Targeted magnetic nanoparticle hyperthermia for the treatment of oral cancer

INTRODUCTION: Patients with oral squamous cell carcinoma currently experience a five year survival rate of approximately 60% with conventional surgical, chemotherapy and radiotherapy treatments. Magnetic hyperthermia offers an alternative treatment method by utilising the heating properties of magnetic nanoparticles to produce thermo-ablation of the tumour site when exposed to an alternating magnetic field. In this study we investigate in vitro if targeted magnetic hyperthermia offers a potential treatment for oral squamous cell carcinoma. MATERIALS AND METHODS: Magnetic iron oxide nanoparticles, with a biocompatible silica coating, were produced and conjugated with antibodies to target integrin αvβ6, a well-characterised oral squamous cell carcinoma biomarker. Utilising the heating properties of the magnetic nanoparticles we exposed them to an alternating magnetic field to produce thermo-ablation of tumour cells either negative for or over-expressing integrin αvβ6. RESULTS: The cell surface biomarker, αvβ6 integrin, was upregulated in tissue biopsies from oral squamous cell carcinoma patients compared to normal tissue. Functionalisation of the silica coating with anti-αvβ6 antibodies enabled direct targeting of the nanoparticles to αvβ6-overexpressing cells and applying thermal therapy significantly increased killing of the targeted tumour cells compared to control cells. CONCLUSION: Combining antibody-targeting magnetic nanoparticles with thermal-ablation offers a promising therapy for the targeted treatment of oral squamous cell carcinoma

Immuno-responsive tissue engineered oral mucosal equivalents containing macrophages

Macrophages play a key role in orchestrating the host immune response towards invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME and to date none have contained primary human macrophages. Here we report the generation of an immuno-competent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of pro-inflammatory cytokine (IL-6, CXCL8 and TNF-α) gene expression and protein secretion as compared to culture as 2D monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate a MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased pro-inflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared to OME alone. Moreover, this pro-inflammatory response was inhibited by pre-treatment with dexamethasone, showing that MDM-OME are also amenable to drug-treatment. Dual-labelled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies

Mucoadhesive electrospun fibre-based technologies for oral medicine

Oral disease greatly affects quality of life, as the mouth is required for a wide range of activities including speech, food and liquid consumption. Treatment of oral disease is greatly limited by the dose forms that are currently available, which suffer from short contact times, poor site specificity, and sensitivity to mechanical stimulation. Mucoadhesive devices prepared using electrospinning offer the potential to address these challenges by allowing unidirectional site-specific drug delivery through intimate contact with the mucosa and with high surface areas to facilitate drug release. This review will discuss the range of electrospun mucoadhesive devices that have recently been reported to address oral inflammatory diseases, pain relief, and infections, as well as new treatments that are likely to be enabled by this technology in the future

A simple rocker-induced mechanical stimulus upregulates mineralization by human osteoprogenitor cells in fibrous scaffolds

Biodegradable electrospun polycaprolactone scaffolds can be used to support bone-forming cells and could fill a thin bony defect, such as in cleft palate. Oscillatory fluid flow has been shown to stimulate bone production in human progenitor cells in monolayer culture. The aim of this study was to examine whether bone matrix production by primary human mesenchymal stem cells from bone marrow or jaw periosteal tissue could be stimulated using oscillatory fluid flow supplied by a standard see-saw rocker. This was investigated for cells in two-dimensional culture and within electrospun polycaprolactone scaffolds. From day 4 of culture onwards, samples were rocked at 45 cycles/min for 1 h/day, 5 days/week (rocking group). Cell viability, calcium deposition, collagen production, alkaline phosphatase activity and vascular endothelial growth factor secretion were evaluated to assess the ability of the cells to undergo bone differentiation and induce vascularisation. Both cell types produced more mineralized tissue when subjected to rocking and supplemented with dexamethasone. Mesenchymal progenitors and primary human mesenchymal stem cells from bone marrow in three-dimensional scaffolds upregulated mineral deposition after rocking culture as assessed by micro-computed tomography and alizarin red staining. Interestingly, vascular endothelial growth factor secretion, which has previously been shown to be mechanically sensitive, was not altered by rocking in this system and was inhibited by dexamethasone. Rocker culture may be a cost effective, simple pretreatment for bone tissue engineering for small defects such as cleft palate

Incorporation of lysozyme into a mucoadhesive electrospun patch for rapid protein delivery to the oral mucosa

The delivery of biopharmaceuticals to the oral mucosa offers a range of potential applications including antimicrobial peptides to treat resistant infections, growth factors for tissue regeneration, or as an alternative to injections for systemic delivery. Existing formulations targeting this site are typically non-specific and provide little control over dose. To address this, an electrospun dual-layer mucoadhesive patch was investigated for protein delivery to the oral mucosa. Lysozyme was used as a model antimicrobial protein and incorporated into poly(vinylpyrrolidone)/Eudragit RS100 polymer nanofibers using electrospinning from an ethanol/water mixture. The resulting fibrous membranes released the protein at a clinically desirable rate, reaching 90 ± 13% cumulative release after 2 h. Dual fluorescent fibre labelling and confocal microscopy demonstrated the homogeneity of lysozyme and polymer distribution. High encapsulation efficiency and preservation of enzyme activity were achieved (93.4 ± 7.0% and 96.1 ± 3.3% respectively). The released lysozyme inhibited the growth of the oral bacterium Streptococcus ratti, providing further evidence of retention of biological activity and illustrating a potential application for treating and preventing oral infections. An additional protective poly(caprolactone) backing layer was introduced to promote unidirectional delivery, without loss of enzyme activity, and the resulting dual-layer patches displayed long residence times using an in vitro test, showing that the adhesive properties were maintained. This study demonstrates that the drug delivery system has great potential for the delivery of therapeutic proteins to the oral mucosa

Development and characterisation of in vitro human oral mucosal equivalents derived from immortalised oral keratinocytes.

Tissue engineered oral mucosal equivalents (OME) are being increasingly used to measure toxicity, drug delivery, and to model oral diseases. Current OME are mainly comprised of normal oral keratinocytes (NOK) cultured on top of a normal oral fibroblasts (NOF)-containing matrix. However, the commercial supply of NOK is limited, restricting widespread use of these mucosal models. In addition, NOK suffer from poor longevity and donor-to-donor variability. Therefore, we constructed, characterised and tested the functionality of oral mucosal equivalents based on commercial TERT2-immortalised oral keratinocytes (FNB6) in order to produce a more readily available alternative to NOK-based OME. FNB6 OME cultured at an air-to-liquid interface for 14 days exhibited expression of differentiation markers cytokeratin 13 in the suprabasal layers and cytokeratin 14 in basal layer of the epithelium. Proliferating cells were restricted to the basal epithelium and there was immuno-positive expression of E-cadherin confirming the presence of established cell-to-cell contacts. The histology and expression of these structural markers paralleled those observed in the normal oral mucosa and NOK-based models. Upon stimulation with TNFα & IL-1, FNB6 OME displayed a similar global gene expression profile to NOK-based OME with increased expression of many common pro-inflammatory molecules such as chemokines (CXCL8), cytokines (IL-6) and adhesion molecules (ICAM-1) when analysed by gene array and qPCR. Similarly, pathway analysis showed that both FNB6 and NOK models initiated similar intracellular signalling upon stimulation. Gene expression in FNB6 OME was more consistent than NOK-based OME that suffered from donor variation in response to stimuli. Mucosal equivalents based on immortalised FNB6 cells are accessible, reproducible and will provide an alternative animal experimental system for studying mucosal drug delivery systems, host-pathogen interactions and drug-induced toxicity

Oxygen mapping of melanoma spheroids using small molecule platinum probe and phosphorescence lifetime imaging microscopy

Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application

Corticosteroid delivery using oral mucosa equivalents for the treatment of inflammatory mucosal diseases

Oral lichen planus (OLP) is an immune‐mediated disease of the oral mucosa with idiopathic aetiology. It is frequently treated with topical corticosteroids (applied as gels, mouthwashes, or sprays); however, the mucosal exposure times of topical corticosteroids are short because of removal by the constant flow of saliva and mechanical forces. In this study we used cell monolayers, as well as oral mucosal equivalents (OMEs) containing activated T‐cells, to examine corticosteroid potency and delivery of clobetasol‐17‐propionate from a novel electrospun mucoadhesive patch. The OMEs displayed tight junctions, desmosomes, hemidesmosomes, and an efficient permeability barrier. Following application of corticosteroids to cells cultured as monolayers, the degree of cytotoxicity measured correlated to the level of potency recognized for each corticosteroid; by contrast, OMEs were largely unaffected by corticosteroid treatment. Permeation of clobetasol‐17‐propionate into and through the OMEs was time‐ and dose‐dependent, regardless of whether this corticosteroid was delivered in liquid form or from a mucoadhesive patch, and both liquid‐ and patch‐delivered clobetasol‐17‐propionate significantly reduced the secretion of interleukin‐2 by activated T‐cells. This study confirms that OMEs are more suitable models than cell monolayers for evaluating toxicity and drug delivery. After topical exposure, clobetasol‐17‐propionate accumulated in OMEs at a higher level than betamethasone‐17‐valerate and hydrocortisone‐17‐valerate, and exerted its immunosuppressive actions following application via the patch delivery system, highlighting the efficacy of this mode of drug delivery to treat OLP

Use of a Rho kinase inhibitor to increase human tonsil keratinocyte longevity for three-dimensional, tissue engineered tonsil epithelium equivalents

The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epithelialized dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to the cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner

Mucoadhesive electrospun patch delivery of lidocaine to the oral mucosa and investigation of spatial distribution in tissue using MALDI-mass spectrometry imaging

Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a non-injectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anaesthetics from this system and their uptake by oral tissue has not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fibre patches, drug release, and subsequent uptake and permeation through porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 minutes (0.16 ± 0.04 mg) compared to from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 minutes, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionisation – mass spectrometry imaging (MALDI-MSI) was used to investigate localisation of lidocaine within oral tissue. Lidocaine in solution as well as from the mucoadhesive patch penetrated into buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 minutes. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anaesthetic drug delivery and may reduce the need for injection