. In this study, reconstruction of the mandibular bone in vivo using poly-lactide-co-glycolide-coated HAp (HAp/PLGA) nanoparticles was monitored with a simultaneous histopathological analysis of distant organs, specifically kidney and liver parenchyma. Heavy metals are among the most prominent environmental pollutants and have a high affinity for the crystal lattice of HAp, where they get incorporated by replacing calcium ions. Lead (Pb) and cadmium (Cd) are two such metals that can be found in food, water and air, but are most commonly present in cigarette smoke, the frequent contaminant of hospital settings in the developing world. The influence of their presence in the repaired bone on the content of calcium (Ca) in the reconstructed bone defect was analyzed, along with the histopathological changes in liver and kidneys. A study performed on 24 female Wistar rats demonstrated that the reconstruction of mandibular bone defects using HAp/PLGA particles induced an increase in the content of Ca in the newly created bone without causing any pathological changes to the liver and the kidneys. The presence of Pb and Cd in the defects reconstructed with HAp/PLGA nanoparticles impeded the regenerative process and led to a severe and irreversible damage to the liver and kidney parenchyma.

The effect of the androstane lung cancer inhibitor content on the cell-selective toxicity of hydroxyapatite-chitosan-PLGA nanocomposites.

An androstane (17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (derivative A)) cancer inhibitor was successfully captured in a carrier made of nano-sized hydroxyapatite (HAp) coated with chitosan-PLGA polymer blends (Ch-PLGA). In our previous studies, we demonstrated that it was convenient to use spherical HAp/Ch-PLGA carriers as vehicles to target the lungs following intravenous administration. In this study, we used emulsification and subsequent freeze-drying to load the spherical HAp/Ch-PLGA carriers with varying contents of the derivative A, in order to examine the selective toxicity towards cancerous/healthy lung cells. The XRD and FT-IR techniques confirmed the drug loading process, and the content of the poorly water soluble derivative A was estimated directly via the DSC technique. The particles were spherical in shape with the d50 distribution varying between 167 and 231 nm, whereas the content of the derivative A ranged from 6.5 to 19.3 wt%. Cell-selective cytotoxicity was examined simultaneously on two cell lines: human lung carcinoma (A549 ATCC CCL 185) and human lung fibroblasts (MRC-5 ATCC CCL 171). All particles exhibited nearly three times larger cytotoxicity towards cancer cells (A549) than towards healthy cells (MRC5), where the particles with the derivative A content of 6.5 wt% allowed for the viability of healthy cells >80%. Ninety-six hours after the treatment of cells with particles with different contents of derivative A (after incubation and recovery), recovery was faster in damaged healthy cells than in cancerous cells.

Chitosan Oligosaccharide Lactate Coated Hydroxyapatite Nanoparticles as a Vehicle for the Delivery of Steroid Drugs and the Targeting of Breast Cancer Cells.

Low targeting efficiency and fast metabolism of antineoplastic drugs are hindrances to effective chemotherapies and there is an ongoing search for better drugs, but also better carriers. Steroid derivatives, 3ß-hydroxy-16-hydroxymino-androst-5-en-17-one (A) and 3ß,17ß-dihydroxy-16-hydroxymino-androst-5-ene (B) as cancer growth inhibitors were chemically synthesized and captured in a carrier composed of hydroxyapatite (HAp) nanoparticles coated with chitosan oligosaccharide lactate (ChOLS). The only difference between the two derivatives is that A has a carbonyl group at the C17 position of the five-membered ring and B has a hydroxyl. This small difference in the structure resulted not only in different physicochemical properties of the A- and B-loaded HAp/ChOSL, but also in different biological activities. The morphology of drug-loaded HAp/ChOSL particles was spherical, but the size depended on the drug identity: d50=138 nm for A-loaded HAp/ChOSL and d50=223 nm for B-loaded HAp/ChOSL. Cell-selective toxicity was tested against human breast carcinoma (MCF7 and MDA-MB-231), human lung carcinoma (A549) and human lung fibroblasts (MRC-5). The small selectivity of pure derivatives A and B toward breast cancer cells became drastically increased when they were delivered using HAp/ChOSL particles. Whereas the ratio of the cytotoxicity imposed onto breast cancer cells and the cytotoxicity imposed onto healthy MRC-5 fibroblasts ranged from 1.5 to 1.7 for pure A and from 1.5 to 2.3 for pure derivative B depending on the concentration, it increased to 5.4 for A-loaded HAp/ChOSL and 5.1 for B-loaded HAp/ChOSL. FACS analysis demonstrated poor uptake of HAp/ChOSL particles by MCF7 cells, suggesting that the drug release occurs extracellularly. The augmented activity of the drugs was most likely due to sustained release, although the favorable positive charge of the carrier, allowing it to adhere to the negatively charged plasma membrane and release the drugs steadily and directly to the hydrophobic cell membrane milieu, was delineated as a possible complementary mechanism.

Selective anticancer activity of hydroxyapatite/chitosan-poly(d,l)-lactide-co-glycolide particles loaded with an androstane-based cancer inhibitor.

In an earlier study we demonstrated that hydroxyapatite nanoparticles coated with chitosan-poly(d,l)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous injection into mice. In this study we utilize an emulsification process and freeze drying to load the composite HAp/Ch-PLGA particles with 17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (A), a chemotherapeutic derivative of androstane and a novel compound with a selective anticancer activity against lung cancer cells. 1H NMR and 13C NMR techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The thermogravimetric and differential thermal analyses coupled with mass spectrometry were used to assess the thermal degradation products and properties of A-loaded HAp/Ch-PLGA. The loading efficiency, as indicated by the comparison of enthalpies of phase transitions in pure A and A-loaded HAp/Ch-PLGA, equaled 7.47wt.%. The release of A from HAp/Ch-PLGA was sustained, neither exhibiting a burst release nor plateauing after three weeks. Atomic force microscopy and particle size distribution analyses were used to confirm that the particles were spherical with a uniform size distribution of d50=168nm. In vitro cytotoxicity testing of A-loaded HAp/Ch-PLGA using MTT and trypan blue dye exclusion assays demonstrated that the particles were cytotoxic to the A549 human lung carcinoma cell line (46±2%), while simultaneously preserving high viability (83±3%) of regular MRC5 human lung fibroblasts and causing no harm to primary mouse lung fibroblasts. In conclusion, composite A-loaded HAp/Ch-PLGA particles could be seen as promising drug delivery platforms for selective cancer therapies, targeting malignant cells for destruction, while having a significantly lesser cytotoxic effect on the healthy cells.

In Vitro Evaluation of Nanoscale Hydroxyapatite-Based Bone Reconstructive Materials with Antimicrobial Properties.

In the field of oral implantology the loss of bone tissue prevents adequate patient care, and calls for the use of synthetic biomaterials with properties that resemble natural bone. Special attention is paid to the risk of infection after the implantation of these materials. Studies have suggested that some nanocontructs containing metal ions have antimicrobial properties. The aim of this study was to examine the antimicrobial and hemolytic activity of cobalt-substituted hydroxyapatite nanoparticles, compared to hydroxyapatite and hydroxyapatite/poly-lactide-co-glycolide. The antibacterial effects of these powders were tested against two pathogenic bacterial strains: Escherichia coi (ATCC 25922) and Staphylococcus aureus (ATCC 25923), using the disc diffusion method and the quantitative antimicrobial test in a liquid medium. The quantitative antimicrobial test showed that all of the tested biomaterials have some antibacterial properties. The effects of both tests were more prominent in case of S. aureus than in E coli. A higher percentage of cobalt in the crystal structure of cobalt-substituted hydroxyapatite nanoparticles led to an increased antimicrobial activity. All of the presented biomaterial samples were found to be non-hemolytic. Having in mind that the tested of cobalt-substituted hydroxyapatite (Ca/Co-HAp) material in given concentrations shows good hemocompatibility and antimicrobial effects, along with its previously studied biological properties, the conclusion can be reached that it is a potential candidate that could substitute calcium hydroxyapatite as the material of choice for use in bone tissue engineering and clinical practices in orthopedic, oral and maxillofacial surgery.

In vitro evaluation of a multifunctional nano drug delivery system based on tigecycline-loaded calcium-phosphate/ poly-DL-lactide-co-glycolide.

Most drug delivery systems as treatment modalities for osteomyelitis have not been evaluated for resistant infections. Tigecycline (TG) is an antimicrobial agent that could be used in the treatment of multi-drug-resistant orthopedic infections. The objective of this in vitro study has been to determine what dosage of TG causes changes in the morphology and number of osteoblasts. We have also investigated whether nanoparticulate tigecycline-loaded calcium-phosphate/poly-DL-lactide-co-glycolide is biocompatible and whether it could release bioactive TG in a controlled manner during the observation time. The cytotoxicity was tested by analyzing the release of lactate dehydrogenase from dead osteoblasts to the medium. Staphylococcus aureus was used to verify the antibacterial effect of the multifunctional drug delivery system. At concentrations as achieved by local application, TG caused high toxic effect and impaired the normal osteoblastic morphology. The nanoparticulate multifunctional drug delivery system showed good compatibility and antibacterial effect during the observation time and thus appears to be suitable for the treatment of osteomyelitis caused by multi-drug-resistant microbes.

Apatite formation on nanomaterial calcium phosphate/poly-DL-lactide-co-glycolide in simulated body fluid.

UNLABELLED: Simulated body fluid (SBF) is an artificial fluid which has ionic composition and ionic concentration similar to human blood plasma. PURPOSE: This paper compares the interaction between the nanomaterial containing calcium phosphate/poly-dl-lactide-co-glycolide (N-CP/PLGA) and SBF, in order to investigate whether and to what extent inorganic ionic composition of human blood plasma leads to the aforementioned changes in the material. METHODS: N-CP/PLGA was incubated for 1, 2, 3, and 5 weeks in SBF. The surface of the material was analyzed on SEM-EDS and FTIR spectrometer, while SBF was subjected to pH and electrical conductivity measurement. RESULTS: Our results indicate that dissolution of the polymer component of the material N-CP/PLGA and precipitation of the material similar to hydroxyapatite on its surface are based on the morphologic changes seen in this material. CONCLUSIONS: The mechanism of the apatite formation on the bioceramic surface was intensively studied and was considered crucial in designing the new biomaterials. The results obtained in this work indicate that N-CP/PLGA may be a good candidate for application to bone regeneration.

Size effect of calcium phosphate coated with poly-DL-lactide- co-glycolide on healing processes in bone reconstruction.

In this article, synthesis and application of calcium phosphate/poly-DL-lactide-co-glycolide (CP/PLGA) composite biomaterial in particulate form, in which each CP granule/particle is coated with PLGA, are described. Two types of the particulate material having different particle sizes were synthesized: one with an average particle diameter between 150 and 250 mum (micron-sized particles, MPs) and the other with an average particle diameter smaller than 50 nm (nanoparticles, NPs). A comparative in vivo analysis was done by reconstructing defects in osteoporotic alveolar bones using both composites. The material, CP granules/particles covered with polymer, was characterized using X-ray structural analysis, scanning electron microscopy, and atomic force microscopy. Changes in reparatory functions of tissues affected by osteoporosis were examined in mice in vivo, using these two kinds of composite materials, with and without autologous plasma. Having defined the target segment, histomorphometric parameters-bone area fraction, area, and mean density-were determined. The best results in the regeneration and recuperation of alveolar bone damaged by osteoporosis were achieved with the implantation of a mixture of nanoparticulate CP/PLGA composite and autologous plasma. After the implantation of microparticulate CP/PLGA, in the form of granules, mixed with autologous plasma, into an artificial defect in alveolar bone, new bone formation was also observed, although its formation rate was slower.

A novel nano drug delivery system based on tigecycline-loaded calciumphosphate coated with poly-DL-lactide-co-glycolide.

The purpose of the study presented in this paper has been to examine the possibility of the synthesis of a new nanoparticulate system for controlled and systemic drug delivery with double effect. In the first step, a drug is released from bioresorbable polymer; in the second stage, after resorption of the polymer, non-bioresorbable calcium phosphate remains the chief part of the particle and takes the role of a filler, filling a bone defect. The obtained tigecycline-loaded calcium-phosphate(CP)/poly(DL-lactide-co-glycolide)(PLGA) nanoparticles contain calcium phosphate coated with bioresorbable polymer. The composite was analyzed by FT-IR, XRD and AFM methods. The average particle size of the nanocomposite ranges between 65 and 95 nm. Release profiles of tigecycline were obtained by UV-VIS spectroscopy in physiological solution at 37 degrees C. Experimental results were analyzed using Peppas and Weibull mathematical models. Based on kinetic parameters, tigecycline release was defined as non-Fickian transport. The cytotoxicity of the nanocomposite was examined on standard cell lines of MC3T3-E1, in vitro. The obtained low values of lactate dehydrogenase (LDH) activity (under 37%) indicate low cytotoxicity level. The behaviour of the composite under real-life conditions was analyzed through implantation of the nanocomposite into living organisms, in vivo. The system with the lowest tigecycline content proved to be an adequate system for local and controlled release. Having in mind the registered antibiotics concentration in other tissues, delivery systems with a higher tigecycline content show both local and systemic effects.