A novel set of dialkynoyl analogues of the cationic, gene delivery lipid DOTAP (1) was synthesized. Structure−activity studies demonstrate that replacement of the cis-double bonds of DOTAP with ...triple bonds in varying positions alters both the physical properties of the resultant cationic liposome−DNA complexes and their biological functionalities, both in vitro and in vivo. Particularly, in vivo studies demonstrate that pDNA transfection of mouse lung endothelial cells with lead analogue DS(14-yne)TAP (4):cholesterol lipoplexes exhibits double the transfection level with less associated toxicity relative to the well-established DOTAP:cholesterol system. In fact, 4:cholesterol delivers up to 3 times the dose of pDNA in mice than can be tolerated by DOTAP, leading to nearly 3 times greater marker-gene expression. X-ray diffraction studies suggest that lipoplexes containing analogue 4 display increased stability at physiological temperatures. Our results thus suggest that analogue 4 is a potentially strong candidate for the gene therapy of lung tumors.
The aim of this study was to explore the preventive effect of probiotic supplements on the development of early childhood caries (ECC). We searched the PubMed, Google Scholar and Cochrane databases ...up to January 15, 2021. The authors screened the hits independently for relevance, extracted outcome data and assessed the risk of bias. We performed a random effects meta-analysis to pool and compare the incidence of ECC in children assigned to test or placebo groups, respectively. The authors included nine randomised controlled trials published between 2001 and 2021, involving 2,363 preschool children. We assessed two publications with a moderate risk of bias and seven with high risk of bias. The median caries incidence in the probiotic test groups was 8.5% compared with 17.5% in the placebo groups and this difference was statistically significant (P<0.001). A pooled random effects meta-analysis on caries incidence on subject level showed a small but statistically significant risk difference in favour of the probiotic intervention (-0.05, 95% confidence interval (CI) -0.10, -0.00; P<0.05). The mean difference in caries increment on tooth/surface level was -0.57, (95% CI -0.91, -0.23; P<0.01). In conclusion, we demonstrated a small but statistically significant preventive effect of probiotic supplements on ECC. However, the certainty of this finding was low due to the risk of bias, heterogeneity and inconsistencies across the studies. Further long-term randomised controlled trials with low risk of bias are required in order to answer the research question with a higher certainty.
Novel carbohydrate-based agents for the stabilization of ternary liposome:mu:DNA (LMD) nonviral vector systems are described. LMD vector systems comprise plasmid DNA (pDNA; D,7.5 kb) expressing a ...reporter gene (in this instance β-galactosidase expressing gene) that is precondensed with the adenoviral core peptide μ (mu, M; MRRAHHRRRRASHRRMRGG) and then further packaged by means of DC−Chol:DOPE (3:2; m/m) cationic liposomes. Final optimized lipid:mu:pDNA ratio is typically 12:0.6:1 (w/w/w). We report the synthesis of a series of nine neoglycolipids prepared by coupling completely unprotected sugar monomers or oligomers (mannose, glucose, galactose, glucuronic acid, maltose, lactose, maltotriose, maltotetraose, and maltoheptaose) through their reducing-residue termini to an aminoxy-functionalized cholesterol-based lipid. Characterization of these novel neoglycolipids by 1H NMR reveals that the coupling reaction has a major configurational preference for the β-anomer. Unusually, even mannose coupling results in a neoglycolipid product with a predominantly β-anomeric conformation (>85%). Formulation of neoglycolipids into LMD vector systems by incubation of LMD particles with neoglycolipid micelles results in the formation of a range of potential stabilized-LMD (sLMD) vector systems. Those potential sLMD systems prepared with longer chain neoglycolipids are found to have enhanced stabilities, with respect to aggregation in high ionic strength buffers, and enhanced transfection efficacies in comparison to the transfection properties of the naked first generation LMD vector system (i.e., gene delivery and expression). By contrast, when LMD vector systems are incubated with poly(ethylene glycol) DSPE-PEG micelles, resulting PEG-LMD vector systems are very stable with respect to colloidal instablility and aggregation in high ionic strength buffers and in serum, but are completely refractory to transfection. These data suggest that oligosaccharides could represent an alternative to PEG as a stealth polymer able to stabilize synthetic nonviral vector systems in some fluids but without impairing transfection efficiency. Furthermore, sLMD systems prepared with longer chain neoglycolipids appear to have sufficient useful characteristics to form the basis of viable second-generation LMD vector systems after further development.
The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; TrojeneTM) and DC-Chol/DOPE (60:40, m/m), were investigated by using a ...combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 ± 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K d = 19 ± 3 μM) and DC-Chol/DOPE liposomes (K d = 2 ± 0.5 μM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (ΔH° = −0.5 and −1.7 kcal/mol, respectively) and increasing system entropy (TΔS = 6 ± 0.3 and 6.2 ± 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome−pDNA binding by CD spectroscopy reveals that CDAN/DOPE−pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE−pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.
Positively-charged gene delivery agents, such as cationic liposomes, typically prepared by mixing a cationic lipid and a neutral lipid in a 1 : 1 molar ratio, exhibit a fundamental flaw: on the one ...hand, the charge encourages cell uptake; on the other hand, the charge leads to aggregation in vivo with anionic serum components. We herein report a more phase-stable analogue of the zwitterionic and fusogenic lipid DOPE that allows for the reduction of the cationic lipid component of the liposome from 50 to 9 mol% with almost no apparent loss in transfection activity. This reduction in charge may induce important in vivo stability whilst still imparting high cell uptake and transgene expression.
Graphic
A facile solid-phase methodology for the production of cholesterol-based polyamines useful in mediating nucleic acid delivery for gene therapy is described. The methodology is compatible with ...a range of polyamines producing a library of lipids in excellent yields (>87%) and purity.
We describe the facile, three-step synthesis of an orthogonally protected, pH-sensitive linker (8), based on maleic acid, and report its application to the preparation of a pH-sensitive phospholipid ...(20) for potential use in drug and gene delivery. In addition, we highlight the benefits of our linker over the use of the commercially available cis-aconitic anhydride (4).
An efficient modification of the Fukuyama-Mitsunobu procedure has been developed whereby primary or secondary amines can be synthesized from alkyl alcohols and the corresponding ...nosyl-protected/activated amine. Most importantly, the use of the DTBAD and diphenylpyridinylphosphine, as Mitsunobu reagents, generates reaction by-products that can be easily removed, providing a remarkably clean product mixture. This improved technique was implemented in the synthesis of a complex lipopeptide designed to target alpha9beta1-integrin proteins predominant on upper airway epithelial cells.
One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD ...(liposome:mu:DNA) that was designed for further modular upgrading with tool-kits of chemical components. First-generation LMD systems were prepared from DC-Chol/DOPE cationic liposomes (DC-Chol=3β-N-(N′,N′-dimethylaminoethane)carbamoyl cholesterol, DOPE=dioleoyl-L-α-phosphatidylethanolamine), μ peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide-targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell-surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target α₉β₁-integrin proteins predominant on upper-airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. “Targeted” LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide-targeted, synthetic, nonviral gene delivery to be realised.
The aim of this study was to compare the concentration of salivary immunoglobulin A (IgA) and the selected interleukins (IL)-1β, IL-6, IL-8 and IL-10 in young individuals with presence and ...non-presence of Lactobacillus reuteri in saliva after a three-week intervention with probiotic lozenges. The study group consisted of 47 healthy individuals aged 18-32 years with no clinical signs of oral inflammation. In a randomised, double-blind, placebo-controlled, cross-over trial participants ingested two lozenges per day containing two strains of the probiotic bacterium L. reuteri or placebo lozenges. The intervention and wash-out periods were three weeks. Stimulated and unstimulated whole saliva was collected at baseline and immediately after termination of the intervention periods. The samples were analysed for total protein, salivary IgA and selected cytokines. In this extended analysis, data were collected by analysing baseline and follow-up saliva samples related to ingestion of the probiotic lozenges for the presence of L. reuteri through DNA-extraction, PCR-amplification and gel-electrophoresis. At baseline, 27% of the individuals displayed presence of L. reuteri and 42% were positive immediately after the three-week probiotic intervention. Individuals with presence of L. reuteri in saliva had significantly higher (P<0.05) concentrations of salivary IgA and %IgA/protein at the termination of the probiotic intake compared with non-presence. No differences in the cytokine levels were observed. In conclusion, detectable levels of L. reuteri in saliva coincided with higher concentrations of salivary IgA and %IgA/protein in stimulated whole saliva after the three-week daily intake of probiotic lozenges. Our findings suggest that monitoring the presence of probiotic candidates in the oral environment is important to interpret and understand their possible immune-modulating role in maintaining oral health.
