Reduction of Pro-Inflammatory Markers in RAW264.7 Macrophages by Polyethylenimines.

The physiological problem of chronic inflammation and its associated pathologies attract ongoing attention with regard to methods for their control. Current systemic pharmacological treatments present problematic side effects. Thus, the possibility of new anti-inflammatory compounds with differing mechanisms of action or biophysical properties is enticing. Cationic polymers, with their ability to act as carriers for other molecules or to form bio-compatible materials, present one such possibility. Although not well described, several polycations such as chitosan and polyarginine, have displayed anti-inflammatory properties. The present work shows the ubiquitous laboratory transfection reagent, polyethylenimine (PEI) and more specifically low molecular weight branched PEI (B-PEI) as also possessing such properties. Using a RAW264.7 murine cell line macrophage as an inflammation model, it is found the B-PEI 700 Da as being capable of reducing the production of several pro-inflammatory molecules induced by the endotoxin lipopolysaccharide. Although further studies are required for elucidation of its mechanisms, the revelation that such a common lab reagent may present these effects has wide-ranging implications, as well as an abundance of possibilities.

The interplay between lysosome, protein corona and biological effects of cationic carbon dots: Role of surface charge titratability.

Carbon dots (CDs) are nanoparticles (NPs) with potential applications in the biomedical field. When in contact with biological fluids, most NPs are covered by a protein corona. As well, upon cell entry, most NP are sequestered in the lysosome. However, the interplay between the lysosome, the protein corona and the biological effects of NPs is still poorly understood. In this context, we investigated the role of the lysosome in the toxicological responses evoked by four cationic CDs exhibiting protonatable or non-protonatable amine groups at their surface, and the associated changes in the CD protein corona. The four CDs accumulated in the lysosome and led to lysosomal swelling, loss lysosome integrity, cathepsin B activation, NLRP3 inflammasome activation, and cell death by pyroptosis in a human macrophage model, but with a stronger effect for CDs with titratable amino groups. The protein corona formed around CDs in contact with serum partially dissociated under lysosomal conditions with subsequent protein rearrangement, as assessed by quantitative proteomic analysis. The residual protein corona still contained binding proteins, catalytic proteins, and proteins involved in the proteasome, glycolysis, or PI3k-Akt KEGG pathways, but with again a more pronounced effect for CDs with titratable amino groups. These results demonstrate an interplay between lysosome, protein corona and biological effects of cationic NPs in link with the titratability of NP surface charges.

Surface charge influences protein corona, cell uptake and biological effects of carbon dots.

Carbon dots are emerging nanoparticles (NPs) with tremendous applications, especially in the biomedical field. Herein is reported the first quantitative proteomic analysis of the protein corona formed on CDs with different surface charge properties. Four CDs were synthesized from citric acid and various amine group-containing passivation reagents, resulting in cationic NPs with increasing zeta (ζ)-potential and density of positive charges. After CD contact with serum, we show that protein corona identity is influenced by CD surface charge properties, which in turn impacts CD uptake and viability loss in macrophages. In particular, CDs with high ζ-potential (>+30 mV) and charge density (>2 µmol mg-1) are the most highly internalized, and their cell uptake is strongly correlated with a corona enriched in vitronectin, fibulin, fetuin, adiponectin and alpha-glycoprotein. On the contrary, CDs with a lower ζ-potential (+11 mV) and charge density (0.01 µmol mg-1) are poorly internalized, while having a corona with a very different protein signature characterized by a high abundance of apolipoproteins (APOA1, APOB and APOC), albumin and hemoglobin. These data illustrate how corona characterization may contribute to a better understanding of CD cellular fate and biological effects, and provide useful information for the development of CDs for biomedical applications.

Cationic Carbon Nanoparticles Induce Inflammasome-Dependent Pyroptosis in Macrophages via Lysosomal Dysfunction.

Carbon nanomaterials, including carbon dots (CDs), form a growing family of engineered nanoparticles (NPs) with widespread applications. As the rapid expansion of nanotechnologies raises safety concerns, interaction of NPs with the immune system is receiving a lot of attention. Recent studies have reported that engineered NPs may induce macrophage death by pyroptosis. Therefore, this study investigated whether cationic CDs induce pyroptosis in human macrophages and assessed the role of inflammasome and lysosome in this process. Cationic CDs were synthetized by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine. The NPs evoked a dose-dependent viability loss in THP-1-derived macrophages. A cell leakage, an increase in IL-1ß secretion and an activation of caspase-1 were also observed in response to the NPs. Inhibition of caspase-1 decreased CD-induced cell leakage and IL-1ß secretion, while restoring cell viability. Besides, CDs triggered swelling and loss of integrity of lysosome, and inhibition of the lysosomal enzyme cathepsin B decreased CD-induced IL-1ß secretion. Thus, our data provide evidence that cationic CDs induce inflammasome-dependent pyroptosis in macrophages via lysosomal dysfunction.

Mucus-producing epithelial models for investigating the activity of gene delivery systems in the lung.

Inhaled transfection particles have to penetrate the mucus layer lining the airways to successfully deliver their therapeutic nucleic acid payload to target cells in the underlying epithelium. However, the in vitro models used for evaluating gene carrier efficiency often disregard this viscous defensive barrier. In this study, the two mucus-secreting cell lines NCI-H292 and Calu-3 were selected to develop a series of epithelial models displaying gradual mucus production. In NCI-H292 models, a gradual increase in the MUC5AC mucin was obtained after cell exposure to inducers. In Calu-3 models, MUC5AC production increased as a function of culture duration (3, 7, 14 days) at the air-liquid interface (ALI). Six DOPC-derived cationic lipids were designed and their pDNA delivery activity was evaluated to validate these cellular models. The strongest impairment of the lipid delivery activity was observed in the Calu-3 14-d ALI model. The MUC5AC production in this model was the greatest and the mucus layer was 20 µm thick. The mucus exhibited a solid viscoelastic behavior, and represented a major hindrance to lipoplex diffusion. The Calu-3 14-d ALI model will be highly useful for accurate evaluation of gene carriers intended for airway administration and characterization of their interactions with the mucus.

A Co-Culture Model of the Human Respiratory Tract to Discriminate the Toxicological Profile of Cationic Nanoparticles According to Their Surface Charge Density.

This study aimed at discriminating with sensitivity the toxicological effects of carbon dots (CDs) with various zeta potential (ζ) and charge density (Qek) in different cellular models of the human respiratory tract. One anionic and three cationic CDs were synthetized as follows: CD-COOH (ζ = -43.3 mV); CD-PEI600 (Qek = 4.70 µmol/mg; ζ = +31.8 mV); CD-PEHA (Qek = 3.30 µmol/mg; ζ = +29.2 mV) and CD-DMEDA (Qek = 0.01 µmol/mg; ζ = +11.1 mV). Epithelial cells (A549) and macrophages (THP-1) were seeded alone or as co-cultures with different A549:THP-1 ratios. The obtained models were characterized, and multiple biological responses evoked by CDs were assessed in the mono-cultures and the best co-culture model. With 14% macrophages, the 2:1 ratio co-culture best mimicked the in vivo conditions and responded to lipopolysaccharides. The anionic CD did not induce any effect in the mono-cultures nor in the co-culture. Among the cationic CDs, the one with the highest charge density (CD-PEI600) induced the most pronounced responses whatever the culture model. The cationic CDs of low charge density (CD-PEHA and CD-DMEDA) evoked similar responses in the mono-cultures, whereas in the co-culture, the three cationic CDs ranked according to their charge density (CD-PEI600 > CD-PEHA > CD-DMEDA), when taking into account their inflammatory effect. Thus, the co-culture system developed in this study appears to be a sensitive model for finely discriminating the toxicological profile of cationic nanoparticles differing by the density of their surface charges.

The pH-Induced Specific Area Changes of Unsaturated Lipids Deposited onto a Bubble Interface.

In this work, we used an original experimental setup to examine the behavior of insoluble monolayers made with pH-sensitive lipids. Two kinds of unsaturated lipids were chosen: a cationic one (lipid 1) bearing an ammonium headgroup and an anionic one (lipid 2) terminated with an acidic phenol group. The lipids were deposited onto an air bubble interface maintained in an aqueous phase and, after stabilization, were subjected to a series of compressions performed at different pH values. These experiments disclosed a gradual increase in the specific area per molecule when lipids were neutralized. Imposing a pH variation at constant bubble volume also provided surface pressure profiles that confirmed this molecular behavior. As complementary characterization, dilatational rheology disclosed a phase transition from a purely elastic monophasic system to a viscoelastic two-phase system. We hypothesized that this unexpected increase in the specific area with lipid neutralization is related to the presence of unsaturations in each of the two branches of the hydrophobic tails that induce disorder, thereby increasing the molecular area at the interface. Application of the two-dimensional Volmer equation of state allowed the generation of quantitative values for the specific areas that showed variations with pH. It also allowed the determination of apparent pKa values, which are affected by both the electrostatic potential within the monolayer and the affinity of the lipid polar head for the aqueous phase.

Combined In Vitro and In Vivo Approaches to Propose a Putative Adverse Outcome Pathway for Acute Lung Inflammation Induced by Nanoparticles: A Study on Carbon Dots.

With the growth of nanotechnologies, concerns raised regarding the potential adverse effects of nanoparticles (NPs), especially on the respiratory tract. Adverse outcome pathways (AOP) have become recently the subject of intensive studies in order to get a better understanding of the mechanisms of NP toxicity, and hence hopefully predict the health risks associated with NP exposure. Herein, we propose a putative AOP for the lung toxicity of NPs using emerging nanomaterials called carbon dots (CDs), and in vivo and in vitro experimental approaches. We first investigated the effect of a single administration of CDs on mouse airways. We showed that CDs induce an acute lung inflammation and identified airway macrophages as target cells of CDs. Then, we studied the cellular responses induced by CDs in an in vitro model of macrophages. We observed that CDs are internalized by these cells (molecular initial event) and induce a series of key events, including loss of lysosomal integrity and mitochondrial disruption (organelle responses), as well as oxidative stress, inflammasome activation, inflammatory cytokine upregulation and macrophage death (cellular responses). All these effects triggering lung inflammation as tissular response may lead to acute lung injury.

Density of surface charge is a more predictive factor of the toxicity of cationic carbon nanoparticles than zeta potential.

BACKGROUND: A positive surface charge has been largely associated with nanoparticle (NP) toxicity. However, by screening a carbon NP library in macrophages, we found that a cationic charge does not systematically translate into toxicity. To get deeper insight into this, we carried out a comprehensive study on 5 cationic carbon NPs (NP2 to NP6) exhibiting a similar zeta (ζ) potential value (from + 20.6 to + 26.9 mV) but displaying an increasing surface charge density (electrokinetic charge, Qek from 0.23 to 4.39 µmol/g). An anionic and non-cytotoxic NP (NP1, ζ-potential = - 38.5 mV) was used as control. RESULTS: The 5 cationic NPs induced high (NP6 and NP5, Qek of 2.95 and 4.39 µmol/g, respectively), little (NP3 and NP4, Qek of 0.78 and 1.35 µmol/g, respectively) or no (NP2, Qek of 0.23 µmol/g) viability loss in THP-1-derived macrophages exposed for 24 h to escalating NP dose (3 to 200 µg/mL). A similar toxicity trend was observed in airway epithelial cells (A549 and Calu-3), with less viability loss than in THP-1 cells. NP3, NP5 and NP6 were taken up by THP-1 cells at 4 h, whereas NP1, NP2 and NP4 were not. Among the 6 NPs, only NP5 and NP6 with the highest surface charge density induced significant oxidative stress, IL-8 release, mitochondrial dysfunction and loss in lysosomal integrity in THP-1 cells. As well, in mice, NP5 and NP6 only induced airway inflammation. NP5 also increased allergen-induced immune response, airway inflammation and mucus production. CONCLUSIONS: Thus, this study clearly reveals that the surface charge density of a cationic carbon NP rather than the absolute value of its ζ-potential is a relevant descriptor of its in vitro and in vivo toxicity.

Dual Gene Delivery Reagents From Antiproliferative Alkylphospholipids for Combined Antitumor Therapy.

Alkylphospholipids (APLs) have elicited great interest as antitumor agents due to their unique mode of action on cell membranes. However, their clinical applications have been limited so far by high hemolytic activity. Recently, cationic prodrugs of erufosine, a most promising APL, have been shown to mediate efficient intracellular gene delivery, while preserving the antiproliferative properties of the parent APL. Here, cationic prodrugs of the two APLs that are currently used in the clinic, miltefosine, and perifosine, are investigated and compared to the erufosine prodrugs. Their synthesis, stability, gene delivery and self-assembly properties, and hemolytic activity are discussed in detail. Finally, the potential of the pro-miltefosine and pro-perifosine compounds M E12 and P E12 in combined antitumor therapy is demonstrated using pUNO1-hTRAIL, a plasmid DNA encoding TRAIL, a member of the TNF superfamily. With these pro-APL compounds, we provide a proof of concept for a new promising strategy for cancer therapy combining gene therapy and APL-based chemotherapy.

rAAV-Mediated Overexpression of SOX9 and TGF-ß via Carbon Dot-Guided Vector Delivery Enhances the Biological Activities in Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Scaffold-assisted gene therapy is a highly promising tool to treat articular cartilage lesions upon direct delivery of chondrogenic candidate sequences. The goal of this study was to examine the feasibility and benefits of providing highly chondroreparative agents, the cartilage-specific sex-determining region Y-type high-mobility group 9 (SOX9) transcription factor or the transforming growth factor beta (TGF-ß), to human bone marrow-derived mesenchymal stromal cells (hMSCs) via clinically adapted, independent recombinant adeno-associated virus (rAAV) vectors formulated with carbon dots (CDs), a novel class of carbon-dominated nanomaterials. Effective complexation and release of a reporter rAAV-lacZ vector was achieved using four different CDs elaborated from 1-citric acid and pentaethylenehexamine (CD-1); 2-citric acid, poly(ethylene glycol) monomethyl ether (MW 550 Da), and N,N-dimethylethylenediamine (CD-2); 3-citric acid, branched poly(ethylenimine) (MW 600 Da), and poly(ethylene glycol) monomethyl ether (MW 2 kDa) (CD-3); and 4-citric acid and branched poly(ethylenimine) (MW 600 Da) (CD-4), allowing for the genetic modification of hMSCs. Among the nanoparticles, CD-2 showed an optimal ability for rAAV delivery (up to 2.2-fold increase in lacZ expression relative to free vector treatment with 100% cell viability for at least 10 days, the longest time point examined). Administration of therapeutic (SOX9, TGF-ß) rAAV vectors in hMSCs via CD-2 led to the effective overexpression of each independent transgene, promoting enhanced cell proliferation (TGF-ß) and cartilage matrix deposition (glycosaminoglycans, type-II collagen) for at least 21 days relative to control treatments (CD-2 lacking rAAV or associated to rAAV-lacZ), while advantageously restricting undesirable type-I and -X collagen deposition. These results reveal the potential of CD-guided rAAV gene administration in hMSCs as safe, non-invasive systems for translational strategies to enhance cartilage repair.

Synthesis and Evaluation of Antitumor Alkylphospholipid Prodrugs.

PURPOSE: Hemolysis is a serious side effect of antitumor alkylphospholipids (APLs) that limits dose levels and is a constraint in their use in therapeutic regimen. Nine prodrugs of promising APLs (miltefosine, perifosine, and erufosine) were synthesized so as to decrease their membrane activity and improve their toxicity profile while preserving their antineoplastic potency. METHODS: The synthesis of the pro-APLs was straightforwardly achieved in one step starting from the parent APLs. The critical aggregation concentration of the prodrugs, their hydrolytic stability under various pH conditions, their blood compatibility and cytotoxicity in three different cell lines were determined and compared to those of the parent antitumor lipids. RESULTS: The APL prodrugs display antitumor activity which is similar to that of the parent alkylphospholipids but without associated hemolytic toxicity. CONCLUSION: The pro-APL compounds may be considered as intravenously injectable derivatives of APLs. They could thus address one of the major issues met in cancer therapies involving antitumor lipids and restricting their utilization to oral and topical administration because of limited maximum tolerated dose.

Erufosine (ErPC3) Cationic Prodrugs as Dual Gene Delivery Reagents for Combined Antitumor Therapy.

Sixteen cationic prodrugs of the antitumor alkylphospholipid (APL) erufosine were rationally synthesized to provide original gene delivery reagents with improved cytotoxicity profile. The DNA complexation properties of these cationic lipids were determined and associated transfection rates were measured. Furthermore, the self-assembly properties of the pro-erufosine compounds were investigated and their critical aggregation concentration was determined. Their hydrolytic stability under pH conditions mimicking the extracellular environment and the late endosome milieu was measured. Hemolytic activity and cytotoxicity of the compounds were investigated. The results obtained in various cell lines demonstrate that the prodrugs of erufosine display antineoplastic activity similar to that of the parent antitumor drug but are not associated with hemolytic toxicity, which is a dose-limiting side effect of APLs and a major obstacle to their use in anticancer therapeutic regimen. Furthermore, by using lipoplexes prepared from a prodrug of erufosine and a plasmid DNA encoding a pro-apoptotic protein (TRAIL), evidence was provided for selective cytotoxicity towards tumor cells while nontumor cells were resistant. This study demonstrates that the combination approach involving well tolerated erufosine cationic prodrugs and cancer gene therapy holds significant promise in tumor therapy.

Physicochemical characteristics that affect carbon dot safety: Lessons from a comprehensive study on a nanoparticle library.

Carbon dots (CDs) are emerging nanomaterial in medicine and pharmacy. To explore the impact of physicochemical characteristics on their safety, we synthesized a library of 35 CDs exhibiting different size, charge, chemical composition and surface coating, using various starting materials (carbon source and passivation reagent) and carbonization procedures. The 35 CDs triggered different levels of viability loss when incubated with human macrophages at 3-200 µg/mL for 24 h. The smaller NPs (10-20 nm) were more toxic that larger ones (40-100 nm), whereas NPs that aggregated in culture medium were more toxic than dispersed ones. A positive correlation was found between CD charge or nitrogen content and toxicity. Furthermore, a greater toxicity was observed for CDs prepared from high molecular weight polyamines, suggesting a role of the CD global density of positive charges, rather than the charge at the CD surface, in the CD toxicity. At last, PEG decoration decreased the toxicity of cationic NPs. In conclusion, the size, aggregation in culture medium, charge, nitrogen content, nature of the passivation agent and synthesis procedure were found to influence CD toxicity, making it difficult to predict CD safety from a single characteristic.

Correction: Influence of carbonization conditions on luminescence and gene delivery properties of nitrogen-doped carbon dots.

[This corrects the article DOI: 10.1039/C8RA09651A.].

Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy.

Cationic DOPC-Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery.

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC-C12 E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12 E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12 E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic-lipid-mediated gene transfer in animals.

DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery.

Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

Characterization of Titratable Amphiphiles in Lipid Membranes by Fluorescence Spectroscopy.

Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent pKa value which may be as large as two pKa units and cannot be satisfactorily predicted.

Efficient in vitro and in vivo pulmonary delivery of nucleic acid by carbon dot-based nanocarriers.

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20-30% yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo transfection efficiency and biocompatibility.