Targeted allergen-specific immunotherapy within the skin improves allergen delivery to induce desensitization to peanut.

Aim: Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy may be limited by allergen uptake through the skin barrier. To enhance allergen uptake into the skin, the authors used peanut-coated microneedles and compared them with EPIT in a peanut allergy mouse model. Methods: Sensitized mice were treated with peanut-coated microneedles or peanut-EPIT and then challenged with peanut to determine protection. Results: Treatment with peanut-coated microneedles was safe and showed enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Protection was associated with reduced Th2 immune responses and mast cell accumulation in the intestine. Conclusion: Peanut-coated microneedles have the potential to present a safe method of improving allergen delivery for cutaneous immunotherapy.

Epicutaneous immunotherapy (EPIT) with peanut has been demonstrated to be safe but efficacy has been varied. The tight barrier provided by the skin may limit the amount of allergen taken up through the skin and thus reduce efficacy. The authors evaluated a microneedle-based approach to improve the amount of allergen deposited into the skin to improve efficacy. Mice were made allergic to peanut and then treated with peanut-coated microneedles or peanut-EPIT. Mice were challenged with peanut to determine suppression of allergic reactivity. In mice, treatment with peanut-coated microneedles was safe and enhanced desensitization to peanut compared with peanut-EPIT administered via a similar schedule. Peanut-coated microneedles may present a novel method of improving allergen immunotherapy delivered through the skin.

Reliability and accuracy of intradermal injection by Mantoux technique, hypodermic needle adapter, and hollow microneedle in pigs.

We compared the ability of three intradermal delivery devices to administer an intended dose to pig skin in vivo and target that dose to the dermal rather than subcutaneous layers. The three devices were a standard hypodermic needle and syringe for the Mantoux technique, an adapter designed to facilitate proper hypodermic needle and syringe use, and a hollow microneedle. Reliability was determined as the percentage of the administered dose that entered the skin, as opposed to remaining in the device or on the skin surface. The intradermal adapter (97.6 ± 1.5 % delivered, mean ± standard deviation), Mantoux technique (95.4 ± 4.9 %), and hollow microneedle (94.9 ± 0.3 %) exhibited similar reliability. Accuracy was determined as the percentage of the dose that entered the skin that localized in the dermis. All three devices achieved similar accuracy: hollow microneedle (99 ± 12 % delivered to the dermis, median ± standard deviation), Mantoux technique (97 ± 16 %), and intradermal adapter (92 ± 21 %). We conclude that intradermal injection by all three methods studied provided reliable delivery to the skin and provided accurate localization of delivery within the dermis. Next-generation designs of these devices have now received clearance from the FDA and are used as medical products and/or in clinical trials.

In vivo high-frequency contrast-enhanced ultrasonography of choroidal melanoma in rabbits: imaging features and histopathologic correlations.

PURPOSE: To evaluate the use of in vivo imaging of rabbit model of choroidal melanoma using high-frequency contrast-enhanced ultrasound (HF-CE-US) with two-dimensional (2D) or three-dimensional (3D) modes and to correlate the sonographic findings with histopathologic characteristics. METHODS: Five New Zealand white rabbits, which were immunosuppressed with daily cyclosporin A (CsA), were inoculated into their right eyes with aliquots of 1.5×10(6)/50 µl of 92.1 human uveal melanoma cells cultured in RPMI. At week 4, the tumour-bearing eyes were imaged using high-frequency ultrasound (HF-US) with microbubble contrast agent to determine the 2D tumour size and relative blood volume and by 3D mode to determine tumour volume. Histologic tumour burden was quantified in enucleated eyes by ImageJ software, and mean vascular density (MVD) was determined by counting vascular channels in periodic acid Schiff (PAS) without haematoxylin sections. RESULTS: Using HF-CE-US, melanomas were visualised as relatively hyperechoic regions in the images. The correlation coefficients of sonographic size and volume compared with histologic area were 0.72 and 0.70, respectively. The sonographic tumour relative blood volume correlated with the histologic tumour vascularity (r(2)=0.92, p=0.04). CONCLUSIONS: There is a positive correlation between in vivo sonographic tumour volume/size and histologic tumour size in our rabbit choroidal melanoma model. HF-CE-US corresponds to MVD and blood volume.

Hollow microneedles for intradermal injection fabricated by sacrificial micromolding and selective electrodeposition.

Limitations with standard intradermal injections have created a clinical need for an alternative, low-cost injection device. In this study, we designed a hollow metal microneedle for reliable intradermal injection and developed a high-throughput micromolding process to produce metal microneedles with complex geometries. To fabricate the microneedles, we laser-ablated a 70 µm × 70 µm square cavity near the tip of poly(lactic acid) (PLA) microneedles. The master structure was a template for multiple micromolded poly(lactic acid-co-glycolic acid) (PLGA) replicas. Each replica was sputtered with a gold seed layer with minimal gold deposited in the cavity due to masking effects. In this way, nickel was electrodeposited selectively outside of the cavity, after which the polymer replica was dissolved to produce a hollow metal microneedle. Force-displacement tests showed the microneedles, with 12 µm thick electrodeposition, could penetrate skin with an insertion force 9 times less than their axial failure force. We injected fluid with the microneedles into pig skin in vitro and hairless guinea pig skin in vivo. The injections targeted 90 % of the material within the skin with minimal leakage onto the skin surface. We conclude that hollow microneedles made by this simple microfabrication method can achieve targeted intradermal injection.

Targeted administration into the suprachoroidal space using a microneedle for drug delivery to the posterior segment of the eye.

PURPOSE: This study seeks to determine the intraocular pharmacokinetics of molecules and particles injected into the suprachoroidal space of the rabbit eye in vivo using a hollow microneedle. METHODS: Suprachoroidal injections of fluorescein and fluorescently tagged dextrans (40 and 250 kDa), bevacizumab, and polymeric particles (20 nm to 10 µm in diameter) were performed using microneedles in New Zealand white rabbits. The fluorescence intensity within the eye was monitored in each animal using an ocular fluorophotometer to determine the distribution of the injected material in the eye over time as compared with intravitreal injection of fluorescein. Fundus photography and histology were performed as well. RESULTS: Molecules and particles injected near the limbus using a microneedle flowed circumferentially around the eye within the suprachoroidal space. By targeting the suprachoroidal space, the concentration of injected materials was at least 10-fold higher in the back of the eye tissues than in anterior tissues. In contrast, intravitreal injection of fluorescein targeted the vitreous humor with no significant selectivity for posterior versus anterior segment tissues. Half-lives in the suprachoroidal space for molecules of molecular weight from 0.3 to 250 kDa ranged from 1.2 to 7.9 hours. In contrast, particles ranging in size from 20 nm to 10 µm remained primarily in the suprachoroidal space and choroid for a period of months and did not clear the eye. No adverse effects of injection into the suprachoroidal space were observed. CONCLUSION: Injection into the suprachoroidal space using a microneedle offers a simple and minimally invasive way to target the delivery of drugs to the choroid and retina.

In vivo ocular fluorophotometry: delivery of fluoresceinated dextrans via transscleral diffusion in rabbits.

PURPOSE: To evaluate the transscleral delivery of fluoresceinated dextrans (FITC-D) with molecular mass up to 70 kDa to the rabbit posterior segment using sub-Tenon injections. METHODS: Eighteen NZW rabbits received a unilateral 200-µL injection of 2 mg/mL sodium fluorescein (NaF), 25 mg/mL 40-kDa FITC-D, or 25 mg/mL 70-kDa FITC-D, with (n = 9) or without (n = 9) immediate euthanatization. In live animals, fluorescence was measured in the retina/choroid and mid-vitreous by fluorophotometry, immediately after injection and after 4, 24, 48, and 72 hours. Euthanatized animals were examined hourly through 5 or 6 hours. RESULTS: In live animals, the average peak NaF concentration in the retina/choroid was 310.2 ng/mL, measured 3 hours after injection. Average 40- and 70-kDa FITC-D concentrations in the retina/choroid peaked at 5409.6 and 2375.6 ng/mL, respectively, 24 hours after injection. Fluorescence returned to baseline levels 6 hours after NaF injection, and 48 and 72 hours after 40- and 70-kDa FITC-D injections, respectively. Rabbits that received NaF followed by euthanatization exhibited a continuous increase in retina/choroid and mid-vitreous fluorescence, beginning 1 hour after injection, whereas FITC-D-injected eyes did not show elevated retina/choroid or mid-vitreous fluorescence through 6 hours. CONCLUSIONS: FITC-D weighing up to 70-kDa, as well as NaF, reached the posterior retina/choroid after sub-Tenon injections in live rabbits. NaF and 40-kDa FITC-D reached higher peak concentrations and were cleared from the eye more rapidly than was 70-kDa FITC-D. There was minimal penetration of NaF and FITC-D into the mid-vitreous in the in vivo experiments.

Mucoadhesive microparticles in a rapidly dissolving tablet for sustained drug delivery to the eye.

PURPOSE: To test the hypothesis that mucoadhesive microparticles formulated in a rapidly dissolving tablet can achieve sustained drug delivery to the eye. METHODS: Mucoadhesive microparticles, smaller than 5 µm were fabricated with poly(lactic-co-glycolic acid) and poly(ethylene glycol) as a core material and mucoadhesion promoter, respectively, and encapsulated pilocarpine as a model drug. These microparticles were embedded in a poly(vinyl alcohol) matrix to form a dry tablet designed to reduce rapid clearance of the microparticles on initial application to the eye. RESULTS: This in vitro drug release study exhibited that for all formulations, approximately 90% of pilocarpine was released during the first 10 minutes, and the remaining 10% was released slowly for 3 hours. In vivo mucoadhesion test on the rabbit eye indicated that mucoadhesive microparticles adhered significantly better to the preocular surface than other formulations. To assess the pharmacodynamics, the most prolonged pilocarpine-induced pupil constriction was observed in rabbit eyes in vivo using a tablet with mucoadhesive microparticles; it lasted up to 330 minutes. CONCLUSIONS: The authors conclude that mucoadhesive microparticles formulated into a dry dosage form is a promising system for sustained drug delivery to the eye.

Suprachoroidal drug delivery to the back of the eye using hollow microneedles.

PURPOSE: In this work, we tested the hypothesis that microneedles provide a minimally invasive method to inject particles into the suprachoroidal space for drug delivery to the back of the eye. METHODS: A single, hollow microneedle was inserted into the sclera, and infused nanoparticle and microparticle suspensions into the suprachoroidal space. Experiments were performed on whole rabbit, pig, and human eyes ex vivo. Particle delivery was imaged using brightfield and fluorescence microscopy as well as microcomputed tomography. RESULTS: Microneedles were shown to deliver sulforhodamine B as well as nanoparticle and microparticle suspensions into the suprachoroidal space of rabbit, pig, and human eyes. Volumes up to 35 µL were administered consistently. Optimization of the delivery device parameters showed that microneedle length, pressure, and particle size played an important role in determining successful delivery into the suprachoroidal space. Needle lengths of 800-1,000 µm and applied pressures of 250-300 kPa provided most reliable delivery. CONCLUSIONS: Microneedles were shown for the first time to deliver nanoparticle and microparticle suspensions into the suprachoroidal space of rabbit, pig and human eyes. This shows that microneedles may provide a minimally invasive method for controlled drug delivery to the back of the eye.