Designing functionalized nanodiamonds with hyaluronic acid-phospholipid conjugates for enhanced cancer cell targeting and fluorescence imaging capabilities.

Nanomedicine aims to develop smart approaches for treating cancer and other diseases to improve patient survival and quality of life. Novel nanoparticles as nanodiamonds (NDs) represent promising candidates to overcome current limitations. In this study, NDs were functionalized with a 200 kDa hyaluronic acid-phospholipid conjugate (HA/DMPE), enhancing the stability of the nanoparticles in water-based solutions and selectivity for cancer cells overexpressing specific HA cluster determinant 44 (CD44) receptors. These nanoparticles were characterized by diffuse reflectance Fourier-transform infrared spectroscopy, Raman spectroscopy, and photoluminescence spectroscopy, confirming the efficacy of the functionalization process. Scanning electron microscopy was employed to evaluate the size distribution of the dry particles, while dynamic light scattering and zeta potential measurements were utilized to evaluate ND behavior in a water-based medium. Furthermore, the ND biocompatibility and uptake mediated by CD44 receptors in three different models of human adenocarcinoma cells were assessed by performing cytofluorimetric assay and confocal microscopy. HA-functionalized nanodiamonds demonstrated the advantage of active targeting in the presence of cancer cells expressing CD44 on the surface, suggesting higher drug delivery to tumors over non-tumor tissues. Even CD44-poorly expressing cancers could be targeted by the NDs, thanks to their good passive diffusion within cancer cells.

Targeting pentamidine towards CD44-overexpressing cells using hyaluronated lipid-polymer hybrid nanoparticles.

Biodegradable nanocarriers possess enormous potential for use as drug delivery systems that can accomplish controlled and targeted drug release, and a wide range of nanosystems have been reported for the treatment and/or diagnosis of various diseases and disorders. Of the various nanocarriers currently available, liposomes and polymer nanoparticles have been extensively studied and some formulations have already reached the market. However, a combination of properties to create a single hybrid system can give these carriers significant advantages, such as improvement in encapsulation efficacy, higher stability, and active targeting towards specific cells or tissues, over lipid or polymer-based platforms. To this aim, this work presents the formulation of poly(lactic-co-glycolic) acid (PLGA) nanoparticles in the presence of a hyaluronic acid (HA)-phospholipid conjugate (HA-DPPE), which was used to anchor HA onto the nanoparticle surface and therefore create an actively targeted hybrid nanosystem. Furthermore, ionic interactions have been proposed for drug encapsulation, leading us to select the free base form of pentamidine (PTM-B) as the model drug. We herein report the preparation of hybrid nanocarriers that were loaded via ion-pairing between the negatively charged PLGA and HA and the positively charged PTM-B, demonstrating an improved loading capacity compared to PLGA-based nanoparticles. The nanocarriers displayed a size of below 150 nm, a negative zeta potential of -35 mV, a core-shell internal arrangement and high encapsulation efficiency (90%). Finally, the ability to be taken up and exert preferential and receptor-mediated cytotoxicity on cancer cells that overexpress the HA specific receptor (CD44) has been evaluated. Competition assays supported the hypothesis that PLGA/HA-DPPE nanoparticles deliver their cargo within cells in a CD44-dependent manner.

Freeze Drying of Polymer Nanoparticles and Liposomes Exploiting Different Saccharide-Based Approaches.

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-co-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.

An ex vivo experimental system to track fluorescent nanoparticles inside skeletal muscle.

The development of novel nanoconstructs for biomedical applications requires the assessment of their biodistribution, metabolism and clearance in single cells, organs and entire organisms in a living environment. To reduce the number of in vivo experiments performed and to refine the methods used, in accordance with the 3Rs principle, this work proposes an ex vivo experimental system to monitor, using fluorescence microscopy, the distribution of nanoparticles in explanted murine skeletal muscle maintained in a bioreactor that can preserve the structural and functional features of the organ for long periods of time. Fluorescently-labelled liposomes and poly(lactide-co-glycolide) (PLGA)-based nanoparticles were injected into the intact soleus muscle (in the distal region close to the tendon) immediately after explants, and their distribution was analysed at increasing incubation times in cross cryosections from the proximal region of the belly. Both nanocarriers were clearly recognized in the muscle and were found to enter and migrate inside the myofibres, whereas their migration in the connective tissue seemed to be limited. In addition, some fluorescent signals were observed inside the macrophages, demonstrating the physiological clearance of the nanocarriers that did not enter the myofibres. Our ex vivo system therefore provides more information than previous in vitro experiments on cultured muscle cells, highlighting the need for the appropriate functionalization of nanocarriers if myofibre targeting is to be improved.

Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming.

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial DharmaFECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80-100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA downregulation. DE-DOPE/miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo.

Hyaluronated and PEGylated Liposomes as a Potential Drug-Delivery Strategy to Specifically Target Liver Cancer and Inflammatory Cells.

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.

Nanotechnological approaches for pentamidine delivery.

Pentamidine (PTM), which is a diamine that is widely known for its antimicrobial activity, is a very interesting drug whose mechanism of action is not fully understood. In recent years, PTM has been proposed as a novel potential drug candidate for the treatment of mental illnesses, myotonic dystrophy, diabetes, and tumors. Nevertheless, the systemic administration of PTM causes severe side effects, especially nephrotoxicity. In order to efficiently deliver PTM and reduce its side effects, several nanosystems that take advantage of the chemical characteristics of PTM, such as the presence of two positively charged amidine groups at physiological pH, have been proposed as useful delivery tools. Polymeric, lipidic, inorganic, and other types of nanocarriers have been reported in the literature for PTM delivery, and they are all in different development phases. The available approaches for the design of PTM nanoparticulate delivery systems are reported in this review, with a particular emphasis on formulation strategies and in vitro/in vivo applications. Furthermore, a critical view of the future developments of nanomedicine for PTM applications, based on recent repurposing studies, is provided. Created with BioRender.com.

L-Carnitine Functionalization to Increase Skeletal Muscle Tropism of PLGA Nanoparticles.

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-co-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.

Nanomedicine for Gene Delivery and Drug Repurposing in the Treatment of Muscular Dystrophies.

Muscular Dystrophies (MDs) are a group of rare inherited genetic muscular pathologies encompassing a variety of clinical phenotypes, gene mutations and mechanisms of disease. MDs undergo progressive skeletal muscle degeneration causing severe health problems that lead to poor life quality, disability and premature death. There are no available therapies to counteract the causes of these diseases and conventional treatments are administered only to mitigate symptoms. Recent understanding on the pathogenetic mechanisms allowed the development of novel therapeutic strategies based on gene therapy, genome editing CRISPR/Cas9 and drug repurposing approaches. Despite the therapeutic potential of these treatments, once the actives are administered, their instability, susceptibility to degradation and toxicity limit their applications. In this frame, the design of delivery strategies based on nanomedicines holds great promise for MD treatments. This review focuses on nanomedicine approaches able to encapsulate therapeutic agents such as small chemical molecules and oligonucleotides to target the most common MDs such as Duchenne Muscular Dystrophy and the Myotonic Dystrophies. The challenge related to in vitro and in vivo testing of nanosystems in appropriate animal models is also addressed. Finally, the most promising nanomedicine-based strategies are highlighted and a critical view in future developments of nanomedicine for neuromuscular diseases is provided.

Treatment of acute lung inflammation by pulmonary delivery of anti-TNF-α siRNA with PAMAM dendrimers in a murine model.

To improve the efficacy of nucleic acid-based therapeutics, e.g., small interfering RNA (siRNA), transfection agents are needed for efficient delivery into cells. Several classes of dendrimers have been found useful as transfection agents for the delivery of siRNA because their surface can readily be functionalized, and the size of the dendriplexes they form with siRNA is within the range of conventional nanomedicine. In this study, commercially available generation 3 poly(amidoamine) (PAMAM) dendrimer was investigated for pulmonary delivery of siRNA directed against tumor necrosis factor (TNF) α for the treatment of acute lung inflammation. Delivery efficiency was assessed in vitro in the RAW264.7 macrophage cell line activated with lipopolysaccharide (LPS), and efficacy was evaluated in vivo in a murine model of LPS-induced lung inflammation upon pre-treatment with TNF-α siRNA. The PAMAM dendrimer-siRNA complexes (dendriplexes) displayed strong siRNA condensation and high cellular uptake in macrophages compared with non-complexed siRNA. Q-PCR analyses showed that the dendriplexes mediated efficient and specific TNF-α silencing in vitro, as compared to non-complexed siRNA and dendriplexes with negative control siRNA. Also in vivo, the PAMAM dendriplexes induced efficacious TNF-α siRNA inhibition, as compared to non-complexed siRNA, upon pulmonary administration to mice with LPS-induced lung inflammation. Hence, these data suggest that PAMAM dendrimers are promising for the local delivery of TNF-α siRNA in the treatment of lung inflammation via pulmonary administration.

Exploiting Lipid and Polymer Nanocarriers to Improve the Anticancer Sonodynamic Activity of Chlorophyll.

Sonodynamic therapy is an emerging approach that uses low-intensity ultrasound to activate a sonosensitizer agent triggering its cytotoxicity for selective cancer cell killing. Several molecules have been proposed as sonosensitizer agents, but most of these, as chlorophyll, are strongly hydrophobic with a low selectivity towards cancer tissues. Nanocarriers can help to deliver more efficiently the sonosensitizer agents in the target tumor site, increasing at the same time their sonodynamic effect, since nanosystems act as cavitation nuclei. Herein, we propose the incorporation of unmodified plant-extracted chlorophyll into nanocarriers with different composition and structure (i.e., liposomes, solid lipid nanoparticles and poly(lactic-co-glycolic acid) nanoparticles) to obtain aqueous formulations of this natural pigment. The nanocarriers have been deeply characterized and then incubated with human prostatic cancer cells (PC-3) and spheroids (DU-145) to assess the influence of the different formulations on the chlorophyll sonodynamic effect. The highest sonodynamic cytotoxicity was obtained with chlorophyll loaded into poly(lactic-co-glycolic acid) nanoparticles, showing promising results for future clinical investigations on sonodynamic therapy.

Pentamidine-Loaded Lipid and Polymer Nanocarriers as Tunable Anticancer Drug Delivery Systems.

Initially developed as a synthetic analogue of insulin, pentamidine (PTM) is an antimicrobial drug that has recently shown in vitro and in vivo anticancer activity. Nevertheless, systemic administration of PTM causes severe side effects, especially nephrotoxicity. Here we propose the association of PTM to different biocompatible nanosystems in order to compare the physicochemical characteristics of the loaded nanocarriers and their influence on the drug cytotoxicity toward cancer cells. In particular, PTM (as free base or with different counterions) was encapsulated into liposomes and poly(lactide-co-glycolide) (PLGA) nanoparticles and all the formulations have been deeply characterized concerning mean diameter, polydispersity index, zeta potential, stability, morphology, PTM loading, and drug release profile. The anticancer activity was evaluated on a human ovarian cancer cell line over 72 h. Results showed that PTM is efficiently loaded into liposomes with a transmembrane citrate or sulfate gradient; concerning PLGA nanoparticles, important association occurred, thanks to ionic interactions between the drug and the polymer. The in vitro studies confirmed the anticancer activity of PTM, which was gradually released with different profiles depending on the drug form and the nanocarrier structure.

Anti-Inflammatory Effect of Anti-TNF-α SiRNA Cationic Phosphorus Dendrimer Nanocomplexes Administered Intranasally in a Murine Acute Lung Injury Model.

Inflammation is an essential component of many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), or acute lung injury. Our purpose was to design efficient carriers for lung delivery of small interfering RNA (siRNA) targeting tumor necrosis factor (TNF-α) in an acute lung injury model. To achieve this goal, two different types of phosphorus-based dendrimers with either pyrrolidinium or morpholinium as terminal protonated amino groups were selected for their better biocompatibility compared to other dendrimers. Dendriplexes containing pyrrolidinium surface groups demonstrated a stronger siRNA complexation, a higher cellular uptake, and enhanced in vitro silencing efficiency of TNF-α in the lipopolysaccharide (LPS)-activated mouse macrophage cell line RAW264.7, compared to morpholinium-containing dendriplexes. The better performance of the pyrrolidium dendriplexes was attributed to their higher pKa value leading to a stronger siRNA complexation and improved protection against enzymatic degradation resulting in a higher cellular uptake. The superior silencing effect of the pyrrolidinium dendriplexes, compared to noncomplexed siRNA, was confirmed in vivo in an LPS-induced murine model of short-term acute lung injury upon lung delivery via nasal administration. These data suggest that phosphorus dendriplexes have a strong potential in lung delivery of siRNA for treating inflammatory lung diseases.