Hyaluronic acid (HA) is widely used in anticancer drug delivery, since it is biocompatible, biodegradable, non-toxic, and non-immunogenic; moreover, HA receptors are overexpressed on many tumor ...cells. Exploiting this ligand-receptor interaction, the use of HA is now a rapidly-growing platform for targeting CD44-overexpressing cells, to improve anticancer therapies. The rationale underlying approaches, chemical strategies, and recent advances in the use of HA to design drug carriers for delivering anticancer agents, are reviewed. Comprehensive descriptions are given of HA-based drug conjugates, particulate carriers (micelles, liposomes, nanoparticles, microparticles), inorganic nanostructures, and hydrogels, with particular emphasis on reports of preclinical/clinical results.
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Hyaluronic acid (HA) is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of ...epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.
•A validated HPLC-UV analytical method for widely used antimicrobials is described.•The method uses a small volume of plasma and the same protocol for all analytes.•The method had the advantage of ...simple and economic preparation and instrumentation.•The method is suitable for Therapeutic Drug Monitoring or experimental trials.
Critically ill patients are often affected by several pathophysiological conditions requiring antibiotic administration and, frequently, extracorporeal therapy that significantly alter the normal pharmacokinetics of drugs. Therapeutic drug monitoring (TDM) may assist to establish the correct antibiotic dosage, but a TDM service is usually available only for some aminoglycosides and glycopeptides. The aim of this study is the validation of an HPLC-UV method for the simultaneous quantification of meropenem, vancomycin, piperacillin and tazobactam in human plasma samples.
The analytes were extracted from 250 μL of human plasma by the addition of acetonitrile for protein precipitation. After evaporation to dryness of the solvent, samples were reconstituted with 250 μL of mobile phase, and 100 μL were injected in HPLC. Chromatographic analysis was performed using a Kinetex C18 column and an UV/Vis detector set at 220 and 298 nm. The mobile phase was a mixture of phosphate buffer 0.1 M pH 3.15 and methanol in gradient, delivered at 1 mL/min.
The method was validated over clinical concentration ranges. For all the analytes, the lower limit of quantification was 1 μg/mL, and the calibration curves were linear between 1 and 100 μg/mL, with coefficients of determination ≥ 0.999. Intra-day precision was < 4%, while inter-day precision was < 7% for each analyte.
The applicability of the method has been evaluated by analysing plasma samples collected from 4 critically ill patients undergoing continuous renal replacement therapy. Moreover, the analysis of vancomycin with VANC Flex® confirmed a good correlation between the results of HPLC-UV and commercially available kits usually used by TDM service.
The method we developed only requires a small volume of plasma and uses the same sample preparation protocol, stationary phase and elution conditions for all analytes. This method offers the additional advantages of simple and rather inexpensive sample preparation and instrumentation, features that make this method an easy implementation for a general TDM laboratory.
Boron/nitrogen co-doped carbon nano-onions (BN-CNOs) are spherical nanoparticles that consist of multiple inter-nestled fullerene layers, giving them an onion-like internal structure. They have ...potential as nanocarriers due to their small size, aqueous dispersibility, and biocompatibility. The non-covalent attachment of a biocompatible polymer to BN-CNOs is a simple and effective method of creating a scaffold for a novel nanocarrier system as it allows for increased aqueous dispersibility whilst preventing the immune system from recognising the particle as a foreign object. The non-covalent approach also preserves the electronic and structural properties of the BN-CNOs. In this study, we attached a hyaluronic acid-phospholipid (HA-DMPE) conjugate polymer to the BN-CNO’s surface to improve its hydrophilicity and provide targetability toward HA-receptor overexpressing cancer cells. To this end, various ratios of HA-DMPE to BN-CNOs were investigated. The resulting supramolecular systems were characterised via UV-Vis absorption and FTIR spectroscopy, dynamic light scattering, and zeta potential techniques. It was found that the HA-DMPE conjugate polymer was permanently wrapped around the BN-CNO nanoparticle surface. Moreover, the resulting BN-CNO/HA-DMPE supramolecular systems displayed enhanced aqueous solubility compared to unfunctionalised BN-CNOs, with excellent long-term stability observed in aqueous dispersions.
Drug efflux transporters, in particular P-glycoprotein (Pgp), limit the success of chemotherapy. We previously found that synthetic doxorubicin conjugated with nitric oxide (NO)-releasing group ...overcomes resistance by inducing a NO-mediated inhibition of Pgp. Here we produced the first liposomal formulations of this nitrooxy-doxorubicin decorated with folic acid (FA), termed LNDF, in order to improve their active targeting against Pgp-expressing tumors. Folate was inserted onto liposomes surface using two different methods and the formulations were compared with respect to their technological features and in vitro behavior. By analyzing human and murine breast cancer cells with different expression of FA receptor (FAR) and Pgp, we demonstrated that LNDF are internalized in a FAR-dependent manner and achieve maximal anti-tumor efficacy against FAR-positive/Pgp-positive cells. Upon uptake of LNDF, nitrooxy-doxorubicin was delivered within nucleus, where it induced cell cycle arrest and DNA damages, and mitochondria, where it impaired the mitochondrial energy metabolism and triggered mitochondria-dependent apoptosis. LNDF reduced the growth of FAR-positive/Pgp-positive tumors and prevented tumor formation in mice, whereas doxorubicin and Caelyx® failed. LNDF cardiotoxicity was comparable to Caelyx®. The sensitivity to LNDF was maintained in tumors exposed to repeated cycles of the drug and in cells derived from the exposed tumors, excluding the onset of secondary resistance.
By combining an innovative multitarget cargo drug, conceived to achieve high efficacy against Pgp-expressing cells, and appropriate strategies of liposome formulation and decoration, we produced a therapeutic tool that may represent a significant advancement in the treatment of FAR-positive/Pgp-positive tumors.
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The aim of this work was the preparation, characterization, and preliminary evaluation of the targeting ability toward pancreatic adenocarcinoma cells of liposomes containing the gemcitabine ...lipophilic prodrug 4-(N)-lauroyl-gemcitabine, C12GEM. Hyaluronic acid (HA) was selected as targeting agent since it is biodegradable, biocompatible, and can be chemically modified and its cell surface receptor CD44 is overexpressed on various tumors.
For this purpose, conjugates between a phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and HA of two different low molecular weights 4800Da (12 disaccharidic units) and 12,000Da (32 disaccharidic units), were prepared, characterized, and introduced in the liposomes during the preparation.
Different liposomal formulations were prepared and their characteristics were analyzed: size, Z potential, and TEM analyses underline a difference in the HA-liposomes from the non-HA ones. In order to better understand the HA-liposome cellular localization and to evaluate their interaction with CD44 receptor, confocal microscopy studies were performed. The results demonstrate that HA facilitates the recognition of liposomes by MiaPaCa2 cells (CD44+) and that the uptake increases with increase in the polymer molecular weight.
Finally, the cytotoxicity of the different preparations was evaluated and data show that incorporation of C12GEM increases their cytotoxic activity and that HA-liposomes inhibit cell growth more than plain liposomes.
Altogether, the results demonstrate the specificity of C12GEM targeting toward CD44-overexpressing pancreatic adenocarcinoma cell line using HA as a ligand.
Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and ...treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression.
Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the ...presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide-
-glycolide) (PLGA) nanoparticles and liposomes was evaluated. In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting.
Muscular dystrophies are a group of rare genetic pathologies, encompassing a variety of clinical phenotypes and mechanisms of disease. Several compounds have been proposed to treat compromised ...muscles, but it is known that pharmacokinetics and pharmacodynamics problems could occur. To solve these issues, it has been suggested that nanocarriers could be used to allow controlled and targeted drug release. Therefore, the aim of this study was to prepare actively targeted poly(lactide-
-glycolide) (PLGA) nanoparticles (NPs) for the treatment of muscular pathologies. By taking advantage of the high affinity for carnitine of skeletal muscle cells due to the expression of Na
-coupled carnitine transporter (OCTN), NPs have been actively targeted via association to an amphiphilic derivative of L-carnitine. Furthermore, pentamidine, an old drug repurposed for its positive effects on myotonic dystrophy type I, was incorporated into NPs. We obtained monodispersed targeted NPs, with a mean diameter of about 100 nm and a negative zeta potential. To assess the targeting ability of the NPs, cell uptake studies were performed on C2C12 myoblasts and myotubes using confocal and transmission electron microscopy. The results showed an increased uptake of carnitine-functionalized NPs compared to nontargeted carriers in myotubes, which was probably due to the interaction with OCTN receptors occurring in large amounts in these differentiated muscle cells.
In this paper, mesoporous silica nanoparticles (MSNs) were studied as vehicles for the delivery of the antitumoral drug gemcitabine (GEM) and of its 4-(N)-acyl derivatives, (4-(N)-valeroyl-(C5GEM), ...4-(N)-lauroyl-(C12GEM) and 4-(N)-stearoyl-gemcitabine (C18GEM)). The loading of the GEM lipophilic prodrugs on MSNs was explored with the aim to obtain both a physical and a chemical protection of GEM from rapid plasmatic metabolization. For this purpose, MSNs as such or with grafted aminopropyl and carboxyethyl groups were prepared and characterized. Then, their different drug loading capacity in relation to the nature of the functional group was evaluated. In our experimental conditions, GEM was not loaded in any MSNs, while C12GEM was the most efficiently encapsulated and employed for further evaluation. The results showed that loading capacity increased with the presence of functional groups on the nanoparticles; similarly, the presence of functional groups on MSNs' surface influenced the drug release profile. Finally, the cytotoxicity of the different preparations was evaluated and data showed that C12GEM loaded MSNs are less cytotoxic than the free drug with an activity that increased with the incubating time, indicating that all these systems are able to release the drug in a controlled manner. Altogether, the results demonstrate that these MSNs could be an interesting system for the delivery of anticancer drugs.
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