Micropuncture closure following high density microarray patch application in healthy subjects

Abstract Background The high‐density microarray patch (HD‐MAP) promises to be a robust vaccination platform with clear advantages for future global societal demands for health care management. The method of action has its base not only in efficient delivery of vaccine but also in the reliable induction of a local innate physical inflammatory response to adjuvant the vaccination process. The application process needs to induce levels of reactivity, which are acceptable to the vaccine, and from which the skin promptly recovers. Materials and methods 1 × 1 cm HD‐MAP patches containing 5000, 250‐μm long microprojections were applied to the skin in 12 healthy volunteers. The return of skin barrier function was assessed by transepidermal water loss (TEWL) and reaction to topical histamine challenge. Results Skin barrier recovery by 48 h was confirmed for all HD‐MAP sites by recovered resistance to the effects of topical histamine application. Conclusions Our previous observation, that the barrier disruption indicator TEWL returns to normal by 48 h, is supported by this paper's demonstration of return of skin resistance to topical histamine challenge in twelve healthy subjects.

The HD-MAP, constituted by a non-dissolvable liquid crystal polymer, is a small 1 × 1 cm patch containing 5000 microprojections with a length of 250 μm on to which vaccine is dried (Figure 1). Each microprojection is conically shaped with a diameter of 120 μm at base and 25 μm at the tip and has an approximated penetration depth of 100 μm.
When an HD-MAP is applied to the skin, vaccine is deposited into the skin. This approach has been successfully demonstrated in pre-clinical studies in mice and rats for a wide range of vaccine types including split virion, 3 subunit vaccine, 4 protein conjugate, 5 inactivated virus 6 and DNA. 7 More recently the potential of the HD-MAP has been demonstrated in phase 1 studies with influenza in humans 8,9 and a dengue fever vaccine candidate in mice. 10 The HD-MAP has several key advantages for use in mass vaccination settings such as: vaccines dried onto the HD-MAP are thermostable 9 not requiring refrigeration; they are easy to apply and do not require highly trained medical personal to administer the vaccine; the vaccine is delivered to the layer of the skin rich in antigen presenting cells resulting in a potentially faster and stronger immune response with a fractional dose when compared to needle-based vaccine delivery. 11 The HD-MAP is applied to the skin using an applicator, which accelerates the HD-MAP towards the skin where the microprojections puncture the stratum corneum and deposit the vaccine in the epidermis and upper dermis. The action of the microprojections penetrating the skin results in a regular array of micropunctures affecting the skin barrier. We have recently demonstrated that transepidermal water loss (TEWL) peaks immediately following HD-MAP removal and then subsequently falls to pre-application levels within 48 h following application, indicating that water vapour is no longer able to escape the skin. 12 Increased TEWL is regarded as one of the most important parameters for demonstration of breached skin barrier integrity. 13,14 Decreasing TEWL values indicate return of barrier function towards normal. 15 An alternative method to demonstrate the skin's return of barrier function is offered by observation of the effect of the application of the vasoactive agent histamine. When applied to normal skin, no reaction is observed. If the barrier is defective, itch, flare and wheal are experienced. If vaccination is to be carried out in a resource poor setting, full understanding of the details of the recovery period including the return of practical barrier function is of great interest in regard to application safety as well as appropriate care for the HD-MAP application site following vaccination. In this study, we confirmed by TEWL the adequate HD-MAP application bilaterally to the forearm and deltoid regions. Skin barrier recovery by 48 h was confirmed for both sites by recovered resistance to the effects of topical histamine application.

Study design
Twelve healthy participants without known skin disease were recruited to the study (six females, six males, 18-65 years of age). Written informed consent was obtained from each subject participating. They rested for 30 min prior to measurements to ensure that they had

TEWL
Measurements of water vapour loss from the skin were performed

Polarisation reflectance spectroscopy
Red blood cell concentration (RBC) was indirectly observed using Tissue Viability Imager (TiVi) (TiVi700 2.0 Tissue Viability Imager, WheelsBridge AB, Linköping, Sweden) at 2 min, 24, 48 and 72 h as well as before and after the histamine challenges to normal skin and HD-MAP areas. The technique has previously been described in detail. 16 Image analysis and calculation of TiVi-values were made using Wheels-

Histamine itch test
To

Ethics statement
The study 'Minimally invasive studies of the skin's innate reactivitymicroneedle provocation' was approved by the Linköping ethical board (MB 2017-409-31). The study described has been carried out in accordance with the code of ethics of the world medical association (declaration of Helsinki) for experiments involving humans.

Statistical analysis
TEWL and TiVi data before and after histamine challenge were compared using repeated-measures one-way analysis of variance

Quantitative analysis of trans-epidermal water loss following HD-MAP application
Prior to HD-MAP application, baseline readings of 5.7-11.1 g/hm 2 were observed at both the upper arm and forearm application sites.
Following HD-MAP application, all sites showed an increased TEWL.
The TEWL reading was at its highest in the 2-min measurement with readings ranging between 57.3-100.9 g/hm 2 and 81.2-111.7 g/hm 2 for the forearm and upper arm respectively, confirming successful perturbation of the skin's barrier function. TEWL levels at 24 h were lower, although still above baseline for all sites in all patients ( Figure 2). By 48-h TEWL readings had returned to, and remained at, baseline. The same was observed at the 72-h reading in all patients. The kinetics of water loss from the skin were thus similar to those we have previously reported. 12

Kinetics of return of barrier function after histamine provocation
No urtication was seen at any time in any subject.
As shown in Figure 3A  In our previous study of HD-MAP provocation in normal skin, we had concluded that the skin abrogation had returned to normal by 48 h, the chief evidence being return of TEWL to near normal levels. 12 The present study reports the outcome of the topical application of histamine to the provocation area, a challenge demonstrated to produce no itching, erythema or urtication in normal skin. TEWL is an indirect measurement compared to the direct histamine provocation. There is no universal definition for what constitutes an intact skin barrier. We know from the study of other minimal trauma models that in early healing the barrier can constitute one layer of keratinocytes. 17 How thick or well differentiated a keratinocyte layer needs to be to reach full barrier function is unclear. Although histamine provocation may be a more sensitive test for the demonstration of a completely intact barrier, it can be associated with discomfort, and we feel that evaporimetry is the preferred method for following skin barrier recovery (healing). Histamine being a small molecule is likely to be a 'worst case' example of barrier penetration. The present paper shows that after due consideration of individual subject variability, the two methods are in general agreement and support our original contention of micropuncture closure by 48 h.
The strength of an individual's innate immune reactivity influences the degree of response to minimal trauma such as HD-MAP application. The innate immune response also has an important role in the complex process of wound healing in which barrier restoration, the focus of the present paper, is achieved. [18][19][20] We plan further studies to elucidate reactivity and healing in subgroups of varying age which can be suspected of having decreased reactivity and healing capability.

F I G U R E 4
To assess the possible effects of histamine on the degree of erythema within the high-density microarray patch (HD-MAP) application area, TiVi data before and after histamine challenge were compared using repeated-measures one-way ANOVA, mixed effect analysis, α = 0.05. Asterisks mark significant changes The paper further confirms the broad occurrence of reactivity, which is considered necessary for effective vaccination, and the rapid restoration of barrier function against even a small molecule like histamine demonstrates that although this study was conducted on healthy individuals, the vaccination site, at which a maximal area of 1 cm 2 represents skin perturbation, should not be at extended risk of infection even in individuals with any background disease such as diabetes.

CONCLUSION
Our findings support the contention that micropuncture closure after HD-MAP application occurs in most subjects by 48 h.