Sterile inflammation enhances ECM degradation in integrin β1 KO embryonic skin

Epidermal knockout of integrin β1 results in complete disorganization of the basement membrane (BM), resulting in neonatal lethality. Here, we report that this disorganization is exacerbated by an early embryonic inflammatory response involving the recruitment of tissue-resident and monocyte-derived macrophages to the dermal-epidermal junction, associated with increased matrix metalloproteinase activity. Remarkably, the skin barrier in the integrin β1 knockout animals is intact, suggesting that this inflammatory response is initiated in a sterile environment. We demonstrate that the molecular mechanism involves de novo expression of integrin αvβ6 in the basal epidermal cells, which activates a TGF-β1 driven inflammatory cascade resulting in upregulation of dermal NF-κB in a Tenascin C-dependent manner. Importantly, treatment of β1 KO embryos in utero with small molecule inhibitors of TGF-βR1 and NF-κB results in marked rescue of the BM defects and amelioration of immune response, revealing an unconventional immuno-protective role for integrin β1 during BM remodeling

Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery

A number of prior studies have demonstrated that the DNA-binding and gene transfection efficacies of cationic amphiphiles crucially depend on their various structural parameters including hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar headgroups connected by the same ester linker group. The only structural difference between the cationic amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2 cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene delivery efficacies of cationic liposomes

The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles

Understanding the structural parameters of cationic amphiphiles which can influence gene transfer efficiencies of cationic amphiphiles continues to remain important for designing efficient liposomal gene delivery reagents. Previously we demonstrated the influence of structural orientation of the ester linker (widely used in covalently tethering the polar head and the non-polar tails) in modulating in vitro gene transfer efficiencies of cationic amphiphiles. However, our previously described cationic amphiphiles with ester linkers failed to deliver genes under in vivo conditions. Herein we report on the development of a highly serum compatible cationic amphiphile with circulation stable amide linker which shows remarkable selectivity in transfecting mouse lung. We also demonstrate that reversing structural orientation of the amide linker adversely affects both serum compatibility and the lung selective gene transfer property. Dynamic laser light scattering and atomic force microscopic studies revealed smaller average hydrodynamic sizes of the liposomes of transfection efficient lipid than those for the liposomes of transfection incompetent analog (148 ± 1 nm vs 214 ± 4 nm). Average surface potential of the liposomes of transfection competent amphiphiles were found to be significantly higher than that for the liposomes of transfection incompetent analog (10.7 ± 5.4 mV vs 2.8 ± 1.3 mV, respectively). Findings in fluorescence resonance energy transfer and dye entrapment experiments support lower rigidity and higher biomembrane fusogenicity of the liposomes of the transfection efficient amphiphiles. Importantly, cationic lipoplexes of the novel amide-linker based amphiphile exhibited higher mouse lung selective gene transfer properties than DOTAP, one of the widely used commercially available liposomal lung transfection kits. In summary, the present findings demonstrate for the first time that amide linker structural orientation profoundly influences the serum compatibility and lung transfection efficiencies of cationic amphiphiles

Formulation, Pharmacokinetic, and Efficacy Studies of Mannosylated Self-Emulsifying Solid Dispersions of Noscapine.

PURPOSE:To formulate hydroxypropyl methylcellulose-stabilized self-emulsifying solid dispersible carriers of noscapine to enhance oral bioavailability. METHODS:Formulation of noscapine (Nos) self-emulsifying solid dispersible microparticles (SESDs) was afforded by emulsification using an optimized formula of Labrafil M1944, Tween-80, and Labrasol followed by spray-drying with hydroxypropyl methylcellulose (HPMC), with and without mannosamine (Mann-Nos_SESDs and Nos_SESDs respectively); self-microemulsifying liquid dispersions (SMEDDs) with and without mannosamine (Mann-Nos_SMEDDs and Nos_SMEDDs respectively) were also prepared. SMEDDs and SESDs were characterized for size, polydispersity, surface charge, entrapment efficiency, in vitro permeability, in vitro release kinetics, and oral pharmacokinetics in Sprague-Dawley rats (10 mg/kg p.o). The antitumor efficacy of Mann-Nos_SESDs on the basis of chemosensitization to cisplatin (2.0 mg/kg, i.v.) was investigated in a chemorefractory lung tumor Nu/Nu mouse model up to a maximal oral dose of 300 mg/kg. RESULTS:The oil/surfactant/co-surfactant mixture of Labrafil M1944, Tween-80, and Labrasol optimized at weight ratios of 62.8:9.30:27.90% produced stable self-microemulsifying dispersions (SMEDDs) at a SMEDD to water ratio of 1-3:7-9 parts by weight. SMEDDs had hydrodynamic diameters between 231 and 246 nm; surface charges ranged from -16.50 to -18.7 mV; and entrapment efficiencies were between 32 and 35%. SESDs ranged in size between 5.84 and 6.60 μm with surface charges from -10.62 to -12.40 mV and entrapment efficiencies of 30.96±4.66 and 32.05±3.72% (Nos_SESDs and Mann-Nos_SESDs respectively). Mann-Nos_SESDs exhibited saturating uptake across Caco-2 monolayers (Papp = 4.94±0.18 × 10(-6) cm/s), with controlled release of 50% of Nos in 6 hr at pH 6.8 following Higuchi kinetics. Mann-Nos_ SESDs was 40% more bioavailable compared to Nos_SESDs; and was effective in sensitizing H1650 SP cells to Cisplatin in vitro and in an orthotopic lung tumor model of H1650 SP origin. CONCLUSIONS:Mannosylated noscapine self-emulsifying solid dispersions (Mann-Nos_SESDs) are bioavailable and potentiate the antineoplastic effect of cisplatin-based chemotherapy in cisplatin-resistant NSCLC

Design, synthesis of novel lipids as chemical permeation enhancers and development of nanoparticle system for transdermal drug delivery.

In the present study, we designed and developed novel lipids that include (Z)-1-(Octadec-9-en-1-yl)-pyrrolidine (Cy5T), 1, 1-Di-((Z)-octadec-9-en-1-yl)pyrrolidin-1-ium iodide (Cy5), (Z)-1-(Octadec-9-en-1-yl)-piperidine (Cy6T), and 1, 1-Di-((Z)-octadec-9-en-1-yl) piperidin-1-ium iodide (Cy6) to enhance the transdermal permeation of some selected drugs. Firstly, we evaluated the transdermal permeation efficacies of these lipids as chemical permeation enhancers in vehicle formulations for melatonin, ß-estradiol, caffeine, α-MSH, and spantide using franz diffusion cells. Among them Cy5 lipid was determined to be the most efficient by increasing the transdermal permeation of melatonin, ß-estradiol, caffeine, α-MSH, and spantide by 1.5 to 3.26-fold more at the epidermal layer and 1.3 to 2.5-fold more at the dermal layer, in comparison to either NMP or OA. Hence we developed a nanoparticle system (cy5 lipid ethanol drug nanoparticles) to evaluate any further improvement in the drug penetration. Cy5 lipid formed uniformly sized nanoparticles ranging from 150-200 nm depending on the type of drug. Further, Cy5 based nanoparticle system significantly (p<0.05) increased the permeation of all the drugs in comparison to the lipid solution and standard permeation enhancers. There were about 1.54 to 22-fold more of drug retained in the dermis for the Cy5 based nanoparticles compared to OA/NMP standard enhancers and 3.87 to 66.67-fold more than lipid solution. In addition, epifluorescent microscopic analysis in rhodamine-PE permeation studies confirmed the superior permeation enhancement of LEDs (detection of fluorescence up to skin depth of 340 μm) more than lipid solution, which revealed fluorescence up to skin depth of only 260 μm. In summary the present findings demonstrate that i) cationic lipid with 5 membered amine heterocyclic ring has higher permeating efficacy than the 6 membered amine hertocyclic ring. ii) The nanoparticle system prepared with Cy5 showed significant (p<0.05) increase in the permeation of the drugs than the control penetration enhancers, oleic acid and NMP

Enhanced Spacer Length between Mannose Mimicking Shikimoyl and Quinoyl Headgroups and Hydrophobic Region of Cationic Amphiphile Increases Efficiency of Dendritic Cell Based DNA Vaccination: A Structure–Activity Investigation

In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl headgroup are promising DNA vaccine carriers for dendritic cell (DC) transfection. The present structure–activity study reports on the influence of spacer length (between mannose-mimicking headgroups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57BL/6J mice under prophylactic settings

Cationic amphiphiles with fatty acyl chain asymmetry of coconut oil deliver genes selectively to mouse lung

Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhancing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics 2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. Herein, we demonstrate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent

Mannosylated microparticle retaines 50% of oral noscapine in 6 hours.

<p>(a) The dissolution rate and release of total noscapine from HPMC-coated spray-dried Nos_SMEDD and Mann-Nos_SMEDD are shown in simulated gastric (pH 2.0) and intestinal (pH 6.8) buffers from 5 min to 24 hr. Results were presented as mean percent of Nos release vs time. (b) Kinetic release of Nos_SMEDD and Mann-Nos_SMEDD showing Higuchi relationship of drug amount released from solid microparticle into dissolution buffer against square root of time. Straight line equation obtained by linear regression. Both formulations were linear with R² ranging between 0.9905 and 0.9968. Data represents 6 replicates of experiments repeated 2× and differences were not considered significant.</p

Mannosylated microparticle saturates noscapine transcytosis.

<p>Transport of Nos formulations across Caco-2 monolayers. (a) Absorptive apparent permeability (Papp) of Nos solution (2.50±0.17 × 10⁻⁶ cm/s), Nos_SMEDD (3.43±0.13 × 10⁻⁶ cm/s), and Mann-Nos_SMEDD (4.94±0.18 × 10⁻⁶ cm/s). (b) Concentration vs time plot of Nos solution and Mann-Nos_SMEDD across Caco-2 monolayers showing linear pseudo zero order transport and apparent first order transport, respectively. (c) Secretory apparent permeability (Papp) of Nos solution (13.66±1.01 × 10–6 cm/s), Nos_SMEDD (5.50±0.65 × 10–6 cm/s), and Mann-Nos_SMEDD (1.03±0.15 × 10–6 cm/s). (d) Efflux ratios of total Nos from Nos solution, Nos_SMEDD, and Mann-Nos_SMEDD. Efflux ratios were estimated by dividing the secretory apparent permeabilities by the corresponding absorptive apparent permeability for each formulation. Results are presented as means ± SD. Results are from triplicates of at least two experiments per group. Differences in means were analyzed by unpaired t-test; P<0.05 were considered significant (***P < 0.0001, vs Nos solution; ^###P < 0.0001, vs Nos_SMEDD).</p

Mannosylated noscapine microparticles enhance cisplatin sensitivity in NSCLC.

<p>Panel displays anti-tumor effects of Nos-SMEDD and Mann-Nos_SMEDD in nu/nu mice bearing orthotopic lung tumors, alone and in combination with Cisplatin. Fig 5a shows the effect of oral noscapine and the solid microparticle. Fig 5b shows the effect of mannosylation over the solid microparticle. Fig 5c shows dose sensitivity of noscapine. Fig 5d shows the effect of the mannosylated microparticle on cisplatin response,and Fig 5e shows potential toxicity of treatment with respect to mouse body weight. Results are presented at percent means ± SD normalized against untreated control group (n = 4 per group).Differences in means were analyzed by One-way analysis of variance (P<0.05), followed by Bonferroni post-test with 90% confidence intervals (P<0.1). Significance was reached at *P<0.1, **P<0.01, and ***P<0.001.</p