Controlled decoration of nanoceria on the surface of MoS2nanoflowers to improve the biodegradability and biocompatibility inDrosophila melanogastermodel.

In recent years, nanozymes based on two-dimensional (2D) nanomaterials have been receiving great interest for cancer photothermal therapy. 2D materials decorated with nanoparticles (NPs) on their surface are advantageous over conventional NPs and 2D material based systems because of their ability to synergistically improve the unique properties of both NPs and 2D materials. In this work, we report a nanozyme based on flower-like MoS2nanoflakes (NFs) by decorating their flower petals with NCeO2using polyethylenimine (PEI) as a linker molecule. A detailed investigation on toxicity, biocompatibility and degradation behavior of fabricated nanozymes in wild-typeDrosophila melanogastermodel revealed that there were no significant effects on the larval size, morphology, larval length, breadth and no time delay in changing larvae to the third instar stage at 7-10 d for MoS2NFs before and after NCeO2decoration. The muscle contraction and locomotion behavior of third instar larvae exhibited high distance coverage for NCeO2decorated MoS2NFs when compared to bare MoS2NFs and control groups. Notably, the MoS2and NCeO2-PEI-MoS2NFs treated groups at 100µg ml-1covered a distance of 38.2 mm (19.4% increase when compared with control) and 49.88 mm (no change when compared with control), respectively. High-resolution transmission electron microscopy investigations on the new born fly gut showed that the NCeO2decoration improved the degradation rate of MoS2NFs. Hence, nanozymes reported here have huge potential in various fields ranging from biosensing, cancer therapy and theranostics to tissue engineering and the treatment of Alzheimer's disease and retinal therapy.

Uniform iron oxide nanoparticles reduce the required amount of polyethylenimine in the gene delivery to mesenchymal stem cells.

Cationic polyethylenimine (PEI) is regarded as the 'golden standard' of non-viral gene vectors. However, the superiority of PEI with high positive charge density also induces its major drawback of cytotoxicity, which restricts its application for an effective and safe gene delivery to stem cells. To redress this shortcoming, herein, a magnetic gene complex containing uniform iron oxide nanoparticles (UIONPs), plasmid DNA, and free PEI is prepared through electrostatic interactions for the gene delivery to bone marrow-derived mesenchymal stem cells (BM-MSCs). Results show that UIONPs dramatically promote the gene delivery to BM-MSCs using the assistance of magnetic force. In addition, decreasing the free PEI nitrogen to DNA phosphate (N/P) ratio from 10 to 6 has little adverse impact on the transgene expression levels (over 300 times than that of PEI alone at the N/P ratio of 6) and significantly reduces the cytotoxicity to BM-MSCs. Further investigations confirmed that the decrease of free PEI has little influence on the cellular uptake after applying external magnetic forces, but that the reduced positive charge density decreases the cytotoxicity. The present study demonstrates that magnetic gene delivery not only contributes to the enhanced gene expression but also helps to reduce the required amount of PEI, providing a potential strategy for an efficient and safe gene delivery to stem cells.

Polyethylenimine-Bisphosphonate-Cyclodextrin Ternary Conjugates: Supramolecular Systems for the Delivery of Antineoplastic Drugs.

Bisphosphonates (BPs) are bone-binding molecules that provide targeting capabilities to bone cancer cells when conjugated with drug-carrying polymers. This work reports the design, synthesis, and biological evaluation of polyethyleneimine-BP-cyclodextrin (PEI-BP-CD) ternary conjugates with supramolecular capabilities for the loading of antineoplastic drugs. A straightforward, modular, and versatile strategy based on the click aza-Michael addition reaction of vinyl sulfones (VSs) allows the grafting of BPs targeting ligands and ßCD carrier appendages to the PEI polymeric scaffold. The in vitro evaluation (cytotoxicity, cellular uptake, internalization routes, and subcellular distribution) for the ternary conjugates and their doxorubicin inclusion complexes in different bone-related cancer cell lines (MC3T3-E1 osteoblasts, MG-63 sarcoma cells, and MDA-MB-231 breast cancer cells) confirmed specificity, mitochondrial targeting, and overall capability to mediate a targeted drug transport to those cells. The in vivo evaluation using xenografts of MG-63 and MDA-MB-231 cells on mice also confirmed the targeting of the conjugates.

Tumor-Penetrable Nitric Oxide-Releasing Nanoparticles Potentiate Local Antimelanoma Therapy.

Although nitric oxide (NO) has been emerging as a novel local anticancer agent because of its potent cytotoxic effects and lack of off-target side effects, its clinical applications remain a challenge because of the short effective diffusion distance of NO that limits its anticancer activity. In this study, we synthesized albumin-coated poly(lactic-co-glycolic acid) (PLGA)-conjugated linear polyethylenimine diazeniumdiolate (LP/NO) nanoparticles (Alb-PLP/NO NPs) that possess tumor-penetrating and NO-releasing properties for an effective local treatment of melanoma. Sufficient NO-loading and prolonged NO-releasing characteristics of Alb-PLP/NO NPs were acquired through PLGA-conjugated LP/NO copolymer (PLP/NO) synthesis, followed by nanoparticle fabrication. In addition, tumor penetration ability was rendered by the electrostatic adsorption of the albumin on the surface of the nanoparticles. The Alb-PLP/NO NPs showed enhanced intracellular NO delivery efficiency and cytotoxicity to B16F10 murine melanoma cells. In B16F10-tumor-bearing mice, the Alb-PLP/NO NPs showed improved extracellular matrix penetration and spatial distribution in the tumor tissue after intratumoral injection, resulting in enhanced antitumor activity. Taken together, the results suggest that Alb-PLP/NO NPs represent a promising new modality for the local treatment of melanoma.

PEI fluorination reduces toxicity and promotes liver-targeted siRNA delivery.

Polyethyleneimine (PEI) has been extensively investigated as an efficient carrier for nucleic acid delivery. Yet, it suffers from a high toxicity profile that hinders clinical translation. Fluorination has proven to be a valid approach to reduce the cytotoxicity of PEI and improve the in vitro siRNA delivery potency. Hydrophobicity and lipophobicity can be controllably introduced into the side chains of PEI. However, the effect of fluorination on siRNA delivery in vivo, particularly the biodistribution of siRNA polyplex nanoparticles with fluorinated PEIs, has not been extensively explored. Here, we introduce two series of fluorinated PEIs via amidation with ethyl trifluoroacetate and perfluorobutyryl chloride. Fluorination substantially improved the performance of PEI for siRNA delivery by reducing the cytotoxicity to MDA-MB-231 cells. Importantly, fluorinated PEI enabled the major accumulation of siRNA polyplex nanoparticles in the liver while non-fluorinated PEI delivered siRNA nanoparticles mainly to the lungs after intravenous administration to mice. It is envisioned that fluorination may be an important general strategy for lowering toxicity of cationic polymers, and that the fluorination-induced alteration of biodistribution may be applicable for improved delivery to different organs. Graphical abstract.

Supramolecular self-assemblies of engineered polyethylenimines as multifunctional nanostructures for DNA transportation with excellent antimicrobial activity.

In this study, indole-3-butanoic acid (IBA), a biologically and environmentally safe entity, has been grafted onto low and high molecular weight (1.8 and 25 kDa) polyethylenimines (PEI) mainly through primary amines to obtain amphiphilic indole-3-butanoyl-polyethylenimines (IBPs). Two series of IBPs (IBP1.8 and IBP25) were prepared which, on self-assembly in aqueous medium, yielded multifunctional nanomicellar structures (IBP1.8 and IBP25) capable of transporting genetic material in vitro and exhibiting other biological activities. Physicochemical characterization showed the size of IBP1.8 and IBP25 nanostructures in the range of ~332-234 nm and ~283-166 nm, respectively, with zeta potential varying from ~+29-17 mV and ~+37-25 mV. DNA release assay demonstrated higher release of plasmid DNA from IBP nanostructures as compared to native PEIs. Cytotoxicity showed a decreasing pattern with increasing degree of grafting of IBA onto PEIs making these nanostructures non-toxic. pDNA complexes of these nanostructures (both IBPs1.8 and IBPs25) displayed considerably higher transfection efficiency, however, IBP1.8/pDNA complexes performed much better (~7-9 folds) as compared to native PEI/pDNA and Lipofectamine/pDNA complexes on mammalian cells. CLSM analysis revealed that these complexes entered nucleus in sufficient amounts suggesting higher uptake and efficient internalization of the complexes. Besides, these supramolecular nanostructures not only exhibited excellent antimicrobial potential (MIC ~49-100 µg/ml) against clinical as well as resistant pathogenic strains but also found to possess antioxidant property. Overall, the projected low molecular weight PEI-based vectors could serve as more effective multifunctional nanomaterials having promising potential for future gene therapy applications with capability to provide protection against other bacterial infections.

Folic Acid/Peptides Modified PLGA-PEI-PEG Polymeric Vectors as Efficient Gene Delivery Vehicles: Synthesis, Characterization and Their Biological Performance.

Polymeric vectors are safer alternatives for gene delivery owing to their advantages as compared to viral vectors. To improve the stability and transfection efficiency of poly(lactic-co-glycolic acid) (PLGA)- and poly(ethylenimine) (PEI)-based vectors, poly(ethylene glycol) (PEG), folic acid (FA), arginylglycylaspartic acid (RGD) peptides and isoleucine-lysine-valine-alanine-valine (IKVAV) peptides were employed and PLGA-PEI-PEG-FA and PLGA-PEI-PEG-RGD copolymers were synthesized. PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA nanocomplexes (NCs) were formed through bulk mixing. The structure and properties, including morphology, particle size, surface charge and DNA encapsulation, of NCs were studied. Robust NCs with spherical shape, uniform size distribution and slightly positive charge were able to completely bind DNA above their respective N/P ratios. The critical N/P ratio for PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA NCs was identified to be 12:1, 8:1 and 10:1, respectively. The covalent modification of PEI through a combination of biodegradable PLGA, hydrophilic PEG and targeting motifs significantly decreased the cytotoxicity of PEI. The developed NCs showed both N/P ratio and cell type-dependent transfection efficiency. An increase in N/P ratio resulted in increased transfection efficiency, and much improved transfection efficiency of NCs was observed above their respective critical N/P ratios. This study provides a promising means to produce polymeric vectors for gene delivery.

Biocompatibility of magnetic nanoparticles coating with polycations using A549 cells.

Fe3O4 nanoparticles were obtained by chemical coprecipitation of iron chloride and sodium hydroxide. The morphology and sizes of the obtained nanoparticles were characterized using laser Doppler velocimetry, transmission electron and atomic force microscopy. Then the nanoparticles were stabilized by three polycations (polyethylenimine (PEI), poly(allylamine hydrochloride) (PAH), poly(diallyldimethylammonium chloride) (PDADMAC)) to increase their biocompatibility. The cytotoxicity of the obtained polymer-stabilized nanoparticles was studied using a human lung carcinoma cell line (A549). The biodistribution of nanoparticles stabilized by polycations in human lung carcinoma cells was analyzed by transmission electron microscopy, and the toxicity of nanomaterials was evaluated using toxicity tests and flow cytometry. As a result, the most biocompatible nanoparticle-biopolymer complex was identified. PAH stabilized magnetic nanoparticles demonstrated the best biocompatibility, and the PEI-magnetic nanoparticle complex was the most toxic.

A non-viral nano-delivery system targeting epigenetic methyltransferase EZH2 for precise acute myeloid leukemia therapy.

Acute myeloid leukemia (AML), which is common in the elderly population, accounts for poor long-term survival with a high possibility of relapse. The associated lack of currently developed therapeutics is directing the search for new therapeutic targets relating to AML. EZH2 (Enhancer of Zeste Homolog 2) is a histone methyltransferase member of the polycomb-group (PcG) family, and its significant overexpression in AML means it has emerged as a potential epigenetic target. Here, we propose the human serum albumin (HSA) nanoparticle based delivery of small interfering RNA (siRNA), which can target EZH2-expressing genes in AML. EZH2 specific siRNA loaded in a polyethyleneimine (PEI) conjugated HSA nanocarrier can overcome the systemic instability of siRNA and precisely target the AML cell population for increased EZH2 gene silencing. A stable nanosized complex (HSANPs-PEI@EZH2siRNA), achieved via the electrostatic interaction of PEI and EZH2 siRNA, shows increased systemic stability and hemocompatibility, and enhanced EZH2 gene silencing activity in vitro, compared to conventional transfection reagents. HSANPs-PEI@EZH2siRNA-treated AML cells showed downregulated EZH2, which is associated with a reduced level of Bmi-1 protein, and H3K27me3 and H2AK119ub modification. The ubiquitin-mediated proteasomal degradation pathway plays a critical role in the downregulation of associated proteins following HSANPs-PEI@EZH2siRNA exposure to AML cells. c-Myb is the AML-responsive transcription factor that directly binds on the EZH2 promoter and was downregulated in HSANPs-PEI@EZH2siRNA-treated AML cells. The systemic exposure to HSANPs-PEI@EZH2siRNA of AML engrafted immunodeficient nude mice displayed efficient EZH2 gene silencing and a reduced AML cell population in peripheral blood and bone marrow. The present study demonstrates a non-viral siRNA delivery system for epigenetic targeting based superior anti-leukemic therapy.

The Effects of Polymer Coating of Gold Nanoparticles on Oxidative Stress and DNA Damage.

Gold nanoparticles (AuNPs) have been widely used in many biological and biomedical applications. In this regard, their surface modification is of paramount importance in order to increase their cellular uptake, delivery capability, and optimize their distribution inside the body. The aim of this study was to examine the effects of AuNPs on cytotoxicity, oxidant/antioxidant parameters, and DNA damage in HepG2 cells and investigate the potential toxic effects of different surface modifications such as polyethylene glycol (PEG) and polyethyleneimine (PEI; molecular weights of 2,000 (low molecular weight [LMW]) and 25,000 (high molecular weight [HMW]). The study groups were determined as AuNPs, PEG-coated AuNPs (AuNPs/PEG), low-molecular weight polyethyleneimine-coated gold nanoparticles (AuNPs/PEI LMW), and high-molecular weight polyethyleneimine-coated gold nanoparticles (AuNPs/PEI HMW). After incubating HepG2 cells with different concentrations of nanoparticles for 24 hours, half maximal inhibitory concentrations (the concentration that kills 50% of the cells) were determined as 166.77, 257.73, and 198.44 µg/mL for AuNPs, AuNPs/PEG, and AuNPs/PEI LMW groups, respectively. Later, inhibitory concentration 30 (IC30, the concentration that kills 30% of the cells) doses were calculated, and further experiments were performed on cells that were exposed to IC30 doses. Although intracellular reactive oxygen species levels significantly increased in all nanoparticles, AuNPs as well as AuNPs/PEG did not cause any changes in oxidant/antioxidant parameters. However, AuNPs/PEI HMW particularly induced oxidative stress as evidence of alterations in lipid peroxidation and protein oxidation. These results suggest that at IC30 doses, AuNPs do not affect oxidative stress and DNA damage significantly. Polyethylene glycol coating does not have an impact on toxicity, however PEI coating (particularly HMW) can induce oxidative stress.

Poly(l-glutamic acid)-Based Zwitterionic Polymer in a Charge Conversional Shielding System for Gene Therapy of Malignant Tumors.

Recently, pH-sensitive polymers have received extensive attention in tumor therapy. However, the rapid response to pH changes is the key to achieving efficient treatment. Here, a novel shielding system with a rapidly pH-responsive polymer (PAMT) is synthesized by click reaction between poly(γ-allyl-l-glutamate) and thioglycolic acid or 2-(Boc-amino)ethanethiol. The zwitterionic biodegradable polymer PAMT, which is negatively charged at physiological pH, can be used to shield positively charged nanoparticles. PAMT is electrostatically attached to the surface of the positively charged PEI/pDNA complex to form a ternary complex. The zwitterionic PAMT-shielded complex exhibits rapid charge conversion when the pH decreases from 7.4 to 6.8. For the in vivo tumor inhibition experiment, PAMT/PEI/shVEGF injected intravenously shows a more significant inhibitory effect on tumor growth. The excellent results are mainly attributed to introduction of the zwitterionic copolymer PAMT, which can shield the positively charged PEI/shVEGF complex in physiological conditions, while the surface potential of the shielded complexes changes to a positive charge in the acidic tumor environment.

Dual-ligand functionalized carbon nanodots as green fluorescent nanosensors for cellular dual receptor-mediated targeted imaging.

The conjugation of ligands to nanoparticles as drug delivery systems that target specific cells is a promising approach for the delivery of therapeutic agents to tumor cells. Herein, we prepared green-emission fluorescent carbon nanodots (CNDs) by a facile hydrothermal method with d-(+)-glucosamine hydrochloride and l-aspartic acid as the precursors, then covalently conjugated with folate (FA), polyethyleneimine (PEI) and hyaluronic acid (HA) to develop dual ligand-decorated nanocarriers (FA-PEI-HA-CNDs) for the targeted imaging of cancer cells. FA-PEI-HA-CNDs integrated the excellent fluorescence property of CNDs, and can be used for the real-time and noninvasive location tracking of cancer cells. The cellular uptake study demonstrated that FA-PEI-HA-CNDs markedly improved the internalization efficiency in A-549 cells via folate/CD44 receptor-mediated endocytosis in comparison with that of the A549 cells pretreated with excess FA, HA, and FA and HA. Therefore, these dual folate/CD44 receptor-targeted CNDs (FA-PEI-HA-CNDs) show promising potential for cancer detection, drug delivery, and individualized treatment as performance platforms.

The influence of surface coatings on the toxicity of silver nanoparticle in rainbow trout.

Silver nanoparticles (nAg) are often produced with different coatings that could influence bioavailability and toxicity in aquatic organisms. The purpose of this study was to examine the influence of 4 surface coatings of nAg of the same core size towards bioavailability and toxicity in juvenile rainbow trout (Oncorhynchus mykiss). Juveniles were exposed to 50 µg/L of 50 nm diameter nAg for 96 h at 15 °C with the following coatings: branched polyethylenimine (bPEI), citrate, polyvinylpyrrolidone (PVP) and silicate (Si). The data revealed that the coatings influenced hepatic Ag loadings in the following trend PVP > citrate > bPEI and Si with estimated bioavailability factors of 28, 18, 6 and 2 L/kg respectively. Hepatic Ag levels were significantly associated with DNA damage and inflammation as determined by arachidonate cyclooxygenase activity. The bPEI and citrate-coated nAg consistently produced the observed effects above in addition to increased mitochondrial electron transport activity and glutathione S-transferase activity. The absence of metallothionein and lipid peroxidation suggests that mechanisms other than the liberation of Ag+ were at play. In conclusion, surface coatings were shown to significantly influence bioavailability and toxic properties of nAg to rainbow trout juveniles.

A biodegradable poly(amido amine) based on the antimicrobial polymer polyhexamethylene biguanide for efficient and safe gene delivery.

Inspired by the excellent membrane affinity of antimicrobial polymers, we synthesized a novel biodegradable poly(amino amine) polymer with pendent side chains that mimic the widely used biocide polyhexamethylene biguanide (PHMB) for gene delivery. Michael addition polymerization was utilized to form the polymer scaffold between N,N'-cystaminebisacrylamide (CBA) and N-Boc-1,6-diaminohexane (Boc-DAH) followed by N-Boc deprotection. Then the exposed primary amino groups were partly (about 75%) transformed into biguanide by an addition reaction with dicyandiamide to obtain the final product CBA-DAH-biguanide (CBA-DAH-BG). The polymer CBA-DAH-BG was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>35 mV) polyplexes that were well resistant to heparin and DNase I. Rapid DNA release was observed in the presence of dithiothreitol (DTT), indicating that CBA-DAH-BG was equipped with biodegradability by the cleavage of disulfide bonds, which was helpful for unpacking DNA and decreasing cytotoxicity. CBA-DAH-BG/pDNA polyplexes were characterized by efficient cellular uptake efficacy, extremely low cytotoxicity, and high transfection efficiency in two cell lines (i.e., NIH/3T3 and U87 MG), compared to 25 kDa polyethyleneimine (PEI) and the intermediate product CBA-DAH that were both devoid of biguanide groups. Of note, clathrin-mediated endocytosis and lipid rafts played an important role in the internalization of the polyplexes. Taken together, this strategy described herein may represent an innovative avenue for the design of more advanced nonviral gene vectors with high transfection efficiency and biocompatibility.

Highly compressive and stretchable poly(ethylene glycol) based hydrogels synthesised using pH-responsive nanogels without free-radical chemistry.

Poly(ethylene glycol) (PEG) based hydrogels are amongst the most studied synthetic hydrogels. However, reports on PEG-based hydrogels with high mechanical strength are limited. Herein, a class of novel, well-defined PEG-based nanocomposite hydrogels with tunable mechanical strength are synthesised via ring-opening reactions of diglycidyl ethers with carboxylate ions. The pH responsive crosslinked polyacid nanogels (NG) in the dispersed phase act as high functionality crosslinkers which covalently bond to the poly(ethylene glycol) diglycidyl ethers (PEGDGE) as the continuous matrix. A series of NG-x-PEG-y-z gels are prepared where x, y and z are concentrations of NGs, PEGDGE and the PEGDGE molecular weight, respectively. The hydrogel compositions and nano-structural homogeneity of the NGs have strong impact on the enhancement of mechanical properties which enables property tuning. Based on this design, a highly compressive PEG-based nanocomposite hydrogel (NG-13-PEG-20-6000) exhibits a compressive stress of 24.2 MPa, compressive fracture strain greater than 98% and a fracture energy density as high as 1.88 MJ m-3. The tensile fracture strain is 230%. This is amongst one of the most compressive PEG-based hydrogels reported to-date. Our chemically crosslinked gels are resilient and show highly recoverable dissipative energy. The cytotoxicity test shows that human nucleus pulposus (NP) cells remained viable after 8 days of culture time. The overall results highlight their potential for applications as replacements for intervertebral discs or articular cartilages.

Glycopolymers/PEI complexes as serum-tolerant vectors for enhanced gene delivery to hepatocytes.

Serum stability is a crucial factor for ideal polymeric gene vectors. In this work, a series of serum-tolerant and low-toxicity glycopolymers/poly(ethyleneimine) (PEI) complexes were designed for gene delivery. Atomic transfer radical polymerization (ATRP) was used to synthesize the comb-shaped random copolymers dextran-g-poly(2-dimethylaminoethyl methacrylate-co-2-lactobionamidoethyl methacrylate) (DDrL). Then DDrLs/PEI were investigated for their use as plasmid DNA (pDNA) vectors, which can completely condense the pDNA into nanoparticles. The DDrLs/PEI/pDNA complexes in serum-containing media showed better stability than PEI/pDNA complexes. in vitro gene transfection studies showed that DDrLs/PEI exhibited a remarkable transfection efficiency enhancement in the presence of serum compared to that in serum-free conditions. Moreover, the transfection level of DDrLs/PEI were two orders of magnitude higher than that of PEI alone in the presence of 30% serum. DDrLs/PEI complexes with galactose enhanced pDNA delivery to hepatocytes, with higher protein expression in ASGPr-presenting HepG2 than in HeLa cells, which lack the receptor. All of the DDrLs/PEI/pDNA complexes had lower cytotoxicity than PEI/pDNA.

Surfactant protein B (SP-B) enhances the cellular siRNA delivery of proteolipid coated nanogels for inhalation therapy.

Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy. STATEMENT OF SIGNIFICANCE: Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model.

Redox-triggered intracellular siRNA delivery.

Gene silencing using small interfering RNA (siRNA) is a promising strategy for the treatment of multiple diseases. However, the low in vivo stability of siRNA, its poor pharmacokinetics and inability to penetrate inside cells limit its employment in the clinic. Here, we present a novel redox-sensitive micellar nanopreparation based on a triple conjugate of polyethylene glycol, polyethyleneimine and phosphatidylethanolamine, PEG-SS-PEI-PE (PSSPD). This non-toxic system efficiently condenses siRNA and specifically downregulates target green fluorescent protein (GFP) only under reducing conditions via intracellular siRNA release after de-shielding of PEG due to increased glutathione (GSH) levels characteristic of cancer cells.

Macrocycle-wrapped polyethylenimine for gene delivery with reduced cytotoxicity.

Due to its outstanding capability to facilitate DNA condensation, transportation and endosomal escape, polyethylenimine (PEI) has been frequently studied for gene delivery. However, its molecular weight (M.W.) dependent transfection efficiency and cytotoxicity has severely limited its clinical application. To resolve this dilemma, a supramolecular strategy was developed for the first time, in which PEI with large M.W. (branched, 25 kDa) that has a satisfactory transfection efficiency, yet high non-specific cytotoxicity for gene delivery was wrapped with macrocyclic cucurbit[7]uril (CB[7]). The successful wrapping of the PEI by the macrocyclic CB[7] was proved by 1H NMR spectroscopy and supported by isothermal titration calorimetry (ITC). The plasmid DNA (pDNA) condensability of PEI was not affected by the supramolecular coating as evidenced from the agarose gel electrophoresis assay. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results demonstrated that the particle size, zeta potential, and morphology of the self-assemblies of PEI/pDNA and PEI/CB[7]/pDNA were comparable. As a consequence of the supramolecular wrapping, the cytotoxicity of PEI was significantly constrained as demonstrated by MTT assay, apoptosis assay, and a hemolysis study. In particular, both the cellular uptake and the gene transfection efficiency results suggest that the supramolecular wrapping of PEI by CB[7] exhibits negligible effects on PEI, thus functioning as an effective non-viral gene delivery vector. This novel supramolecular-wrapping strategy provides new insights for facile alleviation of the non-specific toxicity of PEI and potentially other polycationic gene vectors without compromising their transfection efficiency.

Comparison of Gene Transfection and Cytotoxicity Mechanisms of Linear Poly(amidoamine) and Branched Poly(ethyleneimine) Polyplexes.

PURPOSE: This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity. METHODS: The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10 kDa or 25 kDa branched polyethyleneimine (BPEI) was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transgene expression was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. RESULTS: The luciferase activity was ~3-fold lower after transfection with PAA polyplexes than with BPEI complexes at the optimal polymer to nucleotide ratio (RU:Nt). However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt. CONCLUSIONS: PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH.