In Vitro Superparamagnetic Hyperthermia Employing Magnetite Gamma-Cyclodextrin Nanobioconjugates for Human Squamous Skin Carcinoma Therapy.

In vitro alternative therapy of human epidermoid squamous carcinoma (A431) by superparamagnetic hyperthermia (SPMHT) using Fe3O4 (magnetite) superparamagnetic nanoparticles (SPIONs) with an average diameter of 15.8 nm, bioconjugated with hydroxypropyl gamma-cyclodextrins (HP-γ-CDs) by means of polyacrylic acid (PAA) biopolymer, is presented in this paper. The therapy was carried out at a temperature of 43 °C for 30 min using the concentrations of Fe3O4 ferrimagnetic nanoparticles from nanobioconjugates of 1, 5, and 10 mg/mL nanoparticles in cell suspension, which were previously found by us to be non-toxic for healthy cells (cell viabilities close to 100%), according to ISO standards (cell viability must be greater than 70%). The temperature for the in vitro therapy was obtained by the safe application (without exceeding the biological limit and cellular damage) of an alternating magnetic field with a frequency of 312.4 kHz and amplitudes of 168, 208, and 370 G, depending on the concentration of the magnetic nanoparticles. The optimal concentration of magnetic nanoparticles in suspension was found experimentally. The results obtained after the treatment show its high effectiveness in destroying the A431 tumor cells, up to 83%, with the possibility of increasing even more, which demonstrates the viability of the SPMHT method with Fe3O4-PAA-(HP-γ-CDs) nanobioconjugates for human squamous cancer therapy.

A Preliminary Report Regarding the Morphological Changes of Nano-Enabled Pharmaceutical Formulation on Human Lung Carcinoma Monolayer and 3D Bronchial Microtissue.

Background and Objectives: Nowadays, the development of enabled pharmaceutical nanoparticles of solid lipid type is continuously growing, because they have the potential to be used for targeted drug release leading to an increased effect of chemotherapy, being used in lung cancer nano-diagnosis and nano-therapy. The current study reports the preliminary results obtained regarding the biological effect of a new nano-enabled pharmaceutical formulation in terms of its cytotoxic and biosafety profile. Materials and Methods: The pharmaceutical formulations consist of solid lipid nanoparticles (SLN) obtained via the emulsification-diffusion method by loading green iron oxide nanoparticles (green-IONPs) with a pentacyclic triterpene (oleanolic acid-OA). Further, a complex biological assessment was performed, employing three-dimensional (3D) bronchial microtissues (EpiAirwayTM) to determine the biosafety profile of the SLN samples. The cytotoxic potential of the samples was evaluated on human lung carcinoma, using an in vitro model (A549 human lung carcinoma monolayer). Results: The data revealed that the A549 cell line was strongly affected after treatment with SLN samples, especially those that contained OA-loaded green-IONPs obtained with Ocimum basilicum extract (under 30% viability rates). The biosafety profile investigation of the 3D normal in vitro bronchial model showed that all the SLN samples negatively affected the viability of the bronchial microtissues (below 50%). As regards the morphological changes, all the samples induce major changes such as loss of the surface epithelium integrity, loss of epithelial junctions, loss of cilia, hyperkeratosis, and cell death caused by apoptosis. Conclusions: In summary, the culprit for the negative impact on viability and morphology of 3D normal bronchial microtissues could be the too-high dose (500 µg/mL) of the SLN sample used. Nevertheless, further adjustments in the SLN synthesis process and another complex in vitro evaluation will be considered for future research.

Oleogel Formulations for the Topical Delivery of Betulin and Lupeol in Skin Injuries-Preparation, Physicochemical Characterization, and Pharmaco-Toxicological Evaluation.

The skin integrity is essential due to its pivotal role as a biological barrier against external noxious factors. Pentacyclic triterpenes stand as valuable plant-derived natural compounds in the treatment of skin injuries due to their anti-inflammatory, antioxidant, antimicrobial, and healing properties. Consequently, the primary aim of the current investigation was the development as well as the physicochemical and pharmaco-toxicological characterization of betulin- and lupeol-based oleogels (Bet OG and Lup OG) for topical application in skin injuries. The results revealed suitable pH as well as organoleptic, rheological, and textural properties. The penetration and permeation of Bet and Lup oleogels through porcine ear skin as well as the retention of both oleogels in the skin were demonstrated through ex vivo studies. In vitro, Bet OG and Lup OG showed good biocompatibility on HaCaT human immortalized cells. Moreover, Bet OG exerted a potent wound-healing property by stimulating the migration of the HaCaT cells. The in ovo results demonstrated the non-irritative potential of the developed formulations. Additionally, the undertaken in vivo investigation indicated a positive effect of oleogels treatment on skin parameters by increasing skin hydration and decreasing erythema. In conclusion, oleogel formulations are ideal for the local delivery of betulin and lupeol in skin disorders.

High Efficacy on the Death of Breast Cancer Cells Using SPMHT with Magnetite Cyclodextrins Nanobioconjugates.

In this study, we present the experimental results obtained in vitro on the human breast adenocarcinoma cell line (MCF-7) by applying superparamagnetic hyperthermia (SPMHT) using novel Fe3O4-PAA-(HP-γ-CDs) (PAA is polyacrylic acid and HP-γ-CDs is hydroxypropyl gamma-cyclodextrins) nanobioconjugates previously obtained by us. In the in vitro SPMHT experiments, we used concentrations of 1, 5 and 10 mg/mL of Fe3O4 ferrimagnetic nanoparticles from Fe3O4-PAA-(HP-γ-CDs) nanobioconjugates suspended in culture media containing 1 × 105 MCF-7 human breast adenocarcinoma cells. The harmonic alternating magnetic field used in the in vitro experiments that did not affect cell viability was found to be optimal in the range of 160-378 Gs and at a frequency of 312.2 kHz. The appropriate duration of the therapy was 30 min. After applying SPMHT with these nanobioconjugates under the above conditions, MCF-7 cancer cells died out in a very high percentage, of until 95.11%. Moreover, we studied the field up to which magnetic hyperthermia can be safely applied without cellular toxicity, and found a new upper biological limit H × f ~9.5 × 109 A/m⋅Hz (H is the amplitude and f is the frequency of the alternating magnetic field) to safely apply the magnetic field in vitro in the case of MCF-7 cells; the value was twice as high compared to the currently known value. This is a major advantage for magnetic hyperthermia in vitro and in vivo, because it allows one to achieve a therapy temperature of 43 °C safely in a much shorter time without affecting healthy cells. At the same time, using the new biological limit for a magnetic field, the concentration of magnetic nanoparticles in magnetic hyperthermia can be greatly reduced, obtaining the same hyperthermic effect, while at the same time, reducing cellular toxicity. This new limit of the magnetic field was tested by us in vitro with very good results, without the cell viability decreasing below ~90%.

Enhanced Cytotoxicity and Antimelanoma Activity of Novel Semisynthetic Derivatives of Betulinic Acid with Indole Conjugation.

The prevalence and severity of skin cancer, specifically malignant melanoma, among Caucasians remains a significant concern. Natural compounds from plants have long been explored as potential anticancer agents. Betulinic acid (BI) has shown promise in its therapeutic properties, including its anticancer effects. However, its limited bioavailability has hindered its medicinal applications. To address this issue, two recently synthesized semisynthetic derivatives, N-(2,3-indolo-betulinoyl)diglycylglycine (BA1) and N-(2,3-indolo-betulinoyl)glycylglycine (BA2), were compared with previously reported compounds N-(2,3-indolo-betulinoyl)glycine (BA3), 2,3-indolo-betulinic acid (BA4), and BI. These compounds were evaluated for their effects on murine melanoma cells (B164A5) using various in vitro assays. The introduction of an indole framework at the C2 position of BI resulted in an increased cytotoxicity. Furthermore, the cytotoxicity of compound BA4 was enhanced by conjugating its carboxylic group with an amino acid residue. BA2 and BA3, with glycine and glycylglycine residues at C28, exhibited approximately 2.20-fold higher inhibitory activity compared to BA4. The safety assessment of the compounds on human keratinocytes (HaCaT) has revealed that concentrations up to 10 µM slightly reduced cell viability, while concentrations of 75 µM resulted in lower cell viability rates. LDH leakage assays confirmed cell membrane damage in B164A5 cells when exposed to the tested compounds. BA2 and BA3 exhibited the highest LDH release, indicating their strong cytotoxicity. The NR assay revealed dose-dependent lysosome disruption for BI and 2,3-indolo-betulinic acid derivatives, with BA1, BA2, and BA3 showing the most cytotoxic effects. Scratch assays demonstrated concentration-dependent inhibition of cell migration, with BA2 and BA3 being the most effective. Hoechst 3342 staining revealed that BA2 induced apoptosis, while BA3 induced necrosis at lower concentrations, confirming their anti-melanoma properties. In conclusion, the semisynthetic derivatives of BI, particularly BA2 and BA3, show promise as potential candidates for further research in developing effective anti-cancer therapies.

Fe3O4-PAA-(HP-γ-CDs) Biocompatible Ferrimagnetic Nanoparticles for Increasing the Efficacy in Superparamagnetic Hyperthermia.

In this paper, we present the obtaining of Fe3O4-PAA-(HP-γ-CDs) ferrimagnetic nanobioconjugates (PAA: polyacrylic acid, HP-γ-CDs: hydroxypropyl gamma-cyclodextrins) in a hybrid core-shell biostructure (core: inorganic Fe3O4 nanoparticles, and shell: organic PAA-(HP-γ-CDs)) and their use in superparamagnetic hyperthermia without cellular toxicity and with increased efficacy for future alternative cancer therapy. In order to design the optimal experimental conditions for obtaining nanobioconjugates and then superparamagnetic hyperthermia (SPMHT), we used molecular docking simulation and computational assessment of the maximum specific loss power (SLP) that led to nanoparticles' heating. The nanoparticles and nanobioconjugates obtained were studied and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transformed-infrared spectroscopy (FT-IR), dynamic light scattering (DLS), and magnetic measurements (MMs). The cell viability of the nanoparticles and nanobioconjugates was assessed by means of the MTT assay using human immortalized keratinocytes (HaCaT) as an in vitro model. Superparamagnetic hyperthermia with nanoparticles and nanobioconjugates was obtained experimentally in a magnetic field of 15.92 kA/m and frequency of 312.2 kHz for the magnetic nanoparticle core with a (average) diameter of 15.8 nm, which resulted in the maximum hyperthermic effect that led to a temperature of ~42.5 °C necessary in the therapy of tumors in a short time so as not to affect healthy tissues. The biological screening of Fe3O4-PAA nanoparticles and PAA-(HP-γ-CDs) nanobioconjugates showed no cytotoxic effect on HaCaT cells for a time interval of 24 h, both under standard (37 °C) and hyperthermia conditions (42.5 °C). Thus, Fe3O4-PA-(HP-γ-CDs) ferrimagnetic nanobioconjugates can be used successfully in superparamagnetic hyperthermia without toxicity and with increased efficiency due to the small layer thickness of the PAA-(HP-γ-CDs) shell, which is suitable in this alternative therapeutic technique.

Biosynthesis of Iron Oxide Nanoparticles: Physico-Chemical Characterization and Their In Vitro Cytotoxicity on Healthy and Tumorigenic Cell Lines.

Iron oxide nanoparticles were synthesized starting from two aqueous extracts based on Artemisia absinthium L. leaf and stems, employing a simplest, eco-friendliness and low toxicity method-green synthesis. The nanoparticles were characterized by powder X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FT-IR), X-ray fluorescence analysis (XRF), thermal analysis (TG/DSC), and scanning electron microscopy (SEM). Lack of magnetic properties and the reddish-brown color of all the samples confirms the presence of hematite as majority phase. The FTIR bands located at 435 cm-1 and 590 cm-1, are assigned to Fe-O stretching vibration from hematite, confirming the formation of α-Fe2O3 nanoparticles (NPs). The in vitro screening of the samples revealed that the healthy cell line (HaCaT) presents a good viability (above 80%) after exposure to iron oxide NPs and lack of apoptotic features, while the tumorigenic cell lines manifested a higher sensitivity, especially the melanoma cells (A375) when exposed to concentration of 500 µg/mL iron oxide NPs for 72 h. Moreover, A375 cells elicited significant apoptotic markers under these parameters (concentration of 500 µg/mL iron oxide NPs for a contact time of 72 h).

Development and Characterization of Fe3O4@Carbon Nanoparticles and Their Biological Screening Related to Oral Administration.

The current study presents the effect of naked Fe3O4@Carbon nanoparticles obtained by the combustion method on primary human gingival fibroblasts (HGFs) and primary gingival keratinocytes (PGKs)-relevant cell lines of buccal oral mucosa. In this regard, the objectives of this study were as follows: (i) development via combustion method and characterization of nanosized magnetite particles with carbon on their surface, (ii) biocompatibility assessment of the obtained magnetic nanoparticles on HGF and PGK cell lines and (iii) evaluation of possible irritative reaction of Fe3O4@Carbon nanoparticles on the highly vascularized chorioallantoic membrane of a chick embryo. Physicochemical properties of Fe3O4@Carbon nanoparticles were characterized in terms of phase composition, chemical structure, and polymorphic and molecular interactions of the chemical bonds within the nanomaterial, magnetic measurements, ultrastructure, morphology, and elemental composition. The X-ray diffraction analysis revealed the formation of magnetite as phase pure without any other secondary phases, and Raman spectroscopy exhibit that the pre-formed magnetic nanoparticles were covered with carbon film, resulting from the synthesis method employed. Scanning electron microscopy shown that nanoparticles obtained were uniformly distributed, with a nearly spherical shape with sizes at the nanometric level; iron, oxygen, and carbon were the only elements detected. While biological screening of Fe3O4@Carbon nanoparticles revealed no significant cytotoxic potential on the HGF and PGK cell lines, a slight sign of irritation was observed on a limited area on the chorioallantoic membrane of the chick embryo.

Biocompatible Magnetic Colloidal Suspension Used as a Tool for Localized Hyperthermia in Human Breast Adenocarcinoma Cells: Physicochemical Analysis and Complex In Vitro Biological Profile.

Magnetic iron oxide nanoparticles are the most desired nanomaterials for biomedical applications due to their unique physiochemical properties. A facile single-step process for the preparation of a highly stable and biocompatible magnetic colloidal suspension based on citric-acid-coated magnetic iron oxide nanoparticles used as an effective heating source for the hyperthermia treatment of cancer cells is presented. The physicochemical analysis revealed that the magnetic colloidal suspension had a z-average diameter of 72.7 nm at 25 °C with a polydispersity index of 0.179 and a zeta potential of -45.0 mV, superparamagnetic features, and a heating capacity that was quantified by an intrinsic loss power analysis. Raman spectroscopy showed the presence of magnetite and confirmed the presence of citric acid on the surfaces of the magnetic iron oxide nanoparticles. The biological results showed that breast adenocarcinoma cells (MDA-MB-231) were significantly affected after exposure to the magnetic colloidal suspension with a concentration of 30 µg/mL 24 h post-treatment under hyperthermic conditions, while the nontumorigenic (MCF-10A) cells exhibited a viability above 90% under the same thermal setup. Thus, the biological data obtained in the present study clearly endorse the need for further investigations to establish the clinical biological potential of synthesized magnetic colloidal suspension for magnetically triggered hyperthermia.

Scaffold-Type Structure Dental Ceramics with Different Compositions Evaluated through Physicochemical Characteristics and Biosecurity Profiles.

The design and development of ceramic structures based on 3D scaffolding as dental bone substitutes has become a topic of great interest in the regenerative dentistry research area. In this regard, the present study focuses on the development of two scaffold-type structures obtained from different commercial dental ceramics by employing the foam replication method. At the same time, the study underlines the physicochemical features and the biological profiles of the newly developed scaffolds, compared to two traditional Cerabone® materials used for bone augmentation, by employing both the in vitro Alamar blue proliferation test at 24, 48 and 96 h poststimulation and the in ovo chick chorioallantoic membrane (CAM) assay. The data reveal that the newly developed scaffolds express comparable results with the traditional Cerabone® augmentation masses. In terms of network porosity, the scaffolds show higher pore interconnectivity compared to Cerabone® granules, whereas regarding the biosafety profile, all ceramic samples manifest good biocompatibility on primary human gingival fibroblasts (HGFs); however only the Cerabone® samples induced proliferation of HGF cells following exposure to concentrations of 5 and 10 µg/mL. Additionally, none of the test samples induce irritative activity on the vascular developing plexus. Thus, based on the current results, the preliminary biosecurity profile of ceramic scaffolds supports the usefulness for further testing of high relevance for their possible clinical dental applications.