3.2. Method 2
In Method 2, different vessel geometry has been experimented for both “A” and “B” array: in this way, MNs were produced faster with respect to Method 1. Moreover, changing the dose of exposure radiation enabled different lengths and shapes.
The length of MNs and shape type are reported in
Table 2, where the power density of the UV source is fixed to his maximum,
i.e., 18 mW/cm
2, and the exposure time are 5, 7.5, 10, 12.5, 15 s. Moreover, results using both arrays “A” and “B” are reported (
Table 2).
Table 2.
Fabrication parameters and results for Method 2 with power density 18 mW/cm2.
Table 2.
Fabrication parameters and results for Method 2 with power density 18 mW/cm2.
Sample Name | Exposure Time (s) | Array Type | Height (μm) | Shape |
---|
1A2 | 5 | A | 1340 | cone |
2A2 | 7.5 | A | 1430 | lance |
3A2 | 10 | A | 1450 | lance |
4A2 | 12.5 | A | 1700 | lance |
5A2 | 15 | A | 2240 | lance |
1B2 | 5 | B | 1230 | cone |
2B2 | 7.5 | B | 1430 | cone |
3B2 | 10 | B | 1690 | lance |
4B2 | 12.5 | B | 1700 | lance |
5B2 | 15 | B | 2000 | lance |
In
Figure 2, the collected images of MNs (for samples 1A2, 2A2, 3A2, 4A2, 5A2 and 1B2, 2B2, 3B2, 4B2, 5B2) arrays, and also for single elements, are shown.
Figure 2.
(Scale bar is 500 μm) MNs have been removed by the quartz substrate and images have been captured by a standard optical microscope. The labeling follows the sample names indicated in
Table 2. In the insets, images of MNs arrays on the quartz substrate have been captured by the water contact angle analyzer (WCA) camera. (
a) MNs array obtained by mask array “A”; (
b) MNs array obtained by mask array “B”.
Figure 2.
(Scale bar is 500 μm) MNs have been removed by the quartz substrate and images have been captured by a standard optical microscope. The labeling follows the sample names indicated in
Table 2. In the insets, images of MNs arrays on the quartz substrate have been captured by the water contact angle analyzer (WCA) camera. (
a) MNs array obtained by mask array “A”; (
b) MNs array obtained by mask array “B”.
In this geometry, exposure times were considerably lower than the ones used in Method 1 (fixed at 1000 s). The height of MNs ranged between 1200 and 2200 μm and increases as the exposure time increases, and then the dose increases. MNs fabricated with array A (400 μm diameter) were higher than MNs by array B (250 μm) for equal exposure time. However, the “lancet” shape was demonstrated for both arrays, even with a slight increasing of the exposure time. The effect can be explained by considering that the cross-linked PEGDA is denser than the unexposed one, so that also its refractive index is higher. The ultraviolet light, coming from the mask aligner lamp, is thus confined inside the cross-linked PEGDA (as it happens to light confined in an optical fiber core), so that the light intensity is higher than elsewhere inside the forming MNs. In this way, a variety of MNs shapes and lengths can be finely tuned by changing the process parameters, i.e., the power radiation of the lamp and the exposure time. Moreover, the polymerization process induces a typical nanoporosity of the PEGDA matrix, as well as also in other polymers, that is a very important feature for biomedical applications: MNs could be used as a probe to sense human fluids or deliver biochemical substances, previously absorbed inside the tip, after skin or tissue indentation.
In
Figure 3, we report SEM images of sample 1B2, as an example, where the MN surface roughness was estimated of about 1 μm in value (
Figure 3a) and the curvature radius of about 3 μm was measured (
Figure 3b).
Figure 3.
Scanning electron microscopy (SEM) images of a single MN gold coated: (a) the gold was partially removed in order to show the roughness on surface; and (b) the tip of the MN has a curvature radius of about 2 μm.
Figure 3.
Scanning electron microscopy (SEM) images of a single MN gold coated: (a) the gold was partially removed in order to show the roughness on surface; and (b) the tip of the MN has a curvature radius of about 2 μm.
In order to prove the fabrication of smaller MNs, power radiation of the mask aligner UV lamp, at a fixed exposure time (7.5 s), has been decreased. Results are reported in
Table 3 for the MNs family A2 (array A, Method 2) and B2 (array B, Method 2), as examples.
In
Figure 4, the collected images of MNs realized with different power radiation are shown. It is worth noting that optical powers of 15 and 18 mW/cm
2 gave the same result in term of MN length (about 1450 μm, 7.5 s of time exposure) that implies a sort of saturation effect, which limits this kind of fabrication process.
Table 3.
Fabrication Parameters and results for Method 2 by changing the exposure optical power.
Table 3.
Fabrication Parameters and results for Method 2 by changing the exposure optical power.
Sample Name | Power Radiation (mW/cm2) | Array Type | Exposure Time (s) | Height (μm) | Diameter (μm) |
---|
1A2P | 18 | A | 7.5 | 1450 | 4 |
2A2P | 15 | A | 7.5 | 1450 | 15.5 |
3A2P | 12 | A | 7.5 | 1180 | 21.5 |
4A2P | 9 | A | 7.5 | 940 | 18.0 |
5B2P | 9 | B | 7.5 | 210 | 22.6 |
Figure 4.
Images of the MNs tips: red circles underline the tip curvatures; diameter of curvature is indicated. The labeling follows the sample names in
Table 3. 2A2P, 3A2P and 4A2P samples have been exposed through mask “A” at 15, 12 and 9 mW/cm
2 power radiation, respectively (scale bar is 20 μm). 5B2P has been exposed through mask “B” at 9 mW/cm
2 (scale bar is 100 μm). In the insets, images of MNs arrays on the quartz substrate captured by the WCA camera are shown (scale bar is 500 μm).
Figure 4.
Images of the MNs tips: red circles underline the tip curvatures; diameter of curvature is indicated. The labeling follows the sample names in
Table 3. 2A2P, 3A2P and 4A2P samples have been exposed through mask “A” at 15, 12 and 9 mW/cm
2 power radiation, respectively (scale bar is 20 μm). 5B2P has been exposed through mask “B” at 9 mW/cm
2 (scale bar is 100 μm). In the insets, images of MNs arrays on the quartz substrate captured by the WCA camera are shown (scale bar is 500 μm).
The fabrication procedure optimized in Method 2 allows the production of sharp MNs, which can be useful for skin penetration or indentation in sensing applications [
26,
27], and “lancet” shaped MNs, potentially useful in drug delivery and mechanical interlocking of tissues [
28]. Moreover, considering that during the crosslinking process drugs can be expelled from a gel matrix, encapsulation efficiency decreases by using longer UV exposure. Then, a fast process is desirable.
Fabrication processes were firstly developed on quartz support, but same procedures were repeated using a layer of PEGDA covering the quartz surface, in order to fabricate the MNs array on a flexible support. In the photograph reported in
Figure 5, a MNs array on PEGDA support, easily removed from the quartz by tweezers, is shown. The adhesion of the array on the plastic support is perfect and the structure can be stretched and squeezed by fingers without breaking or damaging the MNs.
Figure 5.
Photo of MNs array on poly(ethylene glycol) diacrylate (PEGDA). The structures are bended by fingers without breaking or damaging the MNs.
Figure 5.
Photo of MNs array on poly(ethylene glycol) diacrylate (PEGDA). The structures are bended by fingers without breaking or damaging the MNs.
Since MNs penetration into the skin is a major issue, mechanical properties of MNs have been tested with Parafilm
® layers, used as artificial skin very similar to pig derma, as reported in ref. [
22].
Figure 6 shows the relative indentation hardness of a Parafilm
® layer (a) compared with the indentation hardness of the central point of a single tip (b). The force spectroscopy plot shows a greater slope, and thus a greater hardness of the MN tip with respect to the artificial skin. This result is essential in quantifying the possibility of a MN in tissue penetration [
22].
The penetration tests, as described in ref. [
22], have been performed by using eight-folded Parafilm
® layer.
Figure 7 and
Figure 8 show the result of penetration tests. In particular, photographic images of the first layer, once separated from the others, reversed and photographed in tilted (45°) view (a) and in lateral (90°) view (b) are reported: it is clear that the MNs penetrated the first layer and impressed their shape by molding the surface.
In
Figure 8, top views of the first five layers of the artificial skin are shown. In particular, in
Figure 8b–f, the holes due to the same MN have been imaged from the first to the fifth layer. Since each Parafilm
® layer is 160 μm thick, the MN 1340 μm long penetrates at least 640 μm, proving a good penetration strength. Shorter MNs gave better results in Parafilm
® penetration, whereas higher MNs tend to bend on themselves without breaking (data not shown here).
We also investigated the capability of the PEGDA-MNs to properly swell on exposure to water solution. The water content in MNs was estimated by thermogravimetric analysis (TGA).
Samples were immersed in PBS solution 10 mM, pH 7.4, at 37 °C. At fixed times (24, 48, 72 and 144 h), samples were dried and analyzed.
As shown in
Figure 9, the water content absorbed by MNs array increased up to 3% in weight during the first 48 h, then the effect saturated.
Figure 6.
Atomic force microscope (AFM) relative indentation hardness measurements of a Parafilm® layer (a) and a MN tip (b).
Figure 6.
Atomic force microscope (AFM) relative indentation hardness measurements of a Parafilm® layer (a) and a MN tip (b).
Figure 7.
Water contact angle analyzer (WCA) images of the first Parafilm® layer after reversing: (a) tilted image and (b) lateral view.
Figure 7.
Water contact angle analyzer (WCA) images of the first Parafilm® layer after reversing: (a) tilted image and (b) lateral view.
Figure 8.
Optical microscope images: (a) (the bar is 500 μm) top view in brightfield of the first Parafilm® layer; and (b–f) (the bar is 200 μm) top view in differential interference contrast (DIC) mode of Parafilm® layers 1–5, respectively.
Figure 8.
Optical microscope images: (a) (the bar is 500 μm) top view in brightfield of the first Parafilm® layer; and (b–f) (the bar is 200 μm) top view in differential interference contrast (DIC) mode of Parafilm® layers 1–5, respectively.
Figure 9.
Water loss at 100 °C of microneedles after immersion in buffer solution at fixed time. Experimental points are reported together with their standard deviation.
Figure 9.
Water loss at 100 °C of microneedles after immersion in buffer solution at fixed time. Experimental points are reported together with their standard deviation.
The water absorption caused a weak deformation of MNs shape, as it can be seen in
Figure 10, where a small inclination of MNs can be observed: 89.6°
vs. 92.1° tilt angles before and after immersion, respectively. This result confirmed that the PEGDA based MNs demonstrated a great stability in time as well as in physiologic environment, and could be exploited in continuous monitoring application.
Figure 10.
Picture of microneedles before (a) and after 48 h (b) immersion in buffer solution. We show the MNs normal axis to the base line and tilt angle. The picture and the tilt angle are obtained using DataPhysics Instruments OCA20 (DataPhysics corporation, Filderstadt, Germany).
Figure 10.
Picture of microneedles before (a) and after 48 h (b) immersion in buffer solution. We show the MNs normal axis to the base line and tilt angle. The picture and the tilt angle are obtained using DataPhysics Instruments OCA20 (DataPhysics corporation, Filderstadt, Germany).
Finally, any appreciable degradation of MNs array in terms of mechanical properties after a period of 3–5 days could not be found. In agreement with our results, Browning
et al. [
29] referred that a 10 wt % solution of PEGDA in sterile water exposed at UV light for 6 min had very low degradation after four weeks in terms of swelling ratio and modulus, and higher degradation occurred after 12 weeks during “
in vivo” experiments. However, the proposed MNs are based on strongly cross-linked PEGDA (which means very low water content), as proved by the small water content after 144 h in buffer PBS at 37 °C. Than, the reactivity of PEGDA in proposed MN is lower than in water solution.