Helen O. McCarthy

Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were <100nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

Hydrogel-forming microneedle arrays for enhanced transdermal drug delivery

Unique microneedle arrays prepared from crosslinked polymers, which contain no drug themselves, are described. They rapidly take up skin interstitial fluid upon skin insertion to form continuous, unblockable, hydrogel conduits from attached patch-type drug reservoirs to the dermal microcirculation. Importantly, such microneedles, which can be fabricated in a wide range of patch sizes and microneedle geometries, can be easily sterilized, resist hole closure while in place, and are removed completely intact from the skin. Delivery of macromolecules is no longer limited to what can be loaded into the microneedles themselves and transdermal drug delivery is now controlled by the crosslink density of the hydrogel system rather than the stratum corneum, while electrically modulated delivery is also a unique feature. This technology has the potential to overcome the limitations of conventional microneedle designs and greatly increase the range of the type of drug that is deliverable transdermally, with ensuing benefits for industry, healthcare providers and, ultimately, patients.

Microneedle-mediated intradermal delivery of 5-aminolevulinic acid: potential for enhanced topical photodynamic therapy.

Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA). In this study, silicon microneedle arrays were used, for the first time, to enhance skin penetration of ALA in vitro and in vivo. Puncturing excised murine skin with 6 x 7 arrays of microneedles 270 microm in height, with a diameter of 240 mum at the base and an interspacing of 750 microm led to a significant increase in transdermal delivery of ALA released from a bioadhesive patch containing 19 mg ALA cm(-2). Microneedle puncture enhanced ALA delivery to the upper regions of excised porcine skin but, at mean depths of 1.875 mm, ALA concentrations were similar to control values, possibly reflecting binding of ALA by tissue components. However, and importantly, in vivo experiments using nude mice showed that microneedle puncture could reduce application time and ALA dose required to induce high levels of the photosensitizer protoporphyrin IX in skin. This clearly has implications for clinical practice, as shorter application times would mean improved patient and clinician convenience and also that more patients could be treated in the same session. As ALA is expensive and degrades rapidly via a second order reaction, reducing the required dose is also a notable advantage.

Nitric oxide—A novel therapeutic for cancer

Much research over the past two decades has focussed on understanding the complex interactions of nitric oxide (NO(.)) in both physiological and pathological processes. As with many other aspects of NO(.) biology, its precise role in tumour pathophysiology has been the cause of intense debate and we now know that it participates in numerous signalling pathways that are crucial to the malignant character of cancer. The available experimental evidence highlights contrasting pro- and anti-tumour effects of NO(.) expression, which appear to be reconciled by consideration of the concentrations involved. This review addresses the complexities of the role of NO(.) in cancer, whilst evaluating various experimental approaches to NO(.)-based cancer therapies, including both inhibition of nitric oxide synthases, and overexpression of NO(.) using donor drugs or nitric oxide synthase gene transfer. The evidence provided strongly supports a role for manipulation of tumour NO(.) either as a stand-alone therapy or in combination with conventional treatments to achieve a significant therapeutic gain.

Design and physicochemical characterisation of novel dissolving polymeric microneedle arrays for transdermal delivery of high dose, low molecular weight drugs

We describe formulation and evaluation of novel dissolving polymeric microneedle (MN) arrays for the facilitated delivery of low molecular weight, high dose drugs. Ibuprofen sodium was used as the model here and was successfully formulated at approximately 50% w/w in the dry state using the copolymer poly(methylvinylether/maleic acid). These MNs were robust and effectively penetrated skin in vitro, dissolving rapidly to deliver the incorporated drug. The delivery of 1.5 mg ibuprofen sodium, the theoretical mass of ibuprofen sodium contained within the dry MN alone, was vastly exceeded, indicating extensive delivery of the drug loaded into the baseplates. Indeed in in vitro transdermal delivery studies, approximately 33 mg (90%) of the drug initially loaded into the arrays was delivered over 24 h. Iontophoresis produced no meaningful increase in delivery. Biocompatibility studies and in vivo rat skin tolerance experiments raised no concerns. The blood plasma ibuprofen sodium concentrations achieved in rats (263 μg ml− 1 at the 24 h time point) were approximately 20 times greater than the human therapeutic plasma level. By simplistic extrapolation of average weights from rats to humans, a MN patch design of no greater than 10 cm2 could cautiously be estimated to deliver therapeutically-relevant concentrations of ibuprofen sodium in humans. This work, therefore, represents a significant progression in exploitation of MN for successful transdermal delivery of a much wider range of drugs.

Hydrogel-Forming Microneedles Prepared from ‘‘Super Swelling’’ Polymers Combined with Lyophilised Wafers for Transdermal Drug Delivery

We describe, for the first time, hydrogel-forming microneedle arrays prepared from “super swelling” polymeric compositions. We produced a microneedle formulation with enhanced swelling capabilities from aqueous blends containing 20% w/w Gantrez S-97, 7.5% w/w PEG 10,000 and 3% w/w Na2CO3 and utilised a drug reservoir of a lyophilised wafer-like design. These microneedle-lyophilised wafer compositions were robust and effectively penetrated skin, swelling extensively, but being removed intact. In in vitro delivery experiments across excised neonatal porcine skin, approximately 44 mg of the model high dose small molecule drug ibuprofen sodium was delivered in 24 h, equating to 37% of the loading in the lyophilised reservoir. The super swelling microneedles delivered approximately 1.24 mg of the model protein ovalbumin over 24 h, equivalent to a delivery efficiency of approximately 49%. The integrated microneedle-lyophilised wafer delivery system produced a progressive increase in plasma concentrations of ibuprofen sodium in rats over 6 h, with a maximal concentration of approximately 179 µg/ml achieved in this time. The plasma concentration had fallen to 71±6.7 µg/ml by 24 h. Ovalbumin levels peaked in rat plasma after only 1 hour at 42.36±17.01 ng/ml. Ovalbumin plasma levels then remained almost constant up to 6 h, dropping somewhat at 24 h, when 23.61±4.84 ng/ml was detected. This work represents a significant advancement on conventional microneedle systems, which are presently only suitable for bolus delivery of very potent drugs and vaccines. Once fully developed, such technology may greatly expand the range of drugs that can be delivered transdermally, to the benefit of patients and industry. Accordingly, we are currently progressing towards clinical evaluations with a range of candidate molecules.

Microneedle arrays permit enhanced intradermal delivery of a preformed photosensitizer

Silicon microneedle (MN) arrays were used to puncture excised murine and porcine skin in vitro and transdermal and intradermal delivery of meso‐tetra (N‐methyl‐4‐pyridyl) porphine tetra tosylate (TMP) investigated using topical application of a bioadhesive patch containing 19 mg TMP cm−2. Animal studies, using nude mice, were then conducted to investigate the in vivo performance of the bioadhesive patch following MN puncture of skin. MN puncture significantly enhanced both intradermal and transdermal delivery of TMP in vitro, though the total amounts of drug delivered (25.22% into porcine skin and 0.07% across murine skin) were still quite small in each case. Notwithstanding this, in vivo experiments showed that MN puncture was capable of permitting a prolonged increase in TMP fluorescence at the site of application. Importantly, fluorescence was negligible at distant sites, meaning systemic delivery of the drug was not sufficient to induce TMP accumulation other than at the application site. In this study we have conclusively demonstrated proof of principle; MN puncture allows true intradermal delivery of a preformed photosensitizer in animal skin models in vitro and in vivo. Importantly, transdermal delivery was much reduced in each case. Increasing MN density would allow increased amounts of photosensitizer to be delivered. However, as MNs create aqueous pores in the stratum corneum, a preformed photosensitizer must possess at least some degree of water solubility in order to permit enhanced intradermal delivery in this way. We believe that use of MN array technology in this way has the potential to significantly improve topical photodynamic therapy of skin tumors.

Hypoxia selects for androgen independent LNCaP cells with a more malignant geno- and phenotype

Hypoxia confers resistance to common cancer therapies, however, it has also has been shown to result in genetic alterations which may allow a survival advantage and increase the tumorigenic properties of cancer cells. Additionally, it may exert a selection pressure, allowing expansion of tumor cells with a more aggressive phenotype. To further assess the role of hypoxia in malignant progression in prostate cancer we exposed human androgen dependent prostate cancer cells (LNCaP) to cycles of chronic hypoxia and isolated a subline, LNCaP‐H1. This article describes the partial characterization of this cell line. The LNCaP‐H1 subline showed altered growth characteristics and exhibited androgen independent growth both in vitro and in vivo. Furthermore, these cells were resistant to mitochondrial‐mediated apoptosis, probably since the endogenous levels of Bax was lower and Bcl‐2 higher than in the parental LNCaP cells. Microarray analysis revealed that a complex array of pathways had differential gene expression between the 2 cell lines, with LNCaP‐H1 cells exhibiting a genetic profile which suggests that they may be more likely metastasize to distant organs, especially bone. This was supported by an in vitro invasion assay, and an in vivo metastasis study. This study shows that hypoxia can select for androgen independent prostate cancer cells which have a survival advantage and are more likely to invade and metastasize.

Influence of skin model on in vitro performance of drug-loaded soluble microneedle arrays

A plethora of studies have described the in vitro assessment of dissolving microneedle (MN) arrays for enhanced transdermal drug delivery, utilising a wide variety of model membranes as a representation of the skin barrier. However, to date, no discussion has taken place with regard to the choice of model skin membrane and the impact this may have on the evaluation of MN performance. In this study, we have, for the first time, critically assessed the most common types of in vitro skin permeation models - a synthetic hydrophobic membrane (Silescol(®) of 75 μm) and neonatal porcine skin of definable thickness (300-350 μm and 700-750 μm) - for evaluating the performance of drug loaded dissolving poly (methyl vinyl ether co maleic acid) (PMVE/MA) MN arrays. It was found that the choice of in vitro skin model had a significant effect on the permeation of a wide range of small hydrophilic molecules released from dissolving MNs. For example, when Silescol(®) was used as the model membrane, the cumulative percentage permeation of methylene blue 24h after the application of dissolvable MNs was found to be only approximately 3.7% of the total methylene blue loaded into the MN device. In comparison, when dermatomed and full thickness neonatal porcine skin were used as a skin model, approximately 67.4% and 47.5% of methylene blue loaded into the MN device was delivered across the skin 24h after the application of MN arrays, respectively. The application of methylene blue loaded MN arrays in a rat model in vivo revealed that the extent of MN-mediated percutaneous delivery achieved was most similar to that predicted from the in vitro investigations employing dermatomed neonatal porcine skin (300-350 μm) as the model skin membrane. On the basis of these results, a wider discussion within the MN community will be necessary to standardise the experimental protocols used for the evaluation and comparison of MN devices.

Cathepsin S from both tumor and tumor-associated cells promote cancer growth and neovascularization

Recent murine studies have demonstrated that tumor‐associated macrophages in the tumor microenvironment are a key source of the pro‐tumorigenic cysteine protease, cathepsin S. We now show in a syngeneic colorectal carcinoma murine model that both tumor and tumor‐associated cells contribute cathepsin S to promote neovascularization and tumor growth. Cathepsin S depleted and control colorectal MC38 tumor cell lines were propagated in both wild type C57Bl/6 and cathepsin S null mice to provide stratified depletion of the protease from either the tumor, tumor‐associated host cells, or both. Parallel analysis of these conditions showed that deletion of cathepsin S inhibited tumor growth and development, and revealed a clear contribution of both tumor and tumor‐associated cell derived cathepsin S. The most significant impact on tumor development was obtained when the protease was depleted from both sources. Further characterization revealed that the loss of cathepsin S led to impaired tumor vascularization, which was complemented by a reduction in proliferation and increased apoptosis, consistent with reduced tumor growth. Analysis of cell types showed that in addition to the tumor cells, tumor‐associated macrophages and endothelial cells can produce cathepsin S within the microenvironment. Taken together, these findings clearly highlight a manner by which tumor‐associated cells can positively contribute to developing tumors and highlight cathepsin S as a therapeutic target in cancer.