Olga Swiech

Introduction: Osteomyelitis (OM) is an inflammation of bone and bone marrow and remains one of the most challenging infectious diseases to treat. This clinical difficulty highlights the need for alternative therapeutic strategies, including targeted local delivery of antibacterial agents. In this study, we report for the first time the use of zinc-bovine serum albumin (ZnBSA) hybrid nanoflowers as potential antibiotic carriers for osteomyelitis treatment. Hybrid nanoflowers are hierarchically structured nanomaterials composed of inorganic components (typically metal phosphates) and organic molecules (such as proteins or enzymes) that self-assemble into flower-like morphologies, exhibiting high surface area and synergistic physicochemical properties arising from organic-inorganic integration. Methods: ZnBSA hybrid nanostructures were synthesized through a rapid and optimized procedure and characterized using scanning electron microscopy, infrared spectroscopy and X-ray diffraction. Ciprofloxacin was loaded into the ZnBSA nanostructures, and drug release behavior was studied by ultraviolet-visable spectroscopy. Antibacterial activity was assessed using optical density measurements and disc diffusion assays. The biocompatibility of ZnBSA loaded with ciprofloxacin was evaluated through in vitro toxicity assays on human red blood cells and dermal fibroblasts, as well as in vivo testing using Danio rerio. Results: The ZnBSA nanostructures demonstrated rapid formation and improved biocompatibility compared to conventional copperbased nanoflowers. Toxicity studies confirmed a favorable safety profile both in vitro and in vivo. Ciprofloxacin-loaded ZnBSA carriers exhibited sustained drug release and effective antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae. Discussion: These findings indicate that ZnBSA hybrid nanostructures represent a safe and effective localized drug delivery system for osteomyelitis treatment. Their biocompatibility, rapid synthesis, sustained antibiotic release, and broad-spectrum antibacterial activity highlight their potential as a promising alternative to existing nanocarrier systems for managing bone infections.

β-Cyclodextrin (CD) derivatives containing an aromatic triazole ring were studied as potential carriers of the following drugs containing an anthraquinone moiety: anthraquinone-2-sulfonic acid (AQ2S); anthraquinone-2-carboxylic acid (AQ2CA); and a common anthracycline, daunorubicin (DNR). UV-Vis and voltammetry measurements were carried out to determine the solubilities and association constants of the complexes formed, and the results revealed the unique properties of the chosen CDs as effective pH-dependent drug complexing agents. The association constants of the drug complexes with the CDs containing a triazole and lipoic acid (βCDLip) or galactosamine (βCDGAL), were significantly larger than that of the native βCD. The AQ2CA and AQ2S drugs were poorly soluble, and their solubilities increased as a result of complex formation with βCDLip and βCDGAL ligands. AQ2CA and AQ2S are negatively charged at pH 7.4. Therefore, they were less prone to form an inclusion complex with the hydrophobic CD cavity than at pH 3 (characteristic of gastric juices) when protonated. The βCDTriazole and βCDGAL ligands were found to form weaker inclusion complexes with the positively charged drug DNR at an acidic pH (pH 5.5) than in a neutral medium (pH 7.4) in which the drug dissociates to its neutral, uncharged form. This pH dependence is favorable for antitumor applications.

β-cyclodextrin (CD) derivatives containing aromatic triazole ring were studied as potential carriers of drugs containing an anthraquinone moiety in the structure: anthraquinone-2-sulfonic acid (AQ2S), anthraquinone-2-carboxylic acid (AQ2CA) and a common anthracycline, daunorubicin (DNR). UV-Vis and voltammetry measurements were carried out to determine the solubilities and stability constants of the complexes formed and revealed the unique properties of the chosen CDs as effective pH dependent drug complexing agents. The stability constants of the drug complexes with the CDs containing triazole: βCDLip and βCDGAL were significantly larger than with the native βCD. The AQ2CA and AQ2S drugs are ill-soluble and their solubilities increased as the result of complex formation with βCDLip and βCDGAL ligands. AQ2CA, AQ2S were negatively charged at pH 7.4 and therefore they were less prone to form inclusion complex with the hydrophobic CD cavity than at pH 3 (characteristic of gastric juices) when they were protonated. βCDTriazole and βCDGAL ligands were found to form weaker inclusion complexes with the positively charged drug DNR at acidic pH (pH 5.5) than in the neutral medium (pH 7.4) when the drug dissociates to the neutral, uncharged form. This pH dependence is favorable for anti-tumor applications.

Lipoic acid derivative of cyclodextrin, βCDLip, was used as the drug carrier for doxorubicin (DOX) and the stability constants of the DOX–βCDLip were determined in the environment of the cell medium. The experiments were performed in neutral (pH 7.6) and acidified (pH 6.3) cell media containing more than forty interferences including: amino acids, vitamins, lipids and proteins. We proved that the pH of the medium has a noticeable impact on the affinity of the drug towards the carrier. At neutral pH, the formation constants of the complex are higher than at pH 6.3, what is characteristic for the cancer cells microenvironment. Furthermore, the values obtained in both cell media are twice smaller than the values obtained previously for the same complex but in the absence of common cell media components indicating that there is a competition between DOX and some hydrophobic medium components for the complex formation with βCDLip. On the other hand at pH 7.6, the amount of free DOX is highly limited due to the fact that most of DOX is still in the complexed form, while at pH 6.3 the cell media ingredients become strong interferences in the formation of the complex between DOX and the drug carrier. The observed behaviour is due to partial protonation of DOX and to competition between the drug and the lipoic side arm of cyclodextrin for the cyclodextrin cavity. The stability constants of the DOX–βCDLip complex in acidic pH are similar to the values for DOX with native β-cyclodextrin, demonstrating that the strengthening effect of DOX–CD complex resulting from the presence of cyclodextrin’s aromatic substituent (Lip) occurs only in the case of neutral pH. The high value of the stability constant of the DOX–βCDLip complex in cell medium at pH 7.6 indicates high selectivity of βCDLip ligand which would be of importance both for the effective drug delivery and for its application in DOX sensing devices.

The influence of the pH of the multicomponent cell medium on the performance of doxorubicin (DOX), an anticancer drug, was studied on the examples of cervical (HeLa) and kidney (A498) cancer cell lines. The change of pH of the cell medium to more acidic led to a decrease of DOX toxicity on both cell lines due to the change of drug permeability across the cell membrane as a result of drug protonation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) studies and lactate dehydrogenase (LDH) release tests have shown low toxicity of the drug, especially in the case of A498 cells, which are characterized by an extremely high glycolytic metabolism. The behavior was ascribed primarily to the increased proton concentration in the peripheral blood follicle in the presence of products of the acidic glycolytic metabolism. It is not observed in the measurements performed in commercially available media since they usually have a neutral pH. In earlier reports on kidney cancer, several mechanisms were discussed, including the metabolism of DOX to its less toxic derivative, doxorubicinol, overexpression of ATP binding cassette subfamily B member 1 (ABCB1) transporters, that remove DOX from the inside of cells; however, there was no focus on the simple but very important contribution of drug protonation described in the present study. Drug pH-dependent equilibria in the cell medium should be considered since changes in the drug form may be an additional reason for multidrug resistance.

A new and convenient synthetic pathway was developed to produce monosubstituted cyclodextrins with high yields. Each of the β-cyclodextrin derivatives described in this work has an aromatic substituent connected with cyclodextrin core by a pH-sensitive hydrazone linker and a carbon chain. Carbon chains differ in lengths having one or three carbon atoms. The correlation between water solubility and linker length was determined using UV–Vis spectroscopy, while the dependence of hydrazone bond hydrolysis on the electrolyte pH was confirmed by cyclic voltammetry. The pH-dependent complex-formation ability between the hydrazone derivative of cyclodextrin and anthracycline drug was examined by square wave voltammetry. The significantly big solubility and the appropriate pH, at which the hydrolysis of the hydrazone bond occurs, make the newly synthesized derivatives attractive for pharmaceutical and medical applications.