Keng Wooi Ng

Sustained Polymeric Delivery of Gene Silencing Antisense ODNs, siRNA, DNAzymes and Ribozymes: In Vitro and In Vivo Studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (d,l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

Intestinal Injury and Endotoxemia in Children Undergoing Surgery for Congenital Heart Disease

RATIONALE Children with congenital heart disease are at risk of gut barrier dysfunction and translocation of gut bacterial antigens into the bloodstream. This may contribute to inflammatory activation and organ dysfunction postoperatively. OBJECTIVES To investigate the role of intestinal injury and endotoxemia in the pathogenesis of organ dysfunction after surgery for congenital heart disease. METHODS We analyzed blood levels of intestinal fatty acid binding protein and endotoxin (endotoxin activity assay) alongside global transcriptomic profiling and assays of monocyte endotoxin receptor expression in children undergoing surgery for congenital heart disease. MEASUREMENTS AND MAIN RESULTS Levels of intestinal fatty acid binding protein and endotoxin were greater in children with duct-dependent cardiac lesions. Endotoxemia was associated with severity of vital organ dysfunction and intensive care stay. We identified activation of pathogen-sensing, antigen-processing, and immune-suppressing pathways at the genomic level postoperatively and down-regulation of pathogen-sensing receptors on circulating immune cells. CONCLUSIONS Children undergoing surgery for congenital heart disease are at increased risk of intestinal mucosal injury and endotoxemia. Endotoxin activity correlates with a number of outcome variables in this population, and may be used to guide the use of gut-protective strategies.

Cardioprotective Effects of Insulin How Intensive Insulin Therapy May Benefit Cardiac Surgery Patients

Interest in the effects of insulin on the heart came with the recognition that hyperglycemia in the context of myocardial infarction is associated with increased risks of mortality, congestive heart failure, or cardiogenic shock.1–3 More recently, instigated by research findings on stress hyperglycemia in critical illness, this interest has been extended to the influence of insulin on clinical outcome after cardiac surgery. Even in nondiabetic individuals, stress hyperglycemia commonly occurs as a key metabolic response to critical illness, eg, after surgical trauma. It is recognized as a major pathophysiological feature of organ dysfunction in the critically ill. The condition stems from insulin resistance brought about by dysregulation of key homeostatic processes, which implicates immune/inflammatory, endocrine, and metabolic pathways.4 It has been associated with adverse clinical outcomes, including increased mortality, increased duration of mechanical ventilation, increased intensive care unit (ICU) and hospital stay, and increased risk of infection.5–8 Hyperglycemia in critical illness is managed with exogenous insulin as standard treatment; however, there is considerable disagreement among experts in the field as to what target blood glucose level is optimal for the critically ill patient. Conventionally, the aim of insulin therapy has been to maintain blood glucose levels below the renal threshold, typically 220 mg/dL (12.2 mmol/L). In recent years, some have advocated tight glycemic control (TGC) with intensive insulin therapy (IIT) to normalize blood glucose levels to within the euglycemic range, typically 80 to 110 mg/dL (4.4–6.1 mmol/L). Current evidence on the applicability of TGC to critical illness in general is inconclusive. Although early studies showed that IIT reduced mortality and morbidity in the ICU,9–11 more recent systematic studies and meta-analyses have largely failed to support some or all of these findings, with some suggesting that IIT may increase the risk …

Development of an ex vivo human skin model for intradermal vaccination: tissue viability and Langerhans cell behaviour.

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Distribution of esterase activity in porcine ear skin, and the effects of freezing and heat separation

Porcine ear skin is widely used to study skin permeation and absorption of ester compounds, whose permeation and absorption profiles may be directly influenced by in situ skin esterase activity. Importantly, esterase distribution and activity in porcine ear skin following common protocols of skin handling and storage have not been characterised. Thus, we have compared the distribution and hydrolytic activity of esterases in freshly excised, frozen, heated and explanted porcine ear skin. Using an esterase staining kit, esterase activity was found to be localised in the stratum corneum and viable epidermis. Under frozen storage and a common heating protocol of epidermal sheet separation, esterase staining in the skin visibly diminished. This was confirmed by a quantitative assay using HPLC to monitor the hydrolysis of aspirin, in freshly excised, frozen or heated porcine ear skin. Compared to vehicle-only control, the rate of aspirin hydrolysis was approximately three-fold higher in the presence of freshly excised skin, but no different in the presence of frozen or heated skin. Therefore, frozen and heat-separated porcine ear skin should not be used to study the permeation of ester-containing permeants, in particular co-drugs and pro-drugs, whose hydrolysis or degradation can be modulated by skin esterases.

Metabolic Profiling of Children Undergoing Surgery for Congenital Heart Disease

Objective:Inflammation and metabolism are closely interlinked. Both undergo significant dysregulation following surgery for congenital heart disease, contributing to organ failure and morbidity. In this study, we combined cytokine and metabolic profiling to examine the effect of postoperative tight glycemic control compared with conventional blood glucose management on metabolic and inflammatory outcomes in children undergoing congenital heart surgery. The aim was to evaluate changes in key metabolites following congenital heart surgery and to examine the potential of metabolic profiling for stratifying patients in terms of expected clinical outcomes. Design:Laboratory and clinical study. Setting:University Hospital and Laboratory. Patients:Of 28 children undergoing surgery for congenital heart disease, 15 underwent tight glycemic control postoperatively and 13 were treated conventionally. Interventions:Metabolic profiling of blood plasma was undertaken using proton nuclear magnetic resonance spectroscopy. A panel of metabolites was measured using a curve-fitting algorithm. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. The data were assessed with respect to clinical markers of disease severity (Risk Adjusted Congenital heart surgery score-1, Pediatric Logistic Organ Dysfunction, inotrope score, duration of ventilation and pediatric ICU-free days). Measurements and Main Results:Changes in metabolic and inflammatory profiles were seen over the time course from surgery to recovery, compared with the preoperative state. Tight glycemic control did not significantly alter the response profile. We identified eight metabolites (3-D-hydroxybutyrate, acetone, acetoacetate, citrate, lactate, creatine, creatinine, and alanine) associated with surgical and disease severity. The strength of proinflammatory response, particularly interleukin-8 and interleukin-6 concentrations, inversely correlated with PICU-free days at 28 days. The interleukin-6/interleukin-10 ratio directly correlated with plasma lactate. Conclusions:This is the first report on the metabolic response to cardiac surgery in children. Using nuclear magnetic resonance to monitor the patient journey, we identified metabolites whose concentrations and trajectory appeared to be associated with clinical outcome. Metabolic profiling could be useful for patient stratification and directing investigations of clinical interventions.

Skin deep: the basics of human skin structure and drug penetration

Drug penetration and permeation through the skin are greatly influenced by the structural properties of the skin and the physicochemical properties of the drug. As such, penetration enhancement techniques largely focus on manipulating these two key factors. A great deal of research has focused on the stratum corneum, the primary skin barrier. In this chapter, we describe the structural properties of human skin, its functions, and the basic principles of drug penetration. The lipid composition and structural organisation of the stratum corneum, as well as the pathways of drug permeation, are highlighted. This chapter should provide a basic understanding of these topics and prepare the reader for advanced discussions in the specialist chapters that follow.

Therapeutic and cytotoxic effects of the novel antipsoriasis codrug, naproxyl-dithranol, on HaCaT cells.

A novel topical codrug, naproxyl-dithranol (Nap-DTH), in which dithranol and naproxen are linked via an ester in a 1:1 ratio to form a single chemical entity, was synthesized. The antiproliferative, anti-inflammatory and toxic effects of Nap-DTH were assessed, at the cellular level, using various in vitro methods. Cultured HaCaT keratinocytes were treated with Nap-DTH, and the cellular effects were compared with those of the parent compounds, individually and as a 1:1 mixture of naproxen:dithranol to mimic 1:1 in situ liberation from Nap-DTH. The results demonstrate that Nap-DTH did not modify proliferation and only exhibited slight toxic effects after 24 h at concentrations >21 μM. At a lower concentration (3.4 μM), Nap-DTH did not alter cell proliferation or inflammation, which suggests that the codrug is therapeutically inert. Relating to this, the 1:1 mixture of naproxen:dithranol exhibited the lowest toxic effect and the highest antiproliferative effect on HaCaT keratinocytes compared to dithranol at the same concentration. Moreover, the 1:1 mixture exhibited a reduced inflammatory effect compared to dithranol alone, as reflected by the upregulation of cyclooxygenase-2 by 45% and 136%, respectively. In spite of the 1:1 mixture showing a greater downregulation of Ki-67 and a 2-fold reduction of proliferating cell nuclear antigen (both cellular markers of proliferation) than dithranol, dithranol showed a much greater induction of cleaved caspase-3 protein expression (upregulated by 287%, compared to 85% for the 1:1 mixture). This suggests that when dithranol was administered with naproxen, inhibition of cell growth plays a more important role in the antiproliferation effects than the induction of apoptotic cell death. These results confirm that the codrug would lead to a better therapeutic profile and fewer adverse effects compared to its parent compounds.

Penetration Enhancement of Topical Formulations

This special issue, which is entitled “Penetration Enhancement of Topical Formulations”, presents a selection of the latest research that elucidates the challenges facing topical formulations for human skin in addition to proposing interesting solutions.[…].

Synthesis of thiolated, PEGylated and POZylated silica nanoparticles and evaluation of their retention on rat intestinal mucosa in vitro

&NA; In this study, we synthesised thiolated silica nanoparticles using 3‐mercaptopropyltrimethoxysilane and functionalised them with either 5 kDa methoxy polyethylene glycol maleimide (PEG) or 5 kDa alkyne‐terminated poly(2‐ethyl‐2‐oxazoline) (POZ). The main objectives of this study are to investigate the effects of pH on the size and &xgr;‐potential of these nanoparticles and evaluate their mucoadhesive properties ex vivo using rat intestinal mucosa. The sizes of thiolated, PEGylated and POZylated silica nanoparticles were 53 ± 1, 68 ± 1 and 59 ± 1 nm, respectively. The size of both thiolated and POZylated nanoparticles significantly increased at pH ≤ 2, whereas no size change was observed at pH 2.5–9 for both these two types of nanoparticles. On the other hand, the size of PEGylated nanoparticles did not change over the studied pH range (1.5–9). Moreover, thiolated nanoparticles were more mucoadhesive in the rat small intestine than both PEGylated and POZylated nanoparticles. After 12 cycles of washing (with a total of 20 mL of phosphate buffer solution pH 6.8), a significantly greater amount of thiolated nanoparticles remained on the intestinal mucosa than FITC‐dextran (non‐mucoadhesive polymer, p < 0.005) and both PEGylated and POZylated nanoparticles (p < 0.05 both). However, both PEGylated and POZylated nanoparticles showed similar retention to FITC‐dextran (p > 0.1 for both). Thus, this study indicates that thiolated nanoparticles are mucoadhesive, whereas PEGylated and POZylated nanoparticles are non‐mucoadhesive in the ex vivo rat intestinal mucosa model. Each of these nanoparticles has potential applications in mucosal drug delivery. Graphical abstract Figure. No caption available.