Peptide-Modified Gemini Surfactants as Delivery System Building Blocks with Dual Functionalities for Glaucoma Treatment: Gene Carriers and Amyloid-Beta (Aß) Self-Aggregation Inhibitors.

Retinal ganglion cell (RGC) neurodegeneration in glaucoma has potential links with amyloid-ß (Aß) deposition. Targeting the Aß pathway was shown to reduce RGC apoptosis and protect RGCs from degeneration. We report exploratory studies on the amyloid Aß40 aggregation inhibition properties of four cell adhesion peptide (CAP)-gemini surfactants that are intended as building blocks for gene carrier nanoparticles for glaucoma treatment. The CAP-gemini surfactants (18-7N(p1-4)-18) were evaluated as potential Aß40 peptide aggregation inhibitors by a fluorescence kinetic assay and for their binding interactions with Aß40 dimers by molecular docking studies. In vitro Aß40 peptide aggregation inhibition studies showed that the 18-7N(p3)-18 and 18-7N(p1)-18 ligands inhibit Aß40 peptide aggregation and prevent the formation of higher order structures. CDOCKER energies and CDOCKER interaction energies demonstrated that the CAP-gemini surfactants formed more stable complexes in the Aß40 dimer assembly and underwent both polar and nonpolar interactions compared to CAP peptides alone. Also, 18-7N(p3)-18 showed a significantly lower CDOCKER energy compared to that of the unmodified gemini surfactant 18-7NH-18 (p < 0.0001) and 18-7N(p4)-18 (p < 0.001), 18-7N(p1)-18, and 18-7N(p2)-18. Similarly, 18-7N(p3)-18 showed a significantly lower CDOCKER interaction energy compared to that of 18-7NH-18, 18-7N(p4)-18 (p < 0.0001), and 18-7N(p2)-18 (p < 0.001), while 18-7N(p3)-18 and 18-7N(p1)-18 showed similar CDOCKER interaction energies. These studies suggest that a combination of both hydrophobic and electrostatic interactions contributes to the anti-Aß40 aggregation activity of CAP-gemini surfactants. CAP-gemini surfactants showed 10-fold better Aß40 peptide aggregation inhibition compared to previously reported values and could provide a new opportunity for glaucoma treatment as dual-functional gene carriers.

Dicationic Amino Substituted Gemini Surfactants and their Nanoplexes: Improved Synthesis and Characterization of Transfection Efficiency and Corneal Penetration In Vitro.

PURPOSE: To formulate and characterize nanoparticles from m-7NH-m gemini surfactants, synthesized by a new improved method, for non-invasive gene delivery including optimization of composition for transfection efficiency and corneal penetration. METHODS: A one-pot, solvent-free, DMAP-free method was developed for the synthesis of m-7NH-m (m = 12-18) gemini surfactant series. Lipoplexes (LPXs) and nanoplexes (NPXs) of gemini surfactant-plasmid DNA were formulated with and without DOPE helper lipid, respectively, at various charge ratios and characterized by dynamic light scattering and zeta potential measurements. Transfection efficiency, cellular toxicity, effect of DOPE and gene expression kinetic studies were carried out in A7 astrocytes by flow cytometry and confocal microscopy. Corneal penetration studies of 18-7NH-18 NPXs were carried out using 3D EpiCorneal® tissue model. RESULTS: The new synthesis method provides a two-fold improved yield and the production of a pure species of m-7NH-m without DMAP and trimeric m-7N(m)-m surfactants as impurities. Structure and purity was confirmed by ESI-MS, 1H NMR spectroscopy and surface tension measurements. Particle size of 199.80 ± 1.83 nm ± S.D. and a zeta potential value of +30.18 ± 1.17 mV ± S.D. was obtained for 18-7NH-18 5:1 ratio NPXs showed optimum transfection efficiency (10.97 ± 0.11%) and low toxicity (92.97 ± 0.57% viability) at the 48-h peak expression. Inclusion of DOPE at 1: 0.5 and 1:1 ratios to gemini surfactant reduced transfection efficiency and increased toxicity. Treatment of EpiCorneal® tissue model showed deep penetration of up to 100 µm with 18-7NH-18 NPXs. CONCLUSION: Overall, 18-7NH-18 NPXs are potential gene delivery systems for ophthalmic gene delivery and for further in vivo studies.

Multipotent stem cell-derived retinal ganglion cells in 3D culture as tools for neurotrophic factor gene delivery system development.

Non-viral neurotrophic factor (NF) gene therapy is a new paradigm in glaucoma treatment with the potential for neuroprotection and regeneration of damaged retinal ganglion cells (RGCs). To improve nanoparticle gene delivery systems and generate a suitable RGC cell model to facilitate in vitro investigations, we have developed mouse multipotent retinal stem cell (MRSC)-derived RGCs (XFC-3 cells) that express key RGC characteristics as demonstrated through biomarker expression profiling and stimuli-inducible neurite extension evaluation. Dicationic gemini surfactant-, single-walled carbon nanotube-, and K2-lipopolyamine polymer-based gene delivery systems were formulated and evaluated in three-dimensional (3D) A7/XFC-3 and XFC-3/XFC-3 co-cultures to validate the model for transfection efficiency (TE) and brain-derived neurotrophic factor (BDNF) bioactivity measurements, which helped identify the K2-NPs as having high TE (63.1% ±â€¯1.4%) and high cell viability (94.4% ±â€¯0.4%). Overall, XFC-3 cells are suitable for the construction of 3D in vivo-like tissue models and enable the screening of RGC-aimed gene delivery systems for neuroprotective treatment of glaucoma.

A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation.

BACKGROUND: Gemini-lipid nanoparticles have been received major attention recently as non-viral delivery systems due to their successful non-invasive gene delivery through tough barriers such as eye and skin. The aim of this study was to evaluate non-viral gene delivery by a series of dicationic gemini surfactant-phospholipid nanoparticles (GL-NPs) and to explore their mechanism of interaction with cellular membranes of murine PAM212 epidermal keratinocytes. METHODS: NPs containing pCMV-tdTomato plasmid encoding red fluorescent protein (RFP) were prepared using 12 different gemini surfactants (m-s-m, with m = 12, 16 and 18C alkyl tail and s = 3 and 7C polymethylene spacer group and 7C substituted spacers with 7NH and 7NCH3) and dioleoylphosphatidylethanolamine helper lipid. RFP gene expression and cell viability status were evaluated using flow cytometry. MitoTracker Deep Red mitochondrial stain and the cell impermeable Sytox red nuclear stain were used as indicators of cell viability and cell membrane integrity, respectively. RESULTS: No significant viability loss was detected in cells transfected with 18-3-18, 18-7-18, 18-7NH-18, and 18-7NCH3-18 NPs, whereas a significant reduction of viability was detected in cells treated with 12-3-12, 12-7-12, 12-7NH-12, 16-7NH-16, or 16-7NCH3-16 GL-NPs. Compared to Lipofectamine Plus, 18-3-18 GL-NPs showed higher transfection efficiency and comparable viability profile by evaluation using MitoTracker Deep Red in PAM212 cells. Flow cytometric analysis of PAM212 cells stained with Sytox red revealed two cell populations with low and high fluorescent intensity, representing cells with partially-porated and highly-porated membranes, respectively. Additional combined staining with MitoTracker and ethidium homodimer showed that that 18-3-18 GL-NPs disturbed cell membrane integrity, while cells were still alive and had mitochondrial activity. CONCLUSION: Taken together, this study demonstrated that 18-3-18 GL-NPs have higher transfection efficiency and comparable viability profile to the commercial Lipofectamine Plus, and the interaction of 18-3-18 GL-NPs with PAM212 cell membranes involves a permeability increase, possibly through the formation of nanoscale pores, which could explain efficient gene delivery. This novel nanoconstruct appears to be a promising delivery system for further skin gene therapy studies in vivo.

Protection of Neonatal Broiler Chickens Following in ovo Delivery of Oligodeoxynucleotides Containing CpG Motifs (CpG-ODN) Formulated with Carbon Nanotubes or Liposomes.

Unformulated oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to stimulate the innate immune system against a variety of bacterial, viral, and protozoan infections in a variety of vertebrate species. We have previously shown that in ovo delivery of unformulated CpG-ODN was able to significantly protect neonatal broiler chickens against Escherichia coli or Salmonella Typhimurium infections. The objectives of this study were to examine the safety and immunoprotective effects of CpG-ODN formulated with 2 types of carbon nanotubes (CNTs) or 2 types of lipid-surfactant (LSC) delivery systems in neonatal broilers against E. coli septicemia. Embryonated eggs, which had been incubated for 18 days, received either 50 µg of CNT-CpG-ODN, 50 µg of LSC-CpG-ODN, 50 µg of unformulated CpG-ODN, or saline. Four days after exposure to CpG-ODN (day 1 posthatch), 1 x 10(4) or 1 x 10(5) colony-forming units of a virulent strain of E. coli isolated from a turkey with septicemia were inoculated subcutaneously in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 8 days following challenge with E. coli. Bacterial isolations and pathologic observations were conducted immediately after birds were dead or euthanatized. The survival rate of birds in groups receiving saline following E. coli infection was 20% to 30%. In contrast, birds receiving CpG-ODN formulations had a significantly higher survival rate of 60% to 80% (P < 0.01). Bacterial loads and clinical scores were significantly lower (P < 0.05) in groups treated with CNT- or LSC-CpG-ODN compared to the groups receiving CpG-ODN or saline. Moreover, there is no evidence of any adverse effects of these formulations in any organs or in growth rates of birds until 42 days of age. This is the first time that CpG-ODN formulated with CNT and LSC have been demonstrated to have an immunomodulatory effect against an E. coli infection in neonatal broiler chickens following in ovo delivery.

Therapeutic applications of nanomedicine in autoimmune diseases: from immunosuppression to tolerance induction.

Autoimmune diseases are chronic, destructive diseases that can cause functional disability and multiple organ failure. Despite significant advances in the range of therapeutic agents, especially biologicals, limitations of the routes of administration, requirement for frequent long-term dosing and inadequate targeting options often lead to suboptimal effects, systemic adverse reactions and patient non-compliance. Nanotechnology offers promising strategies to improve and optimize autoimmune disease treatment with the ability to overcome many of the limitations common to the current immunosuppressive and biological therapies. Here we focus on nanomedicine-based delivery strategies of biological immunomodulatory agents for the treatment of autoimmune disorders including psoriasis, rheumatoid arthritis, systemic lupus erythematous, scleroderma, multiple sclerosis and type 1 diabetes. This comprehensive review details the concepts and clinical potential of novel nanomedicine approaches for inducing immunosuppression and immunological tolerance in autoimmune diseases in order to modulate aberrant and pathologic immune responses. FROM THE CLINICAL EDITOR: The treatment of autoimmune diseases remains a significant challenge. The authors here provided a comprehensive review, focusing on the current status and potential of nanomedicine-based delivery strategies of immunomodulatory agents for the treatment of autoimmune disorders including psoriasis, rheumatoid arthritis, systemic lupus erythematous, scleroderma, multiple sclerosis, and type 1 diabetes.

Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: towards non-invasive glaucoma gene therapy.

Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR: In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

Effect of chemical permeation enhancers on stratum corneum barrier lipid organizational structure and interferon alpha permeability.

The outermost layer of the skin, known as the stratum corneum (SC), is composed of dead corneocytes embedded in an intercellular lipid matrix consisting of ceramides, free fatty acids, and cholesterol. The high level of organization within this matrix protects the body by limiting the permeation of most compounds through the skin. While essential for its protective functions, the SC poses a significant barrier for the delivery of topically applied pharmaceutical agents. Chemical permeation enhancers (CPEs) can increase delivery of small drug compounds into the skin by interacting with the intercellular lipids through physical processes including extraction, fluidization, increased disorder, and phase separation. However, it is not clear whether these same mechanisms are involved in delivery of biotherapeutic macromolecules, such as proteins. Here we describe the effect of three categories of CPEs {solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes [menthol, nerol, camphor, methyl salicylate], and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)]} on the lipid organizational structure of human SC as determined by X-ray scattering studies. Small- and wide-angle X-ray scattering studies were conducted to correlate the degree of structural changes and hydrocarbon chain packing in SC lipids caused by these various classes of CPEs to the extent of permeation of interferon alpha-2b (IFNα), a 19 kDa protein drug, into human skin. With the exception of solvents, propylene glycol and ethanol, all classes of CPEs caused increased disordering of lamellar and lateral packing of lipids. We observed that the highest degree of SC lipid disordering was caused by surfactants (especially SDS, DDAB, and DTAB) followed by terpenes, such as nerol. Interestingly, in vitro skin permeation studies indicated that, in most cases, absorption of IFNα was low and that an increase in SC lipid disorder does not correspond to an increase in IFNα absorption.

BDNF gene delivery to the retina by cell adhesion peptide-conjugated gemini nanoplexes in vivo.

Retinal ganglion cell (RGC) neurodegeneration in glaucoma is not prevented by controlling the elevated intraocular pressure alone. Neuroprotective gene therapy approaches could be an essential part of a combination treatment. Five cell adhesion peptide (CAP)-gemini surfactants (18-7N(p1-5)-18) were synthesized as building blocks for brain-derived neurotrophic factor (BDNF) gene carrier nanoparticles (CAP-NPXs). The composition of CAP-NPXs was optimized, physicochemically characterized and evaluated for in vitro transfection efficiency (TE) in A7 astrocytes, 3D retinal neurospheres and for gene expression in vivo in CD1 mice using RFP reporter gene and BDNF levels after intravitreal (IVT) injection. The IgSF-binding 18-7N(pFASNKL)-18 pNPXs treated cells demonstrated 1.4-fold higher TE compared to integrin-binding 18-7N(pRGD)-18 pNPXs and parent 18-7NH-18 NPXs with overall viability between 86 and 95%. The 18-7N(pFASNKL)-18 pNPXs selectively transfected RGCs in 3D MiEye8 neurospheres. In the in vivo CD1 mouse model 18-7N(pFASNKL)-18 pNPXs administered by IVT injection delivered tdTomato/BDNF plasmid to retinal cells and produced higher gene expression than the 18-7N(pRGD)-18 pNPXs, the parent 18-7NH-18 NPXs and Lipofectamine® 3000 as demonstrated by confocal microscopy of whole mount retinas. The BDNF gene expression, assessed by ELISA, showed significantly high levels of BDNF with 18-7N(pFASNKL)-18 (422.60 ± 42.60 pg/eye), followed by 18-7N(pRGD)-18 pNPXs (230.62 ± 24.47 pg/eye), 18-7NH-18 NPXs (245.90 ± 39.72 pg/eye), Lipofectamine® 3000 (199.99 ± 29.90 pg/eye) and untreated controls (131.33 ± 20.30 pg/eye). In summary, the 18-7N(pFASNKL)-18 pNPXs induced 3.4-fold higher BDNF level compared to controls and 2-fold higher than 18-7N(pRGD)-18 pNPXs. The in vivo efficacy of 18-7N(pFASNKL)-18 NPXs to produce BDNF at pharmacologically relevant levels supports further studies.

Gene therapy strategies for glaucoma from IOP reduction to retinal neuroprotection: Progress towards non-viral systems.

Glaucoma is the result of the gradual death of retinal ganglion cells (RGCs) whose axons form the optic nerve. Elevated intraocular pressure (IOP) is a major risk factor that contributes to RGC apoptosis and axonal loss at the lamina cribrosa, resulting in progressive reduction and eventual anterograde-retrograde transport blockade of neurotrophic factors. Current glaucoma management mainly focuses on pharmacological or surgical lowering of IOP, to manage the only modifiable risk factor. Although IOP reduction delays disease progression, it does not address previous and ongoing optic nerve degeneration. Gene therapy is a promising direction to control or modify genes involved in the pathophysiology of glaucoma. Both viral and non-viral gene therapy delivery systems are emerging as promising alternatives or add-on therapies to traditional treatments for improving IOP control and providing neuroprotection. The specific spotlight on non-viral gene delivery systems shows further progress toward improving the safety of gene therapy and implementing neuroprotection by targeting specific tissues and cells in the eye and specifically in the retina.

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

HPV infections: can they be eradicated using nanotechnology?

Human papillomavirus (HPV) infections are considered the most common sexually transmitted diseases, with more than 450 million people worldwide infected. HPVs can cause anogenital warts and neoplasias. One prevalent cancer type caused by HPV (mostly type 16 and 18) is cervical intraepithelial neoplasia (CIN). Nanotechnology tools employed in the development of vaccines and a noninvasive treatment may provide a significant advancement in the global combat against this disease.

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation.

Introduction: Colorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations. Methods: We generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells. Results: Using flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture. Discussion: We conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity.

CpG-ODN induced antimicrobial immunity in neonatal chicks involves a substantial shift in serum metabolic profiles.

Synthetic CpG-ODNs can promote antimicrobial immunity in neonatal chicks by enriching immune compartments and activating immune cells. Activated immune cells undergo profound metabolic changes to meet cellular biosynthesis and energy demands and facilitate the signaling processes. We hypothesize that CpG-ODNs induced immune activation can change the host's metabolic demands in neonatal chicks. Here, we used NMR-based metabolomics to explore the potential of immuno-metabolic interactions in the orchestration of CpG-ODN-induced antimicrobial immunity. We administered CpG-ODNs to day-old broiler chicks via intrapulmonary (IPL) and intramuscular (IM) routes. A negative control group was administered IPL distilled water (DW). In each group (n = 60), chicks (n = 40) were challenged with a lethal dose of Escherichia coli, two days post-CpG-ODN administration. CpG-ODN administered chicks had significantly higher survival (P < 0.05), significantly lower cumulative clinical scores (P < 0.05), and lower bacterial loads (P < 0.05) compared to the DW control group. In parallel experiments, we compared NMR-based serum metabolomic profiles in neonatal chicks (n = 20/group, 24 h post-treatment) treated with IM versus IPL CpG-ODNs or distilled water (DW) control. Serum metabolomics revealed that IM administration of CpG-ODN resulted in a highly significant and consistent decrease in amino acids, purines, betaine, choline, acetate, and a slight decrease in glucose. IPL CpG-ODN treatment resulted in a similar decrease in purines and choline but less extensive decrease in amino acids, a stronger decrease in acetate, and a considerable increase in 2-hydroxybutyrate, 3-hydroxybutyrate, formic acid and a mild increase in TCA cycle intermediates (all P < 0.05 after FDR adjustment). These perturbations in pathways associated with energy production, amino acid metabolism and nucleotide synthesis, most probably reflect increased uptake of nutrients to the cells, to support cell proliferation triggered by the innate immune response. Our study revealed for the first time that CpG-ODNs change the metabolomic landscape to establish antimicrobial immunity in neonatal chicks. The metabolites highlighted in the present study can help future targeted studies to better understand immunometabolic interactions and pinpoint the key molecules or pathways contributing to immunity.

Topical delivery of interferon alpha by biphasic vesicles: evidence for a novel nanopathway across the stratum corneum.

Noninvasive delivery of macromolecules across intact skin is challenging but would allow for needle-free administration of many pharmaceuticals. Biphasic vesicles, a novel lipid-based topical delivery system, have been shown to deliver macromolecules into the skin. Investigation of the delivery mechanism of interferon alpha (IFN alpha), as a model protein, by biphasic vesicles could improve understanding of molecular transport through the stratum corneum and allow for the design of more effective delivery systems. The interaction of biphasic vesicles with human skin and isolated stratum corneum membrane was investigated by confocal microscopy, differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS and WAXS). Confocal microscopy revealed that biphasic vesicles delivered IFN alpha intercellularly, to a depth of 70 microm, well below the stratum corneum and into the viable epidermis. DSC and SAXS/WAXS data suggest that the interaction of biphasic vesicles with SC lipids resulted in the formation of a three-dimensional cubic Pn3m polymorphic phase by the molecular rearrangement of intercellular lipids. This cubic phase could be an intercellular permeation nanopathway that may explain the increased delivery of IFN alpha by biphasic vesicles. Liposomes and submicrometer emulsion (the individual building blocks of biphasic vesicles) separately and methylcellulose gel, an alternative topical vehicle, did not induce a cubic phase and delivered low amounts of IFN alpha below the stratum corneum. Molecular modeling of the cubic Pn3m phase and lamellar-to-cubic phase transitions provides a plausible mechanism for transport of IFN alpha. It is hypothesized that induction of a Pn3m cubic phase in stratum corneum lipids could make dermal and transdermal delivery of other macromolecules also possible.

Mucosal delivery of CpG-ODN mimicking bacterial DNA via the intrapulmonary route induces systemic antimicrobial immune responses in neonatal chicks.

The transition to antibiotic-free poultry production in the face of pathogenic threats is a very challenging task. We recently demonstrated that mucosal delivery of CpG-ODN alone by the intrapulmonary route (IPL) has potential as an effective alternative to antibiotics in neonatal chicks against Escherichia coli septicemia. How exactly mucosal delivery of CpG-ODN elicits, protective antibacterial immunity remained poorly understood. In this study, CpG-ODN or saline was delivered via the intrapulmonary route to day-old chicks (n = 80/group) using a compressor nebulizer in an acrylic chamber (1 mg/mL CpG-ODN for 15 minutes). In the first part of the study, two days after mucosal CpG-ODN delivery, 40 chicks from each group were challenged subcutaneously with 1 × 105 cfu (n = 20) or 1 × 106 cfu (n = 20) of E. coli and the mortality pattern was monitored for seven days. We found significantly higher survival, better clinical conditions and lower bacterial loads in chicks that received mucosal CpG-ODN. To explore the mechanisms behind this protective immunity, we first looked at the kinetics of the cytokine gene expression (three birds/ group/ time for 10 time-points) in the lungs and spleens. Multiplex gene analysis demonstrated a significant elevation of pro-inflammatory cytokine genes mRNA in the CpG-ODN group. Interleukin (IL)-1ß robustly upregulated many folds in the lung after CpG-ODN delivery. Lipopolysaccharide-induced tumor necrosis factor (LITAF) and IL-18 showed expression for an extended period in the lungs. Anti-inflammatory cytokine IL-10 was upregulated in both lungs and spleen, whereas IL-4 showed upregulation in the lungs. To investigate the kinetics of immune enrichment in the lungs and spleens, we performed flow cytometry, histology, and immunohistochemistry at 24, 48 and 72 hrs after CpG-ODN delivery. CpG-ODN treated lungs showed a significant enrichment with monocytes/macrophages and CD4+ and CD8+ T-cell subsets. Macrophages in CpG-ODN treated group demonstrated mature phenotypes (higher CD40 and MHCII expression). Importantly, mucosal delivery of CpG-ODN via the intrapulmonary route significantly enriched immune compartment in the spleen as well, suggesting a systemic effect in neonatal chicks. Altogether, intrapulmonary delivery of aerosolized CpG-ODN orchestrates protective immunity against E. coli septicemia by not only enhancing mucosal immunity but also the systemic immune responses.

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 µg of CpG-ODN or saline (n = ~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n = 3-4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 µg and 50 µg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1 × 104 or 1 × 105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 µg and 50 µg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.

Enhancement of immunoprotective effect of CpG-ODN by formulation with polyphosphazenes against E. coli septicemia in neonatal chickens.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents and vaccine adjuvants in a variety of bacterial and protozoan diseases in different animal species. The objective of this study was to investigate the immunoprotective effect of formulated CpG-ODN with polyphosphazene, liposome or oil-in-water emulsion against E. coli infections in neonatal chickens. Eighteen-day-old embryonating eggs were inoculated with 50 microg CpG-ODN or formulated CpG-ODN with polyphophazene, liposome or oil-in-water emulsion. Four days after exposure to formulated CpG-ODN or day-1 post-hatch, 1 x 10(4) or 1 x l0(5) cfu of a virulent isolate of E. coli was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for eight days following challenge with E. coli. The survival rate of birds following E. coli infection was 0% in groups receiving either non-CpG-ODN or saline. In contrast, birds receiving either CpG-ODN or CpG-ODN formulated with polyphosphazene had significantly higher survival of 55% (P<0.0001). The relative risk of mortality was significantly reduced for birds treated with CpG-ODN formulated in PCPP (0.25), in PCEP (0.33), or unformulated CpG-ODN (0.39) in comparison to the group treated with saline (p<0.01). Although formulation of CpG-ODN with liposomes or oil-in-water emulsion did not increase the immunoprotective effect against E. coli infection, no adverse reactions or poor hatchability were observed in embryos. This is the first time that CpG-ODN formulated with polyphosphazene has been demonstrated to have an immunoprotective effect against an extra cellular bacterial infection in neonatal broiler chickens following in ovo delivery.

Tuning optimum transfection of gemini surfactant-phospholipid-DNA nanoparticles by validated theoretical modeling.

Gemini nanoparticles (NPs) are a family of non-viral gene delivery systems with potential for applications in non-invasive gene therapy. Translation of these non-viral gene delivery systems requires improvement of transfection efficiency (TE) through fine-tuning of their physicochemical properties such as electric charge and exact ratios of their components. Since high-throughput experimental screening of minute differences in NP compositions is not routinely feasible, we have developed a coarse-grained model to quantitatively study the energetics of the formation of gene delivery complexes with cationic gemini surfactants (G) (m-s-m type) and helper lipids (H) (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DOPE/1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC)), in order to use it as a tool to predict effective compositions. The model is based on the polymorphic structural conformational flip of NPs and incorporates the electrostatic, entropic and elastic energies, to predict the formation energy and stability of different polymorphic structures as a function of the electric charge of cationic surfactants and concentration of constituent helper lipids. Our results show that these two factors are intertwined in determining the behavior of gene delivery vectors. Specifically, we show that increasing H/G lowers free energy per DNA base pair and increases the stability of the complex. At pH 7, low H/G and charge ratio (ρ±), where the lamellar structure is favored, the formation free energy per DNA base pair is between 0 and -14kBT. At higher values of H/G (2-3) and ρ±, where HII and cubic structures are formed, the formation free energy drops down to values ≈-50kBT, indicating the stable existence of these polymorphic structures in the NPs. At pH 5, the structural transformation of NPs in the endosomes to the lamellar/HII structure with free energy values of about -40kBT is beneficial for endosomal escape, and correlates with increased transfection efficiency. The theoretical model is supported by transfection data in A7 astrocytes with a panel of 16-3-16 gemini NPs, which validates the mathematical model and supports the hypothesis that the NP polymorphic phase transition increases transfection efficiency.