[Show abstract][Hide abstract] ABSTRACT: Gene delivery into cells offers opportunities to treat various human genetic diseases. Effective gene delivery is dependent on its stability and ability to transfect across cells. DNA is susceptible to enzymatic degradation and its negatively charge are barriers towards successful transfection. DNA has to be protected from degradation and neutralised. Non-viral vectors are preferred carrier systems, therefore, the use of cyclodextrins with Pluronic®-F127 and folic acid at different concentrations to stabilise the formulation was investigated. Formulations were characterised in fresh and freeze dried forms. DNA stability in formulations was tested by determining the stability of DNA against enzymatic degradation. Degree of DNA inclusion into cyclodextrins was investigated using fluorescence spectroscopy. Thermal behaviour was studied using Differential Scanning Calorimetry (DSC). Incorporation of Pluronic®-F127 produced most stable formulations regarding enzymatic degradation. These formulations show high percentage inclusion. Shift of peaks in FTIR data, appearance of uniform particulate as detected by SEM and changing in the denaturation temperature as demonstrated by DSC data for Pluronic®-F127 containing formulations confirm clear interaction between Pluronic®-F127 and cyclodextrin/ DNA complex. It was noted that γ-cyclodextrin provide better protection and inclusion compared to β-cyclodextrin. Pluronic®-F127 with cyclodextrins is a promising combination to improve stability.
Current Pharmaceutical Biotechnology 08/2014; 15(8). DOI:10.2174/1389201015666140827102134 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We test the hypothesis that anesthesia, measured as pain scores, induced by a novel topical anesthetic putty is non-inferior (margin=1.3) to that provided by conventional lidocaine infiltration for the repair of lacerations.
A randomized controlled trial was conducted in the emergency department (ED) of a local hospital. Participants were randomly allocated to receive either infiltration anesthesia or topical anesthetic putty as per the trial protocol. Pain scores were recorded 15 minutes after infiltration and 30 minutes after topical anesthetic putty application. Median pain scores were compared between groups. Wound evaluation scores were conducted after 7 to 10 days and adverse events were monitored for both groups of participants throughout the study.
One hundred and ten participants were enrolled in the study, with 56 receiving infiltration and 54 receiving topical anesthetic putty. The median difference between the pain scores of the 2 groups was 0 (95% confidence interval -1 to 0). There were no substantial differences between the 2 groups in terms of either the wound evaluation scores or the incidence of adverse events.
The novel topical anesthetic putty was not inferior to infiltration with lidocaine with respect to the pain experienced during suturing, and this putty is a feasible alternative to infiltration anesthesia of lacerations in the ED.
Annals of emergency medicine 01/2014; DOI:10.1016/j.annemergmed.2013.12.012 · 4.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present "one-step application" dissolving and hydrogel-forming microneedle arrays (MN) for enhanced delivery of photosensitisers/precursors. MN (280 μm) prepared from 20% w/w poly(methylvinylether/maelic acid) and crosslinked with glycerol by esterification to form hydrogels upon skin insertion, or allowed to dissolve rapidly in skin, were combined with patches containing 19 mg cm(-2) of 5-aminolevulinic acid (ALA) or meso-tetra (N-methyl-4-pyridyl) porphine-tetra-tosylate (TMP) for drug delivery. Both MN types were mechanically-robust, with compression forces of 20.0 N only causing height reductions of 14%. Application forces as low as 8.0 N per array allowed >95% of the MN in each array type to penetrate excised porcine skin, with the MN penetrating to approximately 220 μm. MN significantly enhanced transdermal delivery of ALA and TMP in vitro, with the hydrogel-forming system comparable to the dissolving system for ALA delivery (approximately 3000 nmol cm(-2) over 6 hours), but superior for delivery of the much larger TMP molecule (approximately 14 nmol cm(-2) over 24 hours, compared to 0.15 nmol cm(-2) ). As this technology clearly has potential in enhanced photodynamic therapy of neoplastic skin lesions, we are currently planning animal studies, to be followed by preliminary human evaluations. GMP manufacturing scale-up is ongoing. This article is protected by copyright. All rights reserved.
Photochemistry and Photobiology 11/2013; 90(3). DOI:10.1111/php.12209 · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: No bioadhesive patch-based system is currently marketed. This is despite an extensive number of literature reports on such systems detailing their advantages over conventional pressure sensitive adhesive-based patches in wet environments and describing successful delivery of a diverse array of drug substances. This lack of proprietary bioadhesive patches is largely due to the fact that such systems are exclusively water-based, meaning drying is difficult. In this paper we describe, for the first time, a novel multiple lamination method for production of bioadhesive patches. In contrast to patches produced using a conventional casting approach, which took 48 hours to dry, bioadhesive films prepared using the novel multiple lamination method were dried in 15 min and were folded into formed patches in a further 10 min. Patches prepared by both methods had comparable physicochemical properties. The multiple lamination method allowed supersaturation of 5-aminolevulinic acid to be achieved in formed patch matrices. However, drug release studies were unable to show an advantage for supersaturation with this particular drug, due to its water high solubility. The multiple lamination method allowed greater than 90% of incorporated nicotine to remain within formed patches, in contrast to the 48% achieved for patches prepared using a conventional casting approach. The procedure described here could readily be adapted for automation by industry. Due to the reduced time, energy and ensuing finance now required, this could lead to bioadhesive patch-based drug delivery systems becoming commercially viable. This would, in turn, mean that pathological conditions occurring in wet or moist areas of the body could now be routinely treated by prolonged site-specific drug delivery, as mediated by a commercially produced bioadhesive patch.
Drug Development and Industrial Pharmacy 11/2012; 39(11). DOI:10.3109/03639045.2012.738683 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In search of strategies to develop deeply penetrating agents for use in Photodynamic Therapy (PDT), we have devised a Quantum Dot-Rose Bengal conjugate that is effective at producing singlet oxygen upon two-photon irradiation. The CdSe/ZnS Quantum Dot, with its high two photon absorption cross section, serves as a two-photon absorbing antenna and transfers its excited state energy to the attached photosensitiser which engages with molecular oxygen to produce cytotoxic singlet oxygen. Thus, we were able to excite the photosensitiser indirectly, which has an absorption maximum of 565 nm, with two-photon irradiation at 800 nm. Given the tissue penetration depth of 800 nm light is at least four times greater than 565 nm light, this offers the opportunity to access much deeper-seated tumours than is currently possible with pharmaceutically approved photosensitisers. Furthermore, the attachment of the photosensitiser to the hydrophilic quantum dot improved the aqueous solubility of the photosensitiser by 48
[Show abstract][Hide abstract] ABSTRACT: A combination of light and ultrasound activation of two conventional photosensitising drugs, methylene blue and rose bengal, was shown to generate higher levels of reactive oxygen species (ROS) and lower LD50 values than either light or ultrasound activation alone.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to assess a novel semisolid material as a potential topical drug delivery system for acute laceration. The objectives were to correlate physical characterization data using rheologic studies and to compare with clinical assessment of performance in an emergency department (ED).
Polyvinyl alcohol (PVA) hydrogels, cross-linked using tetrahydroxyborate (THB), were characterized using texture profile analysis. Formulation samples were applied to acute lacerations presented to the ED and factors, such as in vivo residency time and cohesive removal, were assessed.
Viscosity was shown to be related to mechanical characteristics, whereas adhesiveness depended on the THB concentration. Residence in, and clean removal from, lacerations was evaluated on 29 patients. Formulation F3 (10.0% PVA, 2.5% w/w THB) displayed the most appropriate characteristics for clinical use by scoring highest in qualitative assessments. Other formulations exhibited difficulties in application and removal due to excessive adhesiveness. The release of a model local anesthetic drug was proportional to the concentration of drug incorporated, but was not substantially affected by small changes in the formulation constituents.
Using a combination of pharmaceutical evaluation and clinical assessment, it was shown that cross-linked PVA semisolids are a suitable formulation for drug administration to acute lacerations, with potential for induction of anesthesia prior to wound repair.
Academic Emergency Medicine 04/2011; 18(4):333-9. DOI:10.1111/j.1553-2712.2011.01032.x · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Research into the targeting of drug substances to a specific disease site has enjoyed sustained activity for many decades. The reason for such fervent activity is the considerable clinical advantages that can be gained when the delivery system plays a pivotal role in determining where the drug is deposited. When compared to conventional formulations where no such control exists, such as parenteral and oral systems, the sophisticated targeting device can reduce side effects and limit collateral damage to surrounding normal tissue. No more so is this important than in the area of oncology when dose-limiting side effects are often encountered as an ever present difficulty. In this review, the types of colloidal carrier commonly used in targeted drug delivery are discussed, such as gold and polymeric colloids. In particular, the process of attaching targeting capabilities is considered, with reference to antibody technologies used as the targeting motifs. Nanotechnology has brought together a means to carry both a drug and targeting ligand in self-contained constructs and their applications to both clinical therapy and diagnosis are discussed.
Current Nanoscience 11/2010; 6(6):560-570. DOI:10.2174/157341310793348641 · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In photodynamic therapy (PDT) a combination of visible light and a sensitising drug causes the destruction of selected cells. Aminolaevulinic acid (ALA) has been widely used in topical PDT for over 15 years. However, ALA does not possess favourable physicochemical properties for skin penetration. Consequently, the clearance rates for difficult to treat lesions, such as nodular basal cell carcinomas are relatively low. For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into a bioadhesive patch-based system. In vitro penetration studies into excised porcine skin revealed that ALA patches containing relatively high loadings (226.7 micromol cm(-2)) were associated with significantly greater tissue concentrations (70.7 micromol cm(-3)) than patches containing m-ALA (16.3 micromol cm(-3)) or h-ALA (17.4 micromol cm(-3)). ALA was also found to be the most efficient inducer of protoporphyrin (PpIX) fluorescence in mice, in vivo (maximum mean fluorescence: ALA=236.2 a.u., m-ALA=175.1 a.u., h-ALA=193.5 a.u.). However, when the lipophilic hexylester was formulated in a pressure sensitive adhesive (PSA) patch, significantly higher PpIX levels were achieved compared to all bioadhesive systems tested. Of major importance, PSA patches containing relatively low h-ALA loadings induced high PpIX levels, which were localised to the application area. This study has highlighted the importance of rational selection of both the active agent and the delivery system. Bioadhesive preparations containing ALA are ideal for delivery to moist environments; whereas h-ALA-loaded PSA systems may facilitate enhanced delivery to dry areas of skin. In addition, owing to the relatively low loadings of h-ALA required in PSA patches, the costs of clinical PDT may potentially be reduced.
Journal of photochemistry and photobiology. B, Biology 10/2010; 101(1):59-69. DOI:10.1016/j.jphotobiol.2010.06.012 · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminolaevulinic acid (ALA) is known to poorly penetrate into thick lesions, such as nodular basal cell carcinomas. Short chain ALA esters, possessing increased lipophilicity relative to their hydrophilic parent, have previously been shown to be highly efficient at inducing protoporphyrin IX (PpIX) production in cell culture, at equimolar concentrations. In contrast, in vitro skin permeation and in vivo animal studies, which up to now have compared prodrugs on a % w/w basis, have failed to demonstrate such benefits. For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into an o/w cream preparation. In vitro penetration studies into excised porcine skin revealed that increased levels of h-ALA, compared to ALA and m-ALA were found in the upper skin layers, at all drug loadings studied. Topical application of the formulations to nude murine skin in vivo, revealed that creams containing h-ALA induced significantly higher levels of peak PpIX fluorescence (F(max) = 289.0) at low concentrations compared to m-ALA (F(max) = 159.2) and ALA (F(max) = 191.9). Importantly, this study indicates that when compared on an equimolar basis, h-ALA has improved skin penetration, leading to enhanced PpIX production compared to the parent drug and m-ALA.
[Show abstract][Hide abstract] ABSTRACT: The inclusion of chemical penetration enhancers in a novel patch-based system for the delivery of 5-aminolevulinic acid (ALA) was examined in vitro and in vivo. Poor penetration of ALA has been implicated as the primary factor for low response rates achieved with topical ALA-based photodynamic therapy of thicker neoplastic lesions, such as nodular basal cell carcinomas.
Several chemical permeation enhancers (dimethylsulfoxide, Labrafac CC, Labrafac PG and Labrafil M1944CS) were incorporated into bioadhesive patches tailored to deliver 19 mg ALA/cm(2).
In-vitro depth penetration studies into excised porcine skin showed that high concentrations of ALA (>9 micromol/cm(3)) could be delivered to a depth of 1.875 mm. However, inclusion of permeation enhancers did not significantly increase ALA delivery, relative to the control (P > 0.05). In-vivo studies were in strong agreement with in-vitro results, with formulations containing chemical enhancers showing no improvement in delivery compared with the control.
The patches designed in this work are suited to defineable ALA delivery without the need to immobilise patients for up to 6 h, as is common with the cream-under-occlusion approach. Overall, permeation enhancers were not found to markedly enhance the topical delivery of ALA. However, chemical penetration enhancers may have a greater effect on the delivery of more lipophilic ALA prodrugs, which are thought to primarily permeate the stratum corneum via the intercellular pathway.
[Show abstract][Hide abstract] ABSTRACT: In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion-evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells.
[Show abstract][Hide abstract] ABSTRACT: MethodsIn this study we determined, for the first time, the ability of microorganisms to traverse microneedle-induced holes using
two different in vitro models.
ResultsWhen employing Silescol® membranes, the numbers of Candida albicans, Pseudomonas aeruginosa and Staphylococcus epidermidis crossing the membranes were an order of magnitude lower when the membranes were punctured by microneedles rather than a 21G
hypodermic needle. Apart from the movement of C. albicans across hypodermic needle-punctured membranes, where 40.2% of the microbial load on control membranes permeated the barrier
over 24h, the numbers of permeating microorganisms was less than 5% of the original microbial load on control membranes.
Experiments employing excised porcine skin and radiolabelled microorganisms showed that the numbers of microorganisms penetrating
skin beyond the stratum corneum were approximately an order of magnitude greater than the numbers crossing Silescol® membranes in the corresponding experiments.
Approximately 103 cfu of each microorganism adhered to hypodermic needles during insertion. The numbers of microorganisms adhering to MN arrays
were an order of magnitude higher in each case.
ConclusionWe have shown here that microneedle puncture resulted in significantly less microbial penetration than did hypodermic needle
puncture and that no microorganisms crossed the viable epidermis in microneedle—punctured skin, in contrast to needle-punctured
skin. Given the antimicrobial properties of skin, it is, therefore, likely that application of microneedle arrays to skin
in an appropriate manner would not cause either local or systemic infection in normal circumstances in immune-competent patients.
In supporting widespread clinical use of microneedle-based delivery systems, appropriate animal studies are now needed to
conclusively demonstrate this in vivo. Safety in patients will be enhanced by aseptic or sterile manufacture and by fabricating microneedles from self-disabling
materials (e.g. dissolving or biodegradable polymers) to prevent inappropriate or accidental reuse.
Key Wordsinfection-microneedle arrays-microorganisms-
Pharmaceutical Research 11/2009; 26(11):2513-2522. DOI:10.1007/s11095-009-9967-2 · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim was to enhance aminolevulinic acid (ALA) stability by incorporation into low-melting microparticles prepared using a spray congealing procedure and to evaluate temperature-triggered release, allowing topical bioavailability following melting at skin temperature.
ALA-loaded Witepsol microparticles were prepared using a novel spray congealing technique. Entrapment efficiency was compared with conventional emulsion-based methods and modelled drug release profiles determined using a membrane separation technique. Raised receiver medium temperature was used to determine triggered release. Bioavailability and lipid-mediated enhancement of ALA penetration were determined in excised murine skin.
ALA-loaded Witepsol microparticles were spherical, with a mean diameter of 20 mum. Loading and stability studies demonstrated effective encapsulation, ranging from 91% to 100%, with no evidence of degradation to pyrazine derivatives. ALA release correlated with dissolution medium temperature, triggered at temperatures close to that of skin. Results suggested that molten Witepsol enhanced cutaneous permeation, whereas incorporation of microparticles in a semi-solid vehicle attenuated ALA penetration. Optimal use was direct application under occlusion.
Spray congealing is superior to the emulsion-based procedures with respect to encapsulation efficiency of ALA in Witepsol matrices, providing temperature-triggered release, enhanced stability and improved penetration of ALA through keratinised skin. These features could improve ALA delivery to superficial lesions as part of photodynamic therapy.
Journal of Pharmacy and Pharmacology 10/2009; 61(9):1125-35. DOI:10.1211/jpp/61.09.0001 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) using topical 5-aminolevulinic acid (ALA), a water-soluble precursor of the potent endogenous photosensitiser, protoporphyrin IX (PpIX), is a treatment and diagnostic tool for premalignant and malignant skin cancers. However, to improve drug delivery to deeper skin lesions, more lipophilic ALA esters have been investigated. Owing to the necessity in drug delivery research for efficient and validated assays for ALA esters in solution, this paper aims to describe optimised protocols to quantify the methyl and hexyl esters of ALA. ALA esters were derivatised using acetyl acetone and formaldehyde reagents and analysed using reversed phase HPLC. For the first time, the significance of ALA-impurities in ester samples has been highlighted. Furthermore, it was shown that for a given concentration, peak areas obtained for ALA-esters were significantly smaller than those obtained for ALA (p < 0.05). This may be due to parent drug undergoing the derivatisation reaction more efficiently or because the ALA-derivate is inherently more fluorescent than the ester derivatives. The method was optimised to give acceptable intra-and inter-day variability (CV values < 5%) and the limits of detection and quantification were determined for both drugs. The validated methods were used to determine the release profiles of ALA, m-ALA and h-ALA from an o/w cream formulation. The percentage of drug loading released after five hours across a model membrane was in the order of ALA (45.2%) > m-ALA (38.3%) > h-ALA (33.9%). These findings may explain why, historically, some of the benefits seen with ALA-esters using cell culture models have not been demonstrated in vivo.
The Open Analytical Chemistry Journal 08/2009; 3(1):6-15. DOI:10.2174/1874065000903010006
[Show abstract][Hide abstract] ABSTRACT: Poly(vinyl alcohol)-borate complexes were evaluated as a potentially novel drug delivery platform suitable for in vivo use in photodynamic antimicrobial chemotherapy (PACT) of wound infections. An optimised formulation (8.0%w/w PVA, 2.0%w/w borax) was loaded with 1.0 mg ml(-1) of the photosensitisers Methylene Blue (MB) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Both drugs were released to yield receiver compartment concentrations (>5.0 microg ml(-1)) found to be phototoxic to both planktonic and biofilm-grown methicillin-resistant Staphylococcus aureus (MRSA), a common cause of wound infections in hospitals. Newborn calf serum, used to simulate the conditions prevalent in an exuding wound, did not adversely affect the properties of the hydrogels and had no significant effect on the rate of TMP-mediated photodynamic kill of MRSA, despite appreciably reducing the fluence rate of incident light. However, MB-mediated photodynamic kill of MRSA was significantly reduced in the presence of calf serum and when the clinical isolate was grown in a biofilm. Results support the contention that delivery of MB or TMP using gel-type vehicles as part of PACT could make a contribution to the photodynamic eradication of MRSA from infected wounds.
Journal of photochemistry and photobiology. B, Biology 08/2009; 96(3):223-31. DOI:10.1016/j.jphotobiol.2009.06.010 · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: If wound area is to be used as an indicator of healing, then it is vital that all measurements are accurate and consistent. This depends largely on the measurement tool used. This paper offers an insight into the available evidence.
Journal of Wound Care 07/2009; 18(6):250-3. DOI:10.12968/jowc.2009.18.6.42804 · 1.11 Impact Factor