Stimuli-responsive liposomes for drug delivery.

The ultimate goal of drug delivery is to increase the bioavailability and reduce the toxic side effects of the active pharmaceutical ingredient (API) by releasing them at a specific site of action. In the case of antitumor therapy, association of the therapeutic agent with a carrier system can minimize damage to healthy, nontarget tissues, while limit systemic release and promoting long circulation to enhance uptake at the cancerous site due to the enhanced permeation and retention effect (EPR). Stimuli-responsive systems have become a promising way to deliver and release payloads in a site-selective manner. Potential carrier systems have been derived from a wide variety of materials, including inorganic nanoparticles, lipids, and polymers that have been imbued with stimuli-sensitive properties to accomplish triggered release based on an environmental cue. The unique features in the tumor microenvironment can serve as an endogenous stimulus (pH, redox potential, or unique enzymatic activity) or the locus of an applied external stimulus (heat or light) to trigger the controlled release of API. In liposomal carrier systems triggered release is generally based on the principle of membrane destabilization from local defects within bilayer membranes to effect release of liposome-entrapped drugs. This review focuses on the literature appearing between November 2008-February 2016 that reports new developments in stimuli-sensitive liposomal drug delivery strategies using pH change, enzyme transformation, redox reactions, and photochemical mechanisms of activation. WIREs Nanomed Nanobiotechnol 2017, 9:e1450. doi: 10.1002/wnan.1450 For further resources related to this article, please visit the WIREs website.

Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Preparation and electrochemical behavior of gramicidin-bipolar lipid monolayer membranes supported on gold electrodes.

Gramicidin-containing synthetic bolalipid membranes comprised of 2,2'-di-O-decyl-3,3'-O-1,20-eicosanyl-bis-rac-glycero-1,1'-diphosphocholine (C20BAS) have been synthesized and supported on gold electrodes. Supported membranes were prepared by first depositing a partial bolalipid layer on the electrode using a thioctic acid-modified bolalipid (1'-O-omega-thioctamidetetraethylene glycol-2,2'-di-O-decyl-3,3'-di-O-1,20-eicosanyl-bis-rac-glycero-1-phosphate, SSC20BAS) as an anchoring group, followed by a vesicle fusion step using either pure C20BAS or gramicidin-containing C20BAS (C20BAS-GA) vesicles. The latter configuration was designed to immobilize single, continuously-on channels of gramicidin in the C20BAS membrane. Vesicle deposition to form supported bolalipid monolayer membranes was monitored by impedance spectroscopy and cyclic voltammetry. Impedances were observed to increase with vesicle deposition time. Pretreatment of the impedance electrode with SSC20BAS accelerated the supported monolayer membrane deposition rate. Impedances decreased in a gramicidin concentration-dependent manner when gramicidin was incorporated into the C20BAS membrane. These supported bolalipid membranes are also surprisingly inert to organic solvent exposure (CH(3)CH(2)OH;CH(2)Cl(2)), suggesting that they may serve as robust host matrices for integral membrane protein-based sensors.

Phototriggering of liposomal drug delivery systems.

Over the past several years, photodynamic therapy (PDT) has been approved for the treatment of various cancers. Additional applications of photochemical processes for triggering site-specific drug delivery are in early stages of development at this time. This review focuses on the literature appearing between January 1996-June 2001 that describe new and ongoing studies of phototriggering mechanisms that may ultimately find utility in drug delivery applications.

Chloroaluminum phthalocyanine tetrasulfonate delivered via acid-labile diplasmenylcholine-folate liposomes: intracellular localization and synergistic phototoxicity.

Folate-diplasmenylcholine (1,2-di-O-(Z-1'-hexadecenyl)-sn-glycero-3-phosphocholine; DPPlsC) liposomes have been shown to greatly enhance the potency of water-soluble antitumor agents via a selective folate-mediated uptake and acid-catalyzed endosomal escape mechanism (Rui et al. J. Am. Chem. Soc., 1998; 120:11213--18). This study describes an adaptation of this strategy for the delivery of chloroaluminum phthalocyanine tetrasulfonate ([AlPcS(4)](4-)), a water-soluble sensitizer used in photodynamic therapy, in a binary targeting scheme designed to enhance both its tumor selectivity and phototoxicity. [AlPcS(4)](4-)/DPPlsC:folate liposomes (9.8 microM bulk concentration, 2.5 mM intraliposomal concentration) were substantially more phototoxic to folate-deficient KB cells than 12.5 microM free [AlPcS(4)](4-) after a 30 min irradiation (630-910 nm). Considerable differences in phototoxicity were observed, however, between the commercially-available AlPcS(4)(4-) and an HPLC purified sample of [AlPcS(4)](4-) due to an increased tendency for the latter to aggregate. Experiments with [AlPcS(4)](4-)/DPPC:folate and folate-free [AlPcS(4)](4-)/DPPlsC liposomes (acid-insensitive and non-targeted controls, respectively) showed significantly reduced phototoxicities under the same illumination conditions. Our results imply that higher concentrations of water-soluble sensitizers can be delivered to target cells using the folate receptor-mediated pathway, which can change both the biodistribution and intracellular localization of the sensitizer when acid-labile DPPlsC liposomes are used as the delivery vehicle. Potential advantages of this approach include the use of lower bulk [AlPcS(4)](4-) concentrations, rapid plasma clearance of free [AlPcS(4)](4-), and better phototoxic responses, due to higher intracellular [AlPcS(4)](4-) concentrations combined with reduced collateral photodamage arising from misguided sensitizer accumulation, thereby enhancing the selective phototoxicity of PDT treatments.

Blindness after bilateral neck dissection: case report and review.

The primary objective of this review of the literature is to identify the probable causes of blindness after bilateral radical neck dissections. This case report and literature review also discusses possible preventive measures that may avert this catastrophic outcome. Cases of blindness after bilateral radical neck dissection were identified by an electronic literature search, as well as cross-checking all references of the above-identified papers. Eleven previous cases of blindness after bilateral neck dissection were identified. The most common cause was posterior ischemic optic neuropathy (PION), which was permanent. We present the only case in the literature in which blindness occurred after radical neck dissections separated by a span of 9 years. The cause of blindness in our patient was posterior ischemic optic neuropathy. Contributing factors included anemia, hypotension, and disruption of collateral venous return from the neck.

Synthesis of acid-labile diplasmenyl lipids for drug and gene delivery applications.

The low pH environments characteristic of endosomal compartments and ischemic tissues provide an intrinsic pathway for triggering site-specific contents release from appropriately designed delivery vehicles. Accordingly, research in this group has focused on the design, synthesis and application of novel acid-sensitive lipids that will undergo facile lamellar (L alpha) to hexagonal (HII) phase transitions within these acidic sites. Previously, it has been demonstrated that plasmenylcholine-type lipids have excellent acid hydrolysis and contents release kinetics (Gerasimov et al., Biochim. Biophys. Acta. 1324 (1997) 200-214; Rui et al., J. Am. Chem. Soc. 120 (1998) 11213-11218). This paper describes the synthesis of three new acid sensitive lipids, based on a chiral 1,2-di-O-(1Z',9Z'-octadecadienyl)-sn-glycerol (6) platform, displaying phosphocholine (7), poly(ethyleneoxide) (8), and O-carbamoyl-N-diethylen-etriamine (10) headgroups. Intermediate 6 was obtained in 28% overall yield via a six step synthesis from (S)-(+)-2,2-dimethyl-1,2-dioxolane-4-methanol. Subsequent conversion to the final products was acheived in moderate (7 and 10) to excellent yields (8).

Efficient synthesis of 40- and 48-membered tetraether macrocyclic bisphosphocholines.

An efficient route toward the synthesis of unsaturated (bis-diacetylenic) and saturated 40- and 48-membered macrocyclic biphosphocholines has been developed using 2-phenyl-5-hydroxy-1,3-dioxane as a common glycerol synthon. Ring closure was accomplished using either high-dilution Glaser oxidation of [(Cy3P)2RU==CHPh]Cl2-catalyzed olefin metathesis conditions. Deprotection of benzyl ethers using trimethylsilyl iodide (TMS-I) in the presence of diacetylenic moieties has also been demonstrated for the first time.

Unusual septoplasty complication: Streptococcus viridans endocarditis.

Infection is an infrequently reported complication following septoplasty and septorhinoplasty. Among the recognized but rare infections are toxic shock syndrome, spinal osteomyelitis, meningitis, septic cavernous sinus thrombosis and endocarditis. A high index of suspicion is required to diagnose these infections early and thereby minimize morbidity and mortality. We present a case of endocarditis following septoplasty in a patient who had no identifiable preoperative risk factors but who experienced recurrent fever and chills postoperatively.

Cascade liposomal triggering: light-induced Ca2+ release from diplasmenylcholine liposomes triggers PLA2-catalyzed hydrolysis and contents leakage from DPPC liposomes.

We have previously reported a direct triggering approach [Thompson, D. H., et al. (1996) Biochim. Biophys. Acta 1279, 25-34; Gerasimov, O. V., et al. (1997) Biochim. Biophys. Acta 1324, 200-214] based on the facile degradation of plasmenylcholine and diplasmenylcholine vinyl ether linkages by either photooxidation or low-pH environments. This report describes a novel, cascade-type triggering technique that utilizes liposome photooxidation and contents release to activate an enzyme capable of destabilizing conventional phosphatidylcholine liposomes. Our application of this concept employs a mixture of two different liposome populations, one composed of synthetic diplasmenylcholine (1, 2-dihexadec-1'-enyl-sn-glycero-3-phosphocholine, DPPlsCho) containing Ca2+ as a signaling agent for phospholipase A2 (PLA2) and the second composed of 1, 2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) with encapsulated calcein as the reporter molecule. Bacteriochlorophyll (BChl)-sensitized photorelease of Ca2+ from PLA2-resistant DPPlsCho liposomes activates extravesicular PLA2, thereby promoting catalyzed DPPC hydrolysis in a secondary triggering reaction, leading to calcein release. BChl/DPPlsCho/DHC/DPPE-PEG5000/Ca2+IN (0.5:85:10:5) liposomes can be phototriggered using 800 nm excitation, resulting in Ca2+ release (t50% release = 15 min) that cocatalyzes the release of calcein (t50% release = 40 min) from DPPC liposomes (1.5 mM total lipid in DPPlsCho liposomes, 0.18 mM DPPC, 210 micro M final Ca2+ concentration, 90 units of PLA2/ml, 50 mM calcein, and 36 micro M EDTA). No appreciable calcein release occurs in the absence of either PLA2 or BChl/DPPlsCho/DHC/DPPE-PEG5000/CaIN liposomes. The implications of this cascade triggering technique on drug delivery approaches are briefly discussed.

Spontaneous liposome formation induced by grafted poly(ethylene oxide) layers: theoretical prediction and experimental verification.

Spontaneous liposome formation is predicted in binary mixtures of fluid phase phospholipids and poly(n)ethylene oxide (PEO)-bearing lipids by using single chain mean field theory. The range of stability of the spontaneous liposomes is determined as a function of percentage of PEO-conjugated lipids and polymer molecular weight. These predictions were tested by using cast films of 1, 2-diacyl-sn-glycerophosphocholines (e.g., egg L-alpha-lecithin, 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1, 2-distearoyl-sn-glycero-3-phosphocholine) and 1, 2-dipalmitoyl-sn-glycerophosphatidylethanolamine-PEO conjugates (i.e. , 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxypoly(e thylen e glycol)2000]carboxamide and 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxypoly(e thylen e oxide)5000]carboxamide) that were hydrated above their gel-liquid crystal phase transition temperatures. Particle sizes of the resulting dispersions, analyzed by quasielastic light scattering, solute retention, 31P NMR, and freeze-fracture electron microscopy measurements, confirmed the single chain mean field predictions. These data indicate that thermodynamically stable, unilamellar liposomes are formed spontaneously by simple hydration of fluid phase phospholipid bilayer films containing low molar ratios of PEO-based amphiphiles. They further suggest that the equilibrium size and colloidal properties of fluid phase, PEO-modified liposomes can be predicted by using this theoretical approach. The implication of these results on the design and processing of sterically stabilized liposomes used in drug delivery applications also is described.

Acid-catalyzed plasmenylcholine hydrolysis and its effect on bilayer permeability: a quantitative study.

This laboratory has previously shown (Anderson, V.C. and Thompson, D.H. (1992) Biochim. Biophys. Acta 1109, 33-42; Thompson, D.H., Gerasimov, O.V., Wheeler, J.J., Rui, Y. and Anderson, V.C. (1996) Biochim. Biophys. Acta 1279, 25-34), that plasmenylcholine (1-alk-1'-enyl-2-palmitoyl-sn-glycero-3-phosphocholine; PlsPamCho) liposomes release hydrophilic contents upon photooxidation or acid-catalyzed hydrolysis. We now report the kinetics and chemical mechanism of the acid-catalyzed reaction and its effect on calcein leakage rates. Hydrolysis of the plasmenylcholine vinyl ether linkage generates fatty aldehydes and 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine (lysolipid); HPLC and 1H-NMR experiments establish that the former is readily air-oxidized to fatty acids, while the latter undergoes rapid acid-catalyzed rearrangement to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. Lysolipid formation obeys first order kinetics, yielding observed pseudo-first order rate constants that are pH-dependent. Bimolecular hydrolysis rate constants, k(bi), have also been determined. Calcein release rates from plasmenylcholine liposomes are strongly dependent on both the dihydrocholesterol (DHC) content and the extent of PlsPamCho hydrolysis within the bilayer. DHC-free plasmenylcholine liposomes (38 degrees C, pH 2.5) require < 5% PlsPamCho hydrolysis to effect > 50% calcein release within 10 min. The presence of > or = 25 mol% DHC, however, greatly reduces the observed calcein release rate; nearly 30% PlsPamCho hydrolysis is required to effect 50% calcein release over a 70-min period in 6:4 PlsPamCho/DHC liposomes. Bacteriochlorophyll a-sensitized photooxidation of plasmenylcholine liposomes also produces fatty aldehyde and another intermediate, tentatively described as 1-formyl-2-palmitoyl-sn-glycero-3-phosphocholine, that hydrolyzes to form the 1-hydroxy lysolipid. These results have important implications for the quantitative description of lysolipid effects on membrane permeability and on the design of triggerable liposomes for drug delivery.

Microbiology of otitis externa.

Microbiologic and clinical data from 26 patients with otitis externa were prospectively evaluated. Specimens were processed for aerobic and anaerobic bacteria. Bacterial growth was noted in 23 specimens. A total of 33 aerobic and 2 anaerobic bacteria were recovered. Aerobic bacteria only were isolated in 21 (91%) patients, anaerobic bacteria only in 1 (4%), and mixed aerobic and anaerobic bacteria in 1 (4%). The most common isolates were Pseudomonas aeruginosa (14 instances), Staphylococcus aureus (7), Acinetobacter calcoaceticus (2), Proteus mirabilis (2), Enterococcus faecalis (2), Bacteroides fragilis (1), and Peptostreptococcus magnus (1). One isolate was recovered in 13 (57%) patients, 2 isolates in 8 (35%), and 3 isolates in 2 (9%). These data illustrate the polymicrobial nature of otitis externa in about half of the patients and the role of anaerobic bacteria in 8% of them. Further studies are warranted to evaluate the therapeutic implications of these findings.

Triggerable plasmalogen liposomes: improvement of system efficiency.

A photoactivated liposome release system that is generally applicable for triggered release of encapsulated hydrophilic materials is described. This approach to phototriggered release, derived from the known effects of plasmalogen photooxidation on membrane permeability in whole cells and model membrane systems, relies on producing a lamellar phase change or increase in permeability upon cleaving its constitutive lipids to single-chain surfactants using 630-820 nm light to sensitize the photooxidation of the plasmalogen vinyl ether linkage. Semi-synthetic plasmenylcholine liposomes containing encapsulated calcein and a membrane-bound sensitizer, such as zinc phthalocyanine, tin octabutoxyphthalocyanine, or bacteriochlorophyll a, were prepared by extrusion. Irradiation of air-saturated liposome solutions enhanced membrane permeability toward calcein and Mn2+, and promoted membrane fusion processes compared to non-irradiated or anaerobic controls. Bacteriochlorophyll a sensitization produced the fastest observed photoinitiated release rate from these liposomes (100% calcein release in less than 20 min; 800 nm irradiation at 300 mW); the observed release rate was two orders of magnitude slower for egg lecithin liposomes prepared and irradiated under identical experimental conditions. Liposome aggregation, interlipidic particle formation, and membrane fusion between adjoining liposomes was observed by 31P-NMR, freeze-fracture/freeze-etch TEM, and cryo-TEM as a function of irradiation time. The use of near-infrared sensitizers and the capacity of photolyzed plasmenylcholine liposomes to undergo membrane fusion processes make photodynamic therapy with these liposome-borne sensitizers an attractive adjunct to biochemical targeting methods.

Quantitative bacteriology of tonsils removed from children with tonsillitis hypertrophy and recurrent tonsillitis with and without hypertrophy.

The aerobic and anaerobic bacterial species and their numbers were studied in tonsillar specimens from children who had undergone elective tonsillectomy: 6 patients with recurrent tonsillitis (RT), 9 with recurrent tonsillitis with hypertrophy (RTH), and 8 with obstructive tonsillar hypertrophy (OTH). Mixed flora were present in all tonsils, yielding an average of 6.7 isolates (5.6 aerobic or facultative and 1.1 anaerobic bacteria). The highest recovery rate of organisms per tonsil was in patients with OTH (7.7 per tonsil), compared to 6.3 per tonsil in RT and 5.9 per tonsil in RTH. The predominant aerobic and facultative organisms were Haemophilus influenzae (22 isolates), Neisseria sp (16), Staphylococcus aureus (14), and Eikenella corrodens (14), and the predominant anaerobic bacteria were Fusobacterium sp (8), Bacteroides sp (7), and Prevotella melaninogenica (5). The number of bacteria per gram of tonsillar tissue varied between 10(4) and 10(8). A higher concentration of S aureus and H influenzae was found in hypertrophic tonsils (RTH and OTH) as compared to RT. These findings suggest the presence of an increased bacterial load and supports an etiologic role for H influenzae and S aureus in hypertrophic tonsils with and without inflammation (RTH and OTH). Further studies to elucidate the effect of selective antimicrobial therapy directed at these organisms may offer an alternative management of hypertrophic tonsils.