Therapeutic intradermal delivery of tumor necrosis factor-alpha antibodies using tip-loaded dissolvable microneedle arrays

Abstract

Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here, we evaluated the use of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of anti-TNF-α Ab. MNAs with obelisk shape microneedles that incorporate the antibody cargo in the needle tips were created from carboxymethylcellulose (CMC) using a micromilling/spin-casting fabrication method. We found that anti-TNF-α Ab integrated into MNAs using this room temperature fabrication process maintained conformationally dependent TNF-α binding activity. Further, these MNAs efficiently delivered anti-TNF-α antibodies to the dermis of human skin with clinically applicable release profiles. To evaluate MNA delivered anti-TNF-α Ab function, we applied anti-TNF-α Ab containing MNAs to established psoriasiform lesions on the skin of mice. MNA anti-TNF-α Ab treatment reduced key biomarkers of psoriasiform inflammation including epidermal thickness and IL-1β expression. Taken together, these results demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans, and support the development of MNA mediated antibody delivery for clinical applications.

Statement of significance: Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here we demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans. These results support the development of MNA mediated antibody delivery of therapeutic antibodies for clinical applications.

Keywords: Dissolvable microneedle arrays; Intradermal delivery; Therapeutic antibody; Tumor necrosis factor-alpha.

Fig.1.

Description of the micromilling/spin-casting based fabrication approach used for creating tip-loaded dissolvable microneedle arrays. Three-steps: (a) Mechanical micromilling of mastermolds. (b) Elastomer molding of production molds. (c) Spin-casting to localize anti-TNF-α Abs in the apex of obelisk microneedles and fabricate tip-loaded dissolvable MNAs.

Fig.2.

Tip-loaded dissolvable MNAs created using the micromilling/spin-casting technique for intradermal delivery of anti-TNF-α Abs. (a) ESEM images of the PMMA mastermold. Scale bars on the image of array and individual microneedle correspond to 1 mm and 100 μm, respectively. (b) Optical microscope images of the production mold after tip loading with bioactive cargo. (c) Bright field microscope images of the tip-loaded MNAs. Scale bars on the image of array and individual microneedle correspond to 1 mm and 100 μm, respectively. (d) Inverted fluorescence microscope image of a dissolvable MNA tip-loaded with Cy3-labeled anti-TNF-α Ab. (e) Inverted fluorescence microscope image of the individual tip-loaded dissolvable MNAs. (f) Merged bright field and fluorescence microscope image of the tip portion of the individual microneedle.

Fig. 3.

TNF-α binding affinity for anti-TNF-α Ab using biolayer interferometry. Biolayer interferometry was used to measure binding affinity of fresh or previously encapsulated anti-TNF-α Ab with TNF-α immobilized on the sensor tip to characterize the effects of processing on antibody binding affinity. (a) Characteristic binding curves of fresh and previously encapsulated anti-TNF-α Ab show similar binding kinetics in real time. (b) K_D values extracted from binding curves using a 1:1 binding isotherm model show slight increases in K_D for anti-TNF-α Ab previously encapsulated in MNA (K_D = 50.6 ± 5.84 pM) compared to fresh anti-TNF-α Ab (K_D = 9.33 ± 3.18 pM), corresponding to a slight decrease in binding affinity.

Fig.4.

Microneedle penetration, deposition of cargo and intradermal delivery of anti-TNF-α from tip-loaded CMC-MNAs. (a) ESEM image of the microneedle arrays before application. Scale bars correspond to 100 μm. (b) ESEM image of the microneedle arrays after 30 min of application. Scale bars correspond to 100 μm. (c) Inverted fluorescence microscope image of Cy-3 labeled microneedle traces on living human skin explants. 4 x optical magnification. (d, e, f) Intradermal delivery of anti-TNF-α from tip-loaded CMC MNAs. 20x optical magnification. (g, h, i) Intradermal delivery of anti-TNF-α from topical application with DMSO. 20x optical magnification.(d, g) Fluorescence microscope image of the DAPI stained human skin insertion sites. (e, h) Fluorescence microscope images of the Cy3-labeled antibody cargo. (f, i) Fluorescence microscope composite images that demonstrate delivery cavities penetrating the epidermis and upper dermis, and delivery of fluorescent cargo of the microneedles.

Fig.5.

Intradermal release profiles of anti-TNF-α Ab from tip-loaded CMC-MNAs. Tip-loaded MNAs created as described above were applied to living human skin for the indicated time intervals and then removed. Targeted skin was excised and assayed for Cy3-fluoerescence content by spectrofluorometry. Standard deviation values were from 6 replicates.

Fig.6.

MNAs delivering TNF-α antibodies abrogate the development of psoriasiform dermatitis. Balb/c mice were treated with Aldara for 4 days. On day 1 and 3 mice were treated with MNA patches delivering TNF-α antibody or Aldara alone as a negative control. (a) H&E staining of cutaneous cross-sections collected on day 5. (b) Quantitation of cellular infiltrate. Each symbol represents an individual mouse with 10 high-powered fields averaged per mouse. The bar represents the mean of three mice. Asterisks designate a significant difference between indicated treatment groups, ***p < 0.001, ** p < 0.01. (c) Quantitation of epidermal thickness, bars represent the mean thickness of the epidermis from 15 high-powered fields ± S.D. (d) Fold-change in IL-1β mRNA expression detected on day 5 by qRT-PCR. Fold-change was determined using the 2^−ΔΔCt method in which samples were normalized to GusB endogenous control. Each symbol represents an individual mouse with each sample ran in triplicate. The bar represents the mean of three mice.