Hyaluronic acid-conjugated liposomes loaded with dexamethasone: A promising approach for the treatment of inflammatory diseases.

Dexamethasone is a well-known anti-inflammatory drug readily used to treat many lung diseases. However, its side effects and poor lower airway deposition and retention are significant limitations to its usage. In this work, we developed lipid nanoparticulate platforms loaded with dexamethasone and evaluated their behavior in inflammatory lung models in vitro and in vivo. Dexamethasone-loaded liposomes with an average diameter below 150 nm were obtained using a solvent injection method. Three different formulations were produced with a distinct surface coating (polyethylene glycol, hyaluronic acid, or a mixture of both) as innovative strategies to cross the pulmonary mucus layer and/or target CD44 expressed on alveolar proinflammatory macrophages. Interestingly, while electron paramagnetic spectroscopy showed that surface modifications did not induce any molecular changes in the liposomal membrane, drug loading analysis revealed that adding the hyaluronic acid in the bilayer led to a decrease of dexamethasone loading (from 3.0 to 1.7 w/w%). In vitro experiments on LPS-activated macrophages demonstrated that the encapsulation of dexamethasone in liposomes, particularly in HA-bearing ones, improved its anti-inflammatory efficacy compared to the free drug. Subsequently, in vivo data revealed that while intratracheal administration of free dexamethasone led to an important inter-animals variation of efficacy, dexamethasone-loaded liposomes showed an improved consistency within the results. Our data indicate that encapsulating dexamethasone into lipid nanoparticles is a potent strategy to improve its efficacy after lung delivery.

Interplay between mucus mobility and alveolar macrophage targeting of surface-modified liposomes.

Alveolar macrophages play a crucial role in the initiation and resolution of the immune response in the lungs. Pro-inflammatory M1 alveolar macrophages are an interesting target for treating inflammatory and infectious pulmonary diseases. One commune targeting strategy is to use nanoparticles conjugated with hyaluronic acid, which interact with CD44 overexpressed on the membrane of those cells. Unfortunately, this coating strategy may be countered by the presence on the surface of the nanoparticles of a poly(ethylene glycol) corona employed to improve nanoparticles' diffusion in the lung mucus. This study aims to measure this phenomenon by comparing the behavior in a murine lung inflammation model of three liposomal platforms designed to represent different poly(ethylene glycol) and hyaluronic acid densities (Liposome-PEG, Liposome-PEG-HA and Liposome-HA). In this work, the liposomes were obtained by a one-step ethanol injection method. Their interaction with mucin and targeting ability toward pro-inflammatory macrophages were then investigated in vitro and in vivo in a LPS model of lung inflammation. In vitro, poly(ethylene glycol) free HA-liposomes display a superior targeting efficiency toward M1 macrophages, while the addition of poly(ethylene glycol) induces better mucus mobility. Interestingly in vivo studies revealed that the three liposomes showed distinct cell specificity with alveolar macrophages demonstrating an avidity for poly(ethylene glycol) free HA-liposomes, while neutrophils favored PEGylated liposomes exempt of HA. Those results could be explained by the presence of two forces exercising a balance between mucus penetration and receptor targeting. This study corroborates the importance of considering the site of action and the targeted cells when designing nanoparticles to treat lung diseases.

Targeted nanotherapy with everolimus reduces inflammation and fibrosis in scleroderma-related interstitial lung disease developed by PSGL-1 deficient mice.

BACKGROUND AND PURPOSE: Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice. EXPERIMENTAL APPROACH: Aged PSGL-1 KO (Selplg-/- ) mice were used to assess the therapeutic effects of nanotherapy with everolimus, included in liposomes decorated with high MW hyaluronic acid (LipHA+Ev) and administered intratracheally to specifically target CD44-expressing lung cells. KEY RESULTS: PSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels. CONCLUSIONS AND IMPLICATIONS: In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.

Liposomes Loaded with Everolimus and Coated with Hyaluronic Acid: A Promising Approach for Lung Fibrosis.

Chronic lung allograft dysfunction (CLAD) and interstitial lung disease associated with collagen tissue diseases (CTD-ILD) are two end-stage lung disorders in which different chronic triggers induce activation of myo-/fibroblasts (LFs). Everolimus, an mTOR inhibitor, can be adopted as a potential strategy for CLAD and CTD-ILD, however it exerts important side effects. This study aims to exploit nanomedicine to reduce everolimus side effects encapsulating it inside liposomes targeted against LFs, expressing a high rate of CD44. PEGylated liposomes were modified with high molecular weight hyaluronic acid and loaded with everolimus (PEG-LIP(ev)-HA400kDa). Liposomes were tested by in vitro experiments using LFs derived from broncholveolar lavage (BAL) of patients affected by CLAD and CTD-ILD, and on alveolar macrophages (AM) and lymphocytes isolated, respectively, from BAL and peripheral blood. PEG-LIP-HA400kDa demonstrated to be specific for LFs, but not for CD44-negative cells, and after loading everolimus, PEG-LIP(ev)-HA400kDa were able to arrest cell cycle arrest and to decrease phospho-mTOR level. PEG-LIP(ev)-HA400kDa showed anti-inflammatory effect on immune cells. This study opens the possibility to use everolimus in lung fibrotic diseases, demonstrating that our lipids-based vehicles can vehicle everolimus inside cells exerting the same drug molecular effect, not only in LFs, but also in immune cells.

Hyaluronic Acid-Decorated Liposomes as Innovative Targeted Delivery System for Lung Fibrotic Cells.

Collagen Tissue Disease-associated Interstitial Lung Fibrosis (CTD-ILDs) and Bronchiolitis Obliterans Syndrome (BOS) represent severe lung fibrogenic disorders, characterized by fibro-proliferation with uncontrolled extracellular matrix deposition. Hyaluronic acid (HA) plays a key role in fibrosis with its specific receptor, CD44, overexpressed by CTD-ILD and BOS cells. The aim is to use HA-liposomes to develop an inhalatory treatment for these diseases. Liposomes with HA of two molecular weights were prepared and characterized. Targeting efficiency was assessed toward CTD-ILD and BOS cells by flow cytometry and confocal microscopy and immune modulation by RT-PCR and ELISA techniques. HA-liposomes were internalized by CTD-ILD and BOS cells expressing CD44, and this effect increased with higher HA MW. In THP-1 cells, HA-liposomes decreased pro-inflammatory cytokines IL-1ß, IL-12, and anti-fibrotic VEGF transcripts but increased TGF-ß mRNA. However, upon analyzing TGF-ß release from healthy donors-derived monocytes, we found liposomes did not alter the release of active pro-fibrotic cytokine. All liposomes induced mild activation of neutrophils regardless of the presence of HA. HA liposomes could be also applied for lung fibrotic diseases, being endowed with low pro-inflammatory activity, and results confirmed that higher MW HA are associated to an increased targeting efficiency for CD44 expressing LFs-derived from BOS and CTD-ILD patients.

Hyaluronated liposomes containing H2S-releasing doxorubicin are effective against P-glycoprotein-positive/doxorubicin-resistant osteosarcoma cells and xenografts.

Doxorubicin (dox) is one of the first-line drug in osteosarcoma treatment but its effectiveness is limited by the efflux pump P-glycoprotein (Pgp) and by the onset of cardiotoxicity. We previously demonstrated that synthetic doxs conjugated with a H2S-releasing moiety (Sdox) were less cardiotoxic and more effective than dox against Pgp-overexpressing osteosarcoma cells. In order to increase the active delivery to tumor cells, we produced hyaluronic acid (HA)-conjugated liposomes containing Sdox (HA-Lsdox), exploiting the abundance of the HA receptor CD44 in osteosarcoma. HA-Lsdox showed favorable drug-release profile and higher toxicity in vitro and in vivo than dox or the FDA-approved liposomal dox Caelyx® against Pgp-overexpressing osteosarcoma, displaying the same cardiotoxicity profile of Caelyx®. Differently from dox, HA-Lsdox delivered the drug within the endoplasmic reticulum (ER), inducing protein sulfhydration and ubiquitination, and activating a ER stress pro-apoptotic response mediated by CHOP. HA-Lsdox also sulfhydrated the nascent Pgp in the ER, reducing its activity. We propose HA-Lsdox as an innovative tool noteworthy to be tested in Pgp-overexpressing patients, who are frequently less responsive to standard treatments in which dox is one of the most important drugs.

Uptake and intracellular fate of biocompatible nanocarriers in cycling and noncycling cells.

AIM: To elucidate whether different cytokinetic features (i.e., presence or absence of mitotic activity) may influence cell uptake and distribution of nanocarriers, in vitro tests on liposomes, mesoporous silica nanoparticles, poly(lactide-co-glycolide) nanoparticles and nanohydrogels were carried out on C2C12 murine muscle cells either able to proliferate as myoblasts (cycling cells) or terminally differentiate into myotubes (noncycling cells). MATERIALS & METHODS: Cell uptake and intracellular fate of liposomes, mesoporous silica nanoparticles, poly(lactide-co-glycolide) nanoparticles and nanohydrogels were investigated by confocal fluorescence microscopy and transmission electron microscopy. RESULTS: Nanocarrier internalization and distribution were similar in myoblasts and myotubes; however, myotubes demonstrated a lower uptake capability. CONCLUSION: All nanocarriers proved to be suitably biocompatible for both myoblasts and myotubes. The lower uptake capability of myotubes is probably due to different plasma membrane composition related to the differentiation process.

Pancreatic cancer stem cell proliferation is strongly inhibited by diethyldithiocarbamate-copper complex loaded into hyaluronic acid decorated liposomes.

BACKGROUND: Pancreatic cancer stem cells (CSCs) are responsible for resistance to standard therapy, metastatic potential, and disease relapse following treatments. The current therapy for pancreatic ductal adenocarcinoma (PDAC) preferentially targets the more differentiated cancer cell population, leaving CSCs as a cell source for tumor mass formation and recurrence. For this reason, there is an urgent need to improve current therapies and develop novel CSC-targeted therapeutic approaches. METHODS: Hyaluronic acid (HA) decorated liposomes, containing diethyldithiocarbamate­copper (Cu(DDC)2), able to target the specific CSC marker CD44 receptor were prepared by ion gradient technique and fully characterized. Their antiproliferative effect was evaluated on pancreatic CSCs derived from PDAC cell lines or patients. To clarify the mechanism of action of Cu(DDC)2 liposomes, ROS level neutralization assay in the presence of N-acetyl-L-cysteine was performed. RESULTS: Liposomes showed high encapsulation efficiency and Cryo-TEM analysis revealed the presence of Cu(DDC)2 crystals in the aqueous core of liposomes. In vitro test on pancreatic CSCs derived from PDAC cell lines or patients showed high ROS mediated anticancer activity of HA decorated liposomes. The sphere formation capability of CSCs obtained from patients was drastically reduced by liposomal formulations containing Cu(DDC)2. CONCLUSIONS: The obtained results show that the encapsulation of Cu(DDC)2 complex in HA decorated liposomes strongly increases its anti-proliferative activity on pancreatic CSCs. GENERAL SIGNIFICANCE: This paper describes for the first time the use of HA decorated liposomes containing Cu(DDC)2 against pancreatic CSCs and opens the way to the development of nanomedicine based CSC-targeted therapeutic approaches.

Hyaluronated mesoporous silica nanoparticles for active targeting: influence of conjugation method and hyaluronic acid molecular weight on the nanovector properties.

We have prepared and evaluated the physico-chemical and biological properties of four different hyaluronated mesoporous silica nanoparticles (MSNs) samples (MSN/HA). Hyaluronic acid (HA) with two different molecular weights (200 and 6.4 kDa) was used for the conjugation of aminopropyl-functionalized MSN (NH2-MSN), following two different procedures. Namely, samples HA200A and HA6.4A were prepared by reacting activated HA with NH2-MSN (method A), while samples HA200B and HA6.4B were obtained carrying out HA activation in the presence of the nanoparticles (method B). The four samples showed similar hydrophilicity, but clear differences in the HA loading, textural properties, surface charge and stability of the suspensions. More in detail, conjugation using low molecular weight HA with method A resulted in low HA loading, with consequent scarce effects on dispersity and stability in physiological media. The highest yield and corresponding best performances were obtained with method B using high molecular weight HA. HA loading and molecular weight also influenced in a concerted way the biological response towards the MSNs of CD44 target cancer cells (CD44+) and control cells (CD44-): MDA-MB-231 and A2780, respectively. The absence of cytotoxicity was assessed. Moreover, the targeting ability of the best performing MSN/HA was confirmed by cellular uptake studies.

Folate-targeted liposomal nitrooxy-doxorubicin: An effective tool against P-glycoprotein-positive and folate receptor-positive tumors.

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.

Hyaluronan-decorated liposomes as drug delivery systems for cutaneous administration.

The work aimed to evaluate the feasibility to design hyaluronic acid (HA) decorated flexible liposomes to enhance the skin penetration of nifedipine. Egg phosphatidylcholine (e-PC) based transfersomes (Tween 80) and transethosomes (ethanol) were prepared. HA was reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (HA-DPPE) and two molar ratios (0.5 and 3%) of conjugate with respect to e-PC were tested. The presence of HA significantly increased the packing order of the bilayer (as verified by differential scanning calorimetry), reducing both the encapsulation efficiency and the flexibility of the decorated liposomes in a dose-dependent manner. In fact, at the highest HA content the constant of deformability (K, N/mm) increased and the carriers remained on the skin surface after topical application. The stiffening effect of HA was counterbalanced by the addition of ethanol as fluidizing agent that allowed to maintain the highest HA concentration, meanwhile reducing the K value of the vesicles. HA-transethosomes allowed a suitable nifedipine permeation (J ∼ 30 ng/cm2/h) and significantly improved the drug penetration, favouring the formation of a drug depot in the epidermis. These data suggest the potentialities of HA-transethosomes as drug delivery systems intended for the treatment of cutaneous pathologies and underline the importance of studying the effect of surface functionalization on carrier deformability to rationalize the design of such systems.

Cell uptake and intracellular fate of phospholipidic manganese-based nanoparticles.

During the last decades, several studies have proposed manganese (Mn) complexes as alternative contrast agents for magnetic resonance imaging (MRI). With the nanotechnology surge in recent years, different types of Mn-based nanoparticles (Nps) have been developed. However, to design effective and safe administration procedures, preliminary studies on target cells, aimed at verifying their full biocompatibility and biodegradability, are mandatory. In this study, MnO containing-Nps encapsulated in a phospholipidic shell (PL-MnO Nps) were tested in cultured cells and flow cytometry; confocal and transmission electron microscopy were combined to understand the Nps uptake mechanism, intracellular distribution and degradation pathways, as well as possible organelle alterations. The results demonstrated that PL-MnO Nps undergo rapid and massive cell internalization, and persist free in the cytoplasm before undergoing lysosomal degradation without being cytotoxic or inducing subcellular damage. Based on the results with this cell model in vitro, PL-MnO Nps thus proved to be suitably biocompatible, and may be envisaged as very promising tools for therapeutic and diagnostic applications, as drug carriers or contrast agent for MRI.

Liposomal nitrooxy-doxorubicin: one step over caelyx in drug-resistant human cancer cells.

In this work we prepared and characterized two liposomal formulations of a semisynthetic nitric oxide (NO)-releasing doxorubicin (Dox), called nitrooxy-Dox (NitDox), which we previously demonstrated to be cytotoxic in Dox-resistant human colon cancer cells. Liposomes with 38.2% (Lip A) and 19.1% (Lip B) cholesterol were synthesized: both formulations had similar size and zeta potential values and caused the same intracellular distribution of free NitDox, but Lip B accumulated and released NitDox more efficiently. In Dox-resistant human colon cancer cells, Lip A and Lip B exhibited a more favorable kinetics of drug uptake and NO release, and a stronger cytotoxicity than Dox and free NitDox. While Caelyx, one of the liposomal Dox formulations approved for breast and ovary tumors treatment, was ineffective in Dox-resistant breast/ovary cancer cells, Lip B, and to a lesser extent Lip A, still exerted a significant cytotoxicity in these cells. This event was accompanied in parallel by a higher release of NO, which caused nitration of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two transporters involved in Dox efflux, and impaired their pump activity. By doing so, the efflux kinetics of Dox after treatment with Lip B was markedly slowed down and the intracellular accumulation of Dox was increased in breast and ovary drug-resistant cells. We propose these liposomal formulations of NitDox as new tools with a specific indication for tumors overexpressing Pgp and MRP1.