Anthony P. Raphael

Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research

Multiphoton tomography for in vivo high-resolution multidimensional imaging has been used in clinical investigations and small animal studies. The novel femtosecond laser tomographs have been employed to detect cosmetics and pharmaceutical components in situ as well as to study the interaction of drugs with intratissue cells and the extracellular matrix under physiological conditions. Applications include the intra-tissue accumulation of sunscreen nanoparticles in humans, the monitoring the metabolic status of patients with dermatitis, the biosynthesis of collagen after administration of anti-aging products, and the detection of porphyrins after application of 5-aminolevulinic acid. More than 2000 patients and volunteers in Europe, Australia, and Asia have been investigated with these unique tomographs. In addition, femtosecond laser nanoprocessing microscopes have been employed for targeted delivery and deposition in body organs, optical transfection and optical cleaning of stem cells, as well as for the optical transfer of molecular beacons to track microRNAs. These diverse applications highlight the capacity for multiphoton tomography and femtosecond laser nanoprocessing tools to advance drug delivery research.

The viscoelastic, hyperelastic and scale dependent behaviour of freshly excised individual skin layers

Micro-devices using mechanical means to target skin for improved drug and vaccine delivery have great promise for improved clinical healthcare. Fully realizing this promise requires a greater understanding of key micro-biomechanical properties for each of the different skin layers - that are both the mechanical barriers and biological targets of these devices. Here, we performed atomic force microscopy indentation on a micro-nano scale to quantify separately, in fresh mouse skin, the viscous and elastic behaviour of the stratum corneum, viable epidermis and dermis. By accessing each layer directly, we examined the response to nanoindentation at sub-cellular and bulk-cellular scale. We found that the dermis showed greatest mechanical stiffness (elastic moduli of 7.33-13.48 MPa for 6.62 μm and 1.90 μm diameter spherical probes respectively). In comparison, the stratum corneum and viable epidermis were weaker at 0.75-1.62 MPa and 0.49-1.51 MPa respectively (again with the lower values resulting from indentations with the large probe 6.62 μm). The living cell layer of the epidermis (viable epidermis) showed greatest viscoelasticity - almost fully relaxing from shallow indentation - whilst the other layers reached a plateau after relaxing by around 40%. With small scale (sub-micron) AFM indentation, we directly determined the effects of different layer constituents - in particular, the dermis showed that some indents contacted collagen fibrils and others contacted ground substance/cellular areas. This work has far reaching implications for the design of micro-devices using mechanical means to deliver drugs or vaccines into the skin; providing key characterized mechanical property values for each constituent of the target delivery material.

Microneedle Enhanced Delivery of Cosmeceutically Relevant Peptides in Human Skin

Peptides and proteins play an important role in skin health and well-being. They are also found to contribute to skin aging and melanogenesis. Microneedles have been shown to substantially enhance skin penetration and may offer an effective means of peptide delivery enhancement. The aim of this investigation was to assess the influence of microneedles on the skin penetration of peptides using fluorescence imaging to determine skin distribution. In particular the effect of peptide chain length (3, 4, 5 amino acid chain length) on passive and MN facilitated skin penetration was investigated. Confocal laser scanning microscopy was used to image fluorescence intensity and the area of penetration of fluorescently tagged peptides. Penetration studies were conducted on excised full thickness human skin in Franz type diffusion cells for 1 and 24 hours. A 2 to 22 fold signal improvement in microneedle enhanced delivery of melanostatin, rigin and pal-KTTKS was observed. To our knowledge this is the first description of microneedle enhanced skin permeation studies on these peptides.

Nanocomposite-strengthened dissolving microneedles for improved transdermal delivery to human skin

Delivery of drugs and biomolecules into skin has significant advantages. To achieve this, herein, a nanomaterial-strengthened dissolving microneedle patch for transdermal delivery is reported. The patch comprises thousands of microneedles, which are composed of dissolving polymers, nanomaterials, and drug/biomolecules in their interior. With the addition of nanomaterials, the mechanical property of generally weak dissolving polymers can be dramatically improved without sacrificing dissolution rate within skin. In this experiments, layered double hydroxides (LDH) nanoparticles are incorporated into sodium carboxymethylcellulose (CMC) to form a nanocomposite. The results show that, by adding 5 wt% of LDH nanoparticles into CMC, the mechanical strength significantly increased. Small and densely packed CMC-LDH microneedles penetrate human and pig skin more reliably than pure CMC ones and attractively the nanocomposite-strengthened microneedles dissolve in skin and release payload within only 1 min. Finally, the application of using the nanocomposite-strengthened microneedle arrays is tested for in vivo vaccine delivery and the results show that significantly stronger antibody response could be induced when compared with subcutaneous injection. These data suggest that nanomaterials could be useful for fabricating densely packed and small polymer microneedles that have robust mechanical properties and rapid dissolution rates and therefore potential use in clinical applications.

Depth-resolved characterization of diffusion properties within and across minimally-perturbed skin layers

We examine by both experimental and computational means the diffusion of macromolecules through the skin strata (both the epidermis and dermis). Using mouse skin as a test case, we present a novel high-resolution technique to characterize the diffusion properties of heterogeneous biomaterials using 3D imaging of fluorescent probes, precisely-deposited in minimally-perturbed in vivo skin layers. We find the diffusivity of the delivered macromolecules (70 kDa and 2 MDa rhodamine-dextrans) low within the packed cellular arrangement of the epidermis, while gradually increasing (by ~an order of magnitude) through the dermis--as pores in the fibrillar network enlarge from the papillary to the reticular dermis. Our experimental and computational approaches for investigating the diffusion through skin strata help in the assessment and optimization of controlled delivery of drugs (e.g. vaccines) to specific sites (e.g. antigen presenting cells).

Rapid kinetics to peak serum antibodies is achieved following influenza vaccination by dry-coated densely packed microprojections to skin.

A rapid time to peak serum antibody response following vaccination is particularly important for influenza: the time window between the availability of appropriate antigen and the start of the seasonal epidemic is very short. In this paper, influenza vaccine was delivered to both the epidermis and dermis of mouse skin using densely packed microprojection arrays for vaccination. We found that, after vaccination, around 75% and 90% of the delivered influenza vaccine migrated away from the ear skin within just 2 days and 1 week - respectively. And the time to peak serum antibody response was as early as 2 weeks. This result matches the kinetics achieved by intramuscular injection of liquid vaccine to muscle. Thus, we demonstrate that skin delivery of small vaccine volumes discretely by thousands of densely packed microprojections neither induces delay in kinetics nor interferes with the long-lasting antibody response; compared to conventional intramuscular injection.

Computational characterization of reflectance confocal microscopy features reveals potential for automated photoageing assessment

Skin photoageing results from a combination of factors including ultraviolet (sun) exposure, leading to significant changes in skin morphology and composition. Conventional methods assessing the degree of photoageing, in particular histopathological assessment involve an invasive multistep process. Advances in microscopy have enabled a shift towards non‐invasive in vivo microscopy techniques such as reflectance confocal microscopy (RCM) in this context. Computational image analysis of RCM images has the potential to be of use in the non‐invasive assessment of photoageing. In this report, we computationally characterized a clinical RCM data set from younger and older Caucasians with varying levels of photoageing. We identified several mathematical relationships that related to the degree of photoageing as assessed by conventional scoring approaches (clinical photography, SCINEXA and RCM). Furthermore, by combining the mathematical features into a single computational assessment score, we observed significant correlations with conventional RCM (P < 0.0001) and the other clinical assessment techniques.

Formulations for microprojection/microneedle vaccine delivery: structure, strength and release profiles

To develop novel methods for vaccine delivery, the skin is viewed as a high potential target, due to the abundance of immune cells that reside therein. One method, the use of dissolving microneedle technologies, has the potential to achieve this, with a range of formulations now being employed. Within this paper we assemble a range of methods (including FT-FIR using synchrotron radiation, nanoindentation and skin delivery assays) to systematically examine the effect of key bulking agents/excipients - sugars/polyols - on the material form, structure, strength, failure properties, diffusion and dissolution for dissolving microdevices. We investigated concentrations of mannitol, sucrose, trehalose and sorbitol from 1:1 to 30:1 with carboxymethylcellulose (CMC), although mannitol did not form our micro-structures so was discounted early in the study. The other formulations showed a variety of crystalline (sorbitol) and amorphous (sucrose, trehalose) structures, when investigated using Fourier transform far infra-red (FT-FIR) with synchrotron radiation. The crystalline structures had a higher elastic modulus than the amorphous formulations (8-12GPa compared to 0.05-11GPa), with sorbitol formulations showing a bimodal distribution of results including both amorphous and crystalline behaviour. In skin, diffusion properties were similar among all formulations with dissolution occurring within 5s for our small projection array structures (~100μm in length). Overall, slight variations in formulation can significantly change the ability of our projections to perform their required function, making the choice of bulking/vaccine stabilising agents of great importance for these devices.

The human stratum corneum prevents small gold nanoparticle penetration and their potential toxic metabolic consequences

Nanoparticles are being used inmultiple applications, ranging from biomedical and skin care products (e.g., sunscreen) through to industrial manufacturing processes (e.g., water purification). The increase in exposure has led to multiple reports on nanoparticle penetration and toxicity. However, the correlation between nanoparticle size and its penetration without physical/chemical enhancers through the skin is poorly understood--with studies instead focusing primarily on skin penetration under disrupted conditions. In this paper, we investigate the penetration and metabolic effects of 10 nm, 30 nm, and 60 nm gold nanoparticles within viable excised human skin after 24-hour exposure usingmultiphoton tomograph-fluorescence lifetime imaging microscopy. After 24 hour treatment with the 10, 30, and 60 nm gold nanoparticles, there was no significant penetration detected below the stratum corneum. Furthermore, there were no changes in metabolic output (total NAD(P)H) in the viable epidermis posttreatment correlating with lack of penetration of nanoparticles. These results are significant for estimating topical nanoparticle exposure in humans where other model systems may overestimate the exposure of nanoparticles to the viable epidermis. Our data shows that viable human skin resists permeation of small nanoparticles in a size range that has been reported to penetrate deeply in other skin models.

Zinc oxide nanoparticle removal from wounded human skin

AIM Nanoparticle removal from skin is relevant given the concern over topical nanoparticle toxicity. Zinc oxide nanoparticles (ZnO-NPs) are commonly used in sunscreens and their use is currently debated. This study explores the penetration and removal of ZnO-NPs from injured skin. MATERIALS & METHODS Ex vivo/in vivo human skin was tape-stripped and/or microneedled followed by ZnO-NP application. After 2 h, treated skin was washed three-times using soapy water. Multiphoton tomography assessed the ZnO-NP signal before and after washing. RESULTS Washing once removed over 85 and 83% of ZnO-NP signal from ex vivo intact and tape-stripped skin, respectively (p < 0.05) but only 28% (p = 0.5) was removed from puncture sites. A similar trend was found in vivo with removal of 85 and 93% of ZnO-NP signal from intact and tape-stripped skin, respectively (p < 0.05). CONCLUSION Washing is effective for the removal of ZnO-NPs from superficial layers of intact and tape-stripped skin, but not from puncture wounds.