Efficient Drug Delivery into Skin Using a Biphasic Dissolvable Microneedle Patch with Water-Insoluble Backing

Abstract

Dissolvable microneedle patches (MNPs) enable simplified delivery of therapeutics via the skin. However, most dissolvable MNPs do not deliver their full drug loading to the skin because only some of the drug is localized in the microneedles (MNs), and the rest remains adhered to the patch backing after removal from the skin. In this work, biphasic dissolvable MNPs are developed by mounting water-soluble MNs on a water-insoluble backing layer. These MNPs enable the drug to be contained in the MNs without migrating into the patch backing due to the inability of the drugs to partition into the hydrophobic backing materials during MNP fabrication. In addition, the insoluble backing is poorly wetted upon MN dissolution in the skin, which significantly reduces drug residue on the MNP backing surface after application. These effects enable a drug delivery efficiency of >90% from the MNPs into the skin 5 min after application. This study shows that the biphasic dissolvable MNPs can facilitate efficient drug delivery to the skin, which can improve the accuracy of drug dosing and reduce drug wastage.

1. Introduction

Access to many drugs is limited by the need for administration by injection, which cannot easily be done by patients. Indeed, seven of the top ten selling drugs require injection. In addition to requiring expertise, hypodermic injection is painful and generates biohazardous sharps waste. Because many drugs are not suitable for oral delivery due to enzymatic degradation and low bioavailability, injection is often the only option available to patients, who must either learn to self-inject or seek out others with injection expertise.

To address this concern, we and others have developed microneedle patches (MNPs), which contain an array of micron-scale needles that carry and release drug into the skin. MNPs can be used as an attractive alternative to injection due to their administration in a minimally invasive manner, simplified self-administration, stimulation of little or no pain, and no generation of biohazardous sharps waste.^[1] MNPs have been used to administer a broad variety of pharmaceuticals ranging from small-molecule drugs,^[2] proteins,^[3] and nucleic acids^[4] to nanomedicines including nanoparticles^[5] and vaccines.^[6] The ability of MNPs to deliver such a diversity of compounds into the skin is achieved by penetrating the outer skin barrier layer of the stratum corneum and dissolving the MNs in the skin’s interstitial fluid to release the loaded agents.^[7]

A common design of MNPs involves solid dissolvable microneedles (MNs), which are made of a mixture of drug and water-soluble excipients that dissolve in the skin upon patch application. MNs are made of different combinations of soluble materials including poly(vinyl alcohol) (PVA),^[8] polyvinylpyrrolidone (PVP),^[9] polysaccharides (e.g., hyaluronic acid^[10] and dextran^[11]), gelatin^[12] and other soluble polymers, as well as small molecular saccharides (e.g., sucrose,^[13] sorbitol,^[14] and trehalose^[3a]), among other materials that are safe for use in the body.

Significant limitations for dissolvable MNPs found in the literature include variable and unreliable drug delivery efficiency (generally 40–80%) and large variability of the drug dose delivered because a significant fraction of the drug can remain in the patch backing without being carried into the skin by the MNs.^[15] These limitations are especially problematic for MNPs because MNPs are often limited to delivery of low drug doses due to the nanoliter volumes of each MN. An unreliable delivery efficiency makes the low dose constraint even more problematic.

This shortcoming is in part due to the way dissolvable MNPs are fabricated, which usually involves casting onto a mold. In some cases, the whole MNP is created by a single casting, which incorporates drug into the MNs as well as the patch backing that supports them. More typically, a two-casting method is used, where a first cast containing the drug fills the mold cavities that form the MNs, and a second, drug-free cast creates the patch backing. However, this approach still usually leads to an undesirable delivery efficiency which is due to uncontrolled drug migration into the patch backing during fabrication.

Prior research has developed methods to improve drug localization in dissolvable MNs. For example, MNPs formulated with highly viscous patch backing materials (e.g., due to their higher concentration or molecular weight, or rapid evaporation of solvent) or with hydrophobic backing materials (i.e., to reduce drug solubility in the backing layer) have been designed to reduce drug diffusion and partitioning from the MNs to the backing.^[16] Some MNPs have been designed with each MN being separated from the patch backing by a tiny air bubble or a porous layer, which not only provides a barrier to drug migration but also affords rapid detachment of MNs when applied to the skin.^[17] Another approach involves arrowhead MNs, in which the drug-loaded MN “arrowheads” are fabricated separately from the patch backing containing supportive shafts that are attached to the MNs at a later step in the manufacturing process.^[18] Moreover, some dissolvable MNPs incorporate drug-loaded biodegradable nanoparticles or vesicles in the MNs instead of loading free drug directly with dissolvable formulations, so that the drug is better localized in the MNs and then released into skin.^[5a,19] All these methods share a common feature of a two-layer or biphasic design in which the drug-loaded MNs are either separated or have significantly different properties from the backing layer of the patch. In the above examples, the layer directly contacting the drug portion of the MNs has viscous or hydrophobic materials, an air gap, or separately fabricated shafts to reduce the drug migration to the backing layer.

Inspired by the two-layer designs for dissolvable MNPs, we sought to develop a simple and reliable method to fabricate MNPs with a high and reliable delivery efficiency by effective sequestration of the drug in the MNs using a conventional two-cast manufacturing method without the difficulties of complex designs and fabrication schemes. To do this, we designed biphasic dissolvable MNPs whose water-soluble MNs were mounted on a water-insoluble backing. Due to the immiscibility of MN and backing materials, the migration of drug from MNs to the base can be significantly reduced. This approach should be especially effective for hydrophilic drugs with poor solubility in the backing material. Moreover, we hypothesized that the hydrophobic nature of the backing layer would also reduce the drug residue on the backing layer after application to the skin. To study drug migration in MNs, fluorescent dyes of different molecular weights were used as model drugs loaded into MNs, and microscopic examination was performed to reveal the dye distribution in the MNPs. To study the impact of backing materials on drug residue on the patch after use, a dipping test was carried out to measure the dye remaining on different backings. Furthermore, we studied ex vivo application of these biphasic MNPs on porcine skin to assess release kinetics and delivery efficiency compared with conventional dissolvable MNPs.

2. Results

2.1. Design of MNP Fabrication Process

We fabricated MNPs following a standard protocol in our lab, which is similar to protocols reported by others (Figure 1A). In this design, the MNP was created by first casting an aqueous solution containing drug and excipients in the polydimethylsiloxane (PDMS) mold cavities to form the MNs, followed by casting another aqueous solution containing excipients (without drug) to form the patch backing. The patch backing contains pedestals on which each MN was mounted. We found that this process resulted in the mixing of the first and second cast solutions, allowed migration of drug from the MN into the patch backing (especially in the pedestal portion), and facilitated adhesion of the MN base to the pedestal portion of the patch backing after insertion into skin, all of which is undesirable for efficient drug delivery to the skin (see data below).

Figure 1.

Design of MNP fabrication process using water-soluble or water-insoluble patch backings. In this design, each MN is attached to the patch backing containing pedestals on which the MNs are mounted. While the overall process of fabrication (mold-filling and drying, step 1–3) and skin application (applying on skin and removal, step 3–5) are the same, the two processes yield MNPs with different properties: A) Water-soluble backing causes significant drug migration from MNs to the backing layer during the drying process (step 3), and it also retains drug residue when removing the patch from the skin (step 5). B) Water-insoluble backing limits drug migration from MNs (step 3), and it also prevents retention of dissolved drug on the pedestals of the patch backing when removing from the skin (step 5).

We, therefore, created a new fabrication process to minimize drug contamination in the patch backing (Figure 1B). In this design, the first casting process is unchanged, but the second cast is performed with an organic solution (without drug). The excipients in the second cast solution had to be changed from water-soluble materials (i.e., PVA, sucrose) to materials soluble in 1,4-dioxane (i.e., polystyrene, PS). Using this approach, the first and second cast solutions were immiscible, which is intended to prevent migration of the hydrophilic drug into the backing layer and prevent adhesion of the MN materials to the hydrophobic patch backing pedestals after hydration in the skin.

2.2. MNP Morphology and Application to the Skin

MNPs prepared using hydrophilic and hydrophobic backings had similar morphology, which was determined by the dimensions of PDMS mold using during fabrication. An intact MNP contained 100 MNs in a 10 × 10 matrix in a square area of ≈0.7 cm × 0.7 cm. Each sharp-tipped MN (length ≈ 600 μm) stood on a wider pedestal (height ≈ 300 μm) that was part of the patch backing. The distance between adjacent MN tips was around 650 μm.

Although having the same shape, MNPs using different backing materials had different dye distribution (Figure 1). Imaging showed that MNP fabricated with a water-soluble PVA/sucrose backing contained model drug (i.e., red-colored sulforhodamine B, SRhB) located in both the MNs and the patch backing (including the pedestals), even though the SRhB was present only in the casting solution used to make the MNs and not the patch backing (Figure 2A,C). In contrast, MNPs made using an insoluble PS backing had red color SRhB seen only in the MNs, with a clear interface between the MNs and pedestals (Figure 2B,D). The MNs were darker red colored due to the localization of SRhB, while the backing remained clear due to the existence of little or no SRhB.

Figure 2.

Morphological examination of MNPs before and after application on porcine skin. Representative images of MNPs containing a 10 × 10 array of MNs with A) a hydrophilic backing made of PVA and sucrose or B) a hydrophobic backing made of PS. Representative side views of two MNs mounted on patch backing pedestals made of C) hydrophilic materials showing red dye in the MNs and the pedestals, or D) hydrophobic materials showing localization of the red dye only in the MNs. E) Representative image of pig skin after MNP application ex vivo and staining of micropores with gentian violet. The MN insertion sites in the skin correspond to the 10 × 10 pattern of MN in the MNP.

All MNPs could successfully penetrate porcine skin ex vivo, leaving an array of stained micropores corresponding to the 10 × 10 pattern found on the MNPs (Figure 2E). When the MNPs were examined under the microscope again after application, significant color difference was observed for MNPs using different backing materials (Figure 2A,B). While the MNPs with soluble backing still presented an intense red color of SRhB, the MNPs with insoluble backing had almost no evidence of residual SRhB after usage. This difference was also confirmed in the magnified images of individual MNs (Figure 2C,D). Both types of MNPs successfully dissolved the MNs in the skin to a similar level, but the MNs using insoluble backing had much less dye residue on the backing layer. This result indicated that MNPs using insoluble backing might possess a better delivery ability for the compounds loaded in the MNs.

2.3. Distribution of Drugs in MNPs

Based on the skin application study, we found that drug distribution in the MNPs may be a key factor influencing the efficiency of drug delivery into the skin. As a next step, we included red dye (SRhB) in the first-cast solution and green dye (fluorescein isothiocyanate-labeled dextran, Dex-FITC) in the second-cast solution. By labeling the two solutions that form the different parts of the MNP, we could better characterize the drug distribution in the MNP and image the possible mixing of materials between the first and second cast solutions (Figure 3). Using fluorescence microscopy imaging, we found that SRhB was broadly spread among the MNs, pedestals, and into the backing when using water-soluble backing materials (Figure 3, left column). We did not, however, see significant migration of Dex-FITC into the MNs, possibly because of its low diffusivity due to its large molecular weight of 70 kDa and because of limited ability to diffuse through the MN due to the solidification of MN when adding the second cast solution.

Figure 3.

Representative fluorescence microscopy images of SRhB-loaded MNs using different backings. The MNs were made using casting solution with SRhB with red fluorescence. The water-soluble backings were prepared using casting solution with Dex-FITC and the insoluble backings were prepared using casting solution with Cou-6, both of which have green fluorescence. MNs are shown with imaging by bright field, with only the red-fluorescence channel showing the location of SRhB, with only the green fluorescence channel showing the location of Dex-FITC or Cou-6, or with both the red- and green-fluorescence channels displayed to show the yellow color representing colocalization. These images show that SRhB diffused significantly into the water-soluble backing (left column) but did not migrate into the insoluble backing (right column). Scale bar = 500 μm.

In contrast, when using the water-insoluble backing, the migration of SRhB was limited such that SRhB was only located in the MNs. The clear interface between soluble MNs and insoluble backing pedestals was observed by fluorescence microscopy (Figure 3, right column). The green dye loaded in the second cast solution was coumarin-6 (Cou-6), with a low molecular weight of 350 Da, and it was effectively sequestered in the MNP pedestals and backing layer.

Although SRhB is water-soluble, its aqueous solution is miscible with many organic solvents, including 1,4-dioxane used in this work, yet it did not diffuse from the MNs into the dioxane solution used for the second cast. The same phenomenon was also observed in solution where SRhB could diffuse into pure dioxane but did not enter dioxane solution containing PS (Figure S1, Supporting Information). We believe that this phenomenon was caused by the phase separation of PS solution at the dioxane/water interface due to the insolubility of PS in water. We hypothesize that a hydrophobic polymer layer formed as a metastable subphase at the solvent/nonsolvent interface,^[20] and this PS-rich layer formed a physical barrier to the penetration of hydrophilic SRhB from the water phase into the dioxane phase. In the case of MNP fabrication, the aqueous casting solution forming the MNs was already generally solidified when applying the second-cast dioxane solution, which means that SRhB had to diffuse across a solid/liquid interface and its diffusion would be further limited. This interface created by the two casting solutions appears to play an important role in the isolation of water-soluble drugs in MNs.

In a further study, we prepared MNPs loaded with two different-sized model drugs to assess the role of drug diffusivity on distribution in the MNP. Examination using confocal laser scanning microscopy (CLSM) showed that MNPs loaded with SRhB (MW = 559 Da) followed the same trend as observed in fluorescence microscopy: while SRhB migrated significantly in the MNPs with water-soluble backing, it strictly localized in the MNs of MNPs made using insoluble backing (Figure 4, panel A; Videos S1,S2, Supporting Information). The yellow-colored region seen in the merged images (Figure 4, bottom row) shows the colocalization of red SRhB and green backing in the pedestal region only when using the water-soluble backing. However, when MNPs were loaded with tetanus toxoid (TT, MW ≈ 150 kDa, with a red-fluorescent label), this macromolecule remained primarily in the MNs in all cases (Figure 4, panel B; Videos S3,S4, Supporting Information), in sharp contrast to what we saw for MNPs with SRhB. The color colocalization still existed for the TT MNPs with soluble backing, but to a much less extent, suggesting that only a small amount of TT migrated to the backing due to its much lower diffusivity compared to SRhB. Close examination also shows that in the green channel of confocal images there was faint green color in the shape of MNs for MNPs with soluble backing, suggesting the migration of soluble backing materials into the MN tips due to miscibility. However, no green color exists for MNPs with insoluble backing, which further supports the separation effect enabled by the biphasic design to differentiate the MNs and backing.

Figure 4.

Representative CLSM images of MNs loaded with SRhB (panel A) or Alexa Fluor 568 labeled tetanus toxoid (TT-AF568) (panel B), showing the cross-sections of MNs and distribution of model drugs along the MNs. A) Using water-soluble backing (left column), SRhB in the MNs and Dex-FITC in the backing were colocalized to reveal the migration of SRhB from MNs to the pedestal region of the backing; while using insoluble backing (right column), a clear interface between MNs and backing was observed to indicate no migration of SRhB into the backing. B) Using soluble backing (left column), colocalization of TT-AF568 and Dex-FITC was not as significant as the case of SRhB, indicating little migration of TT-AF568 to the backing; using insoluble backing (right column), a clear interface was still observed to indicate no migration of TT-AF568 into the backing.

2.4. Drug Residue on Different Backing Materials

The physicochemical properties of the backing materials may also impact drug delivery by affecting the drug residue on used MNPs. We assessed this effect by dipping backings with water-soluble and insoluble backing materials into SRhB aqueous solution. We found that the hydrophilic backing created using the water-soluble materials attracted significantly more dye on its surface compared to the hydrophobic backing made of insoluble materials (Figure 5B). This result correlated with the different surface wettability of the two backings. The soluble PVA/sucrose backing could be easily wetted by water (contact angle = 64.7 ± 4.0°) compared with the hydrophobic PS backing (contact angle = 103.6 ± 0.7°) (Figure 5A). In fact, after contacting water, the PVA/sucrose backing was partially dissolved on the surface, leaving a highly viscous adhesive surface that facilitated the attachment of more drug residue from the solution. This result suggests that the PS backing would be expected to attract much less drug dissolved in the skin from the MNs when applying such MNPs to the skin and contacting the skin’s aqueous interstitial fluid.

Figure 5.

Impact of MNP backing type on the drug residue left on the backing. A) Static water contact angle (n = 6) on insoluble PS film (left) or soluble PVA/ sucrose film (right). B) Representative images comparing SRhB residue on insoluble and soluble MNP backings after dipping into aqueous solutions containing various concentrations of polyacrylic acid (PAA) and sucrose; the insoluble backing had much less SRhB residue on the surface. Scale bar = 5000 μm. C) Quantitative comparison of SRhB residue on insoluble and soluble patch backings after dipping into aqueous solutions of PAA and sucrose (100% level is the SRhB amount on the soluble backing from 15% PAA +30% sucrose). *p < 0.001 (Student’s t-test). Data are shown as mean ± SD, n = 4.

When applying MNPs to the skin, the MNs dissolve in the micropores that they generated upon penetrating the skin. This creates a high viscosity within the micropores initially due to the high concentration of dissolved materials. However, as the dissolved materials diffuse out of the micropores and into the skin, the solute concentration as well as the liquid viscosity in those pores gradually decreases. Therefore, in the dipping test, we evaluated SRhB adherence to MNP backings at three different solute concentrations to simulate the environment of dissolved MNs left in the pores of skin. We found that the soluble backing always attracted large and similar amounts of dye regardless of solution composition, and that the insoluble backing tended to take much less dye (from the highest 45 ± 16% to the lowest 11 ± 3%, compared with those on the soluble backing), especially when the solution was more dilute (Figure 5B,C). This trend indicates that drug release from MNPs with insoluble backing might benefit from extended wearing time as the excipient concentration in the pores decreases, while MNPs with soluble backings might not see improvement of the extent of drug release after leaving the patches for a longer time on the skin.

2.5. Delivery Efficiency in Skin

We finally examined the delivery efficiency of MNPs with different backings in skin. Model drugs with three different sizes, including the small molecule SRhB, peptide insulin (INS), and large protein TT, were each loaded into MNPs and delivered ex vivo by applying the MNPs to porcine skin. The amount of drug delivered to the skin was monitored over 20 min to establish the kinetics of drug delivery from MNPs (Figure 6).

Figure 6.

Kinetics of delivering different-sized model drugs into porcine skin ex vivo using MNPs with water-soluble or insoluble backing: A) SRhB, B) INS, and C) TT. Drug dose in all MNPs was 5 μg per MNP. Data are shown as mean ± SD, n = 4 for SRhB, n = 6 for INS and TT.

For the small molecule SRhB, the MNPs with water-soluble backing only delivered 33.6 ± 2.6% of loaded dye after 20 min, which was significantly lower compared to the 97.6 ± 0.5% delivery by MNPs using an insoluble backing (Figure 6A; two-way t-test, p < 0.001). Additionally, MNPs with insoluble backing quickly reached >95% delivery within only 5 min of wearing on the skin. As for the larger peptide INS, MNPs with soluble backing reached 62.5 ± 15.6% delivery at the end of 20 min, but it was still significantly lower than the 94.7 ± 0.6% delivered by MNPs using insoluble backing (Figure 6B; two-way t-test, p = 0.026). When delivering the large macromolecule TT, the delivery efficiency from MNPs with soluble backing increased to 78.5 ± 1.7%, which was still significantly lower than the 92.8 ± 2.1% delivery achieved by MNPs with insoluble backing (Figure 6C; two-way t-test, p < 0.001). Overall, we saw that MNPs with soluble backing had higher delivery efficiency when administering drugs with higher molecular weight, probably because the lower diffusivity of macromolecules kept them better sequestered in the MNs. In contrast, for MNPs with insoluble backing, the molecular weight of drugs had little impact on the delivery efficiency, and the delivery efficiencies were always higher than that of water-soluble backing MNPs.

3. Discussion

This study addressed several factors affecting the efficiency of drug delivery into the skin from dissolving MNPs, including MNP properties like hydrophilicity of the patch backing, application properties like MNP insertion time in the skin, and drug properties like molecular weight. The delivery efficiency was less of a problem for high molecular weight drugs because larger drug molecules migrated less into the backing layer of MNPs, and therefore most drug localized in the MNs and could be delivered when the MNs dissolved in the skin. However, even for the largest model drug studied (i.e., TT) with the least diffusion out of the MNs, its delivery could not exceed 80% after 20 min. This reduced delivery efficiency may further be explained by drug residue on the patch backing, where the PVA/sucrose backing surface retained dissolved drug after contacting water, due to its high wettability. This limitation cannot be overcome by increasing molecular weight or better localizing drug within the MNs.

When MNPs were fabricated using a water-insoluble backing, they were able to achieve a delivery efficiency that exceeded 90%, and even increased above 95% when delivering the lower molecular weight drugs. This extremely high delivery efficiency is important for a successful MNP drug delivery system, since it can enable more accurate drug dosing, which can be important for achieving good drug efficacy, safety, and side effect profile. In addition, a time frame as short as 5 min was needed for these MNPs to achieve a high delivery (>90% for SRhB and INS, >85% for TT), which means a short MNP wearing time on the skin, and thus may improve compliance of users.

Our biphasic dissolvable MNPs were inspired by two-layer MNP concepts used by other MNP designs in the literature. Like the high viscosity layer formed by the MN casting solution,^[16a] or the air bubble or porous layer to separate MNs and backing,^[17a] or the shafts in arrowhead MNPs,^[18a] or the biodegradable nanoparticles embedded in dissolvable MN matrix,^[5a,19] a water-insoluble backing layer also differentiates from the soluble MNs and limits drug migration.^[16b] A similar concept has also been used to develop long-acting biodegradable MNPs, except the properties of MNs and patch backing were switched. For example, MNs made from water-insoluble poly(lactic-co-glycolic acid) can load hydrophobic drugs and can be mounted on a water-soluble backing.^[21]

This study has limitations. For example, it only considered water-soluble drugs. Amphiphilic or lipophilic drugs might be less diffusible out of MNs and into the patch backing. We also considered only one MN formulation, one water-soluble backing formulation and one insoluble backing formulation, although we expect that the principles learned in this study should be broadly applied to other MNP formulations. We compared MNs with the same geometry while MNs with other shapes might have different behavior. Moreover, delivery efficiency was determined in pig skin ex vivo; future study should examine drug delivery using biphasic MNPs in human skin and in vivo.

4. Conclusion

In this study, we sought to increase drug delivery efficiency from dissolvable MNPs by i) limiting drug diffusion from MNs into the patch backing and ii) reducing drug retention on the patch backing upon dissolution. We accomplished this by developing a biphasic dissolvable MNP that mounted water-soluble MNs on an insoluble PS backing, and compared the performance of this MNP design with a conventional dissolvable MNP design using a water-soluble PVA/sucrose backing. The performance was assessed in terms of the morphology of MNs, drug distribution in MNPs, and wettability of backing materials, as these are factors that influence the drug delivery efficiency of MNPs.

We found that, compared to MNPs with water-soluble backing, those with insoluble backing localized the drug significantly better in the MNs and generated much less drug residue after being applied to the skin. These two effects combined to enable high drug delivery efficiency above 90% within 5 min for a low molecular weight compound (SRhB or INS) and within 20 min for a high molecular weight model drug (TT), thereby enabling a short MNP wear time on the skin. While MNPs with soluble backing had variable delivery efficiency when administering drugs with different molecular weights, the biphasic MNPs with water-insoluble backing showed a high delivery efficiency regardless of the drug molecule size. We conclude that this biphasic MNP design using a water-insoluble backing enables drug delivery to the skin with increased efficiency and speed, which can be valuable to improve drug efficacy, safety, and cost effectiveness of MNP-based drug delivery systems.

5. Experimental Section

Fabrication of Drug-Loaded MNPs:

MNPs were prepared using a two-step molding process on polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) molds with minor modification on an established method.^[22] The first casting solution contained 3% w/v polyacrylic acid (PAA, Polysciences, Warrington, PA), 6% w/v sucrose (Sigma, St. Louis, MO) and 1 mg mL⁻¹ model drug (SRhB, INS or TT), which was prepared by mixing the different components at the desired ratio in deionized (DI) water. This solution was cast on PDMS molds under vacuum to facilitate filling the solution into the mold cavities. After 20 min, excess solution was removed, and the filled molds were centrifuged at 5000 rcf for 20 min to dry the casting solution forced into the mold cavities to form the MNs. The second casting solution was then cast on the filled PDMS molds under vacuum to create the MNP backing, which included pedestals on which each MN was mounted. To achieve MNPs with hydrophilic or water-soluble backing (as a control group), an aqueous solution consisting of 18% w/w PVA 4–88 (EMD Millipore, Billerica, MA) and 18% w/w sucrose was used; while the MNPs with hydrophobic backing were prepared with a casting solution comprised of an organic solution containing 20% w/w PS (Sigma, St. Louis, MO) in 1,4-dioxane. After the second filling, the molds were kept under vacuum for another 3 h to dry the solution at room temperature (20–25 °C), and then further dried at 40 °C overnight before demolding the MNPs using adhesive tape. All MNPs were stored with desiccant at room temperature until use.

Three different model drugs were loaded separately into MNs. SRhB sodium salt (MW = 580 Da, Sigma, St. Louis, MO) was dissolved in DI water at 2 mg mL⁻¹ as the stock solution. Human recombinant INS (MW ≈ 5.8 kDa, Life Technologies, Carlsbad, CA) was dissolved in 0.01 M HCl in DI water at 10 mg mL⁻¹ as the stock solution. TT (MW ≈ 150 kDa, generously provided by Serum Institute of India, Pune, India) stock solution was of 3480 Lf mL⁻¹ or 8.7 mg mL⁻¹ in phosphate buffer. The drug stock solutions were used to make the first cast solution containing 1 mg mL⁻¹ drug which was described above. To load TT, the PAA was partially neutralized in advance by mixing 25% w/v PAA solution with 10 M NaOH at volumetric ratio of 4:1.

In some cases, MNPs were prepared for imaging by fluorescence microscopy using the same method as above, except green fluorescence was added into the backing layer. The water-soluble backing was made using the same PVA/sucrose solution containing 0.4 mg mL⁻¹ Dex-FITC (MW ≈ 70 kDa, Sigma, St. Louis, MO), while the water-insoluble backing was made from the same PS solution containing 0.005 mg mL⁻¹ coumarin-6 (Cou-6, MW = 350 Da, Sigma, St. Louis, MO).

Examination of MNPs and their Application on Skin:

MNPs with either water-soluble or insoluble backing were examined under a microscope (Olympus SZX16, Tokyo, Japan) after fabrication. To assess their ability to be inserted into skin was examined by applying the MNPs on porcine skin. The porcine skin was thawed at room temperature from frozen storage at −80 °C, and it was cleaned by shaving the hairs on the surface and removing the fat beneath the skin. MNPs were pressed against the skin by thumb for 30 s and were then left in place for additional 20 min (unless otherwise indicated). After removal of MNPs, 1% gentian violet solution (Humco, Texarkana, TX) was dropped on the insertion site on the skin to stain the micropores generated by the MNP. Both the used MNPs and stained skin were examined again under the microscope to assess the successful insertion of MNs.

Examination of Drug Distribution in MNs:

The distribution of SRhB in SRhB-loaded MNs was examined by fluorescent microscopy. SRhB loaded in the first casting solution was the tracer in MNs. Dex-FITC or Cou-6 loaded in the second casting solution was the tracer of water-soluble or insoluble backing, respectively. A single row of MNs was cut from each MNP and mounted horizontally under a fluorescence microscope for observation.

The distribution of drugs (SRhB or TT) in MNs was examined and compared by two-photon CLSM. While the small molecule, SRhB, itself could be directly seen as the tracer, macromolecular TT was labeled with Alexa Fluor 568 succinimidyl ester (Thermo Fisher, Waltham, MA) to obtain TT-AF568 as the tracer. The CLSM was performed by a Zeiss 710 confocal scan head mounted on an AxioObserver Z1 inverted microscope with a motorized stage and automatic focus control (LSM 710 NLO, Carl Zeiss AG, Jena, Germany). For the multi-photon imaging, the microscope was coupled with a multi-photon excitation source provided by a Chameleon titanium/sapphire pulsed infrared laser (Coherent, Santa Clara, CA). The dyes in the MNs and backings were simultaneously excited by the chameleon laser at 700 nm, and the emission signals were collected by a Zeiss objective (5X/0.16 NA Plan-Apochromat), BP 500–550 IR and BP 550–690 IR filters, and a photomultiplier tube detector. ZEN 2.6 software (Carl Zeiss) was used for image acquisition and processing. Laser power and pinhole threshold were kept constant between all samples while the gain was minimally adjusted to compensate for over- or under-saturation. 3D-images were produced by z-stack optical slices taken from the backing to the needle tip of MNPs at 5 μm intervals.

Examination of Drug Residue on Different Backing Materials:

A dipping study was designed to assess the residual drug content on MNP backing after application on skin. In this study, two types of backing materials (PVA/sucrose or PS) were made into dome-shaped solid blocks (base diameter ≈ 1 cm, height ≈ 0.7 cm) to simulate the MNP backing after use. These solid blocks were made by filling the corresponding second cast solution into PDMS molds and drying on a 40 °C hot plate. The dome blocks were mounted on a motorized force test stand (ESM301, Mark-10, Copiague, NY) and dipped into 2 mL of aqueous solution containing 1 mg mL⁻¹ SRhB (simulating drugs) and various contents of PAA/sucrose (5%/10%, 10%/20%, or 15%/30%, simulating dissolved formulations) till the base of dome was around 2 mm away from the solution surface. The domes were kept merged in the solution for 1 min prior to taking out. SRhB adhered to the dome blocks during this process was collected by washing the dome blocks in 1 mL phosphate-buffered saline (Mediatech, Manassas, VA) with mild shaking for 5 min. The amount of SRhB coated on the dome blocks were measured by a microplate reader (BioTek, Synergy H4, Winooski, VT).

The wettability of different MNP backings was also studied by measuring the contact angle of water on films made of the corresponding backing materials. The hydrophilic and hydrophobic films were drop cast from the aqueous and organic casting solutions described above. Films were dried on a 40 °C hot plate for one day and then stored with desiccant until measurement. The water contact angle was measured on a contact angle goniometer (Rame-Hart, model 300, Succasunna, NJ), and measurements were repeated at six different locations on the film.

Kinetics of Drug Release in the Skin:

The kinetic behavior of MNPs delivering different model drugs was studied by applying drug-loaded MNPs on shaved porcine skin ex vivo and measuring the drug content delivered after the application. Briefly, the MNPs were pressed against the skin by thumb for 30 s, and then left in place for 0, 1, 3, 5, 10, or 20 min to allow dissolution of the MNs and release of drugs into the skin. After the specified time, the MNPs were removed from the skin and dissolved in water to measure the residual drug content. Meanwhile, unused MNPs were also dissolved in water to measure the drug loading in full patches, and its difference from the residual drug content in the used MNPs was calculated as the delivered drug dose. The delivery efficiency was calculated as the percentage of the drug loading in full MNPs that was delivered to the skin.

The quantification of SRhB was achieved by measuring the absorbance of the dye at 563 nm from UV–vis spectroscopy; the content of INS was measured by a commercial human insulin enzyme-linked immunosorbent assay (ELISA) kit (RAB0327, Sigma Aldrich, St. Louis, MO); and TT was quantified by ELISA.^[23] Briefly, the microplate was pre-coated with TT by incubating diluted TT stock at 4 °C overnight, and then blocked with 3% bovine serum albumin in 0.05 M carbonate buffer (pH 9.6); the TT samples and standards were loaded together with horseradish peroxidase linked anti-tetanus antibodies (Alpha Diagnostic, San Antonio, Texas) into the microplate and incubated at 4 °C overnight; after washing the microplate for multiple times, 3,3′,5,5′-tetramethylbenzidine substrate was added to produce color for reading.

Statistics:

All quantitative measurements were repeated at least four times and the data were presented as mean ± SD. F-test was performed to examine the standard deviations between data sets before performing the Student’s t-test to compare between groups. The difference was considered significant as p < 0.05.

Data Availability Statement

Research data are not shared.