(W1130-04-21) Fluorescently Labeled P407 Enters Micropores Generated Using Microneedles

Purpose: The objective of this study is to confirm the migration of poloxamer as a liquid and a gel formulation into micropores generated using microneedles, with the goal of forming a gel depot in microneedle-treated skin. Presently, Poloxamer 407 (P407) was reacted with 5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF), a fluorescent label, and the extent of the reaction was analyzed using UV spectrophotometry and HPLC. Changes to the gelation properties were analyzed using rheology and migration of P407 formulations into micropores on MN-treated skin was analyzed using confocal microscopy.

Methods: P407  and 5-DTAF solutions were prepared and mixed at a molar ratio of 1:2 (P407:5-DTAF) in the dark for 12 hours. The product was isolated using dialysis and reconstituted. UV absorbance was measured to determine P407 and 5-DTAF concentrations throughout the reaction. P407 concentration was measured using a colorimetric assay by conjugating P407 with cobalt thiocyanate. Cobalt absorbance (and the corresponding concentration of P407) was measured at 623nm while absorbance of 5-DTAF was measured at 492 nm. A gradient HPLC method was performed on samples from all stages of the reaction including P407 solution, 5-DTAF solution, reactant mixture (“Reactants mixed”), before dialysis (“RXN undialyzed”), and after dialysis (“RXN dialyzed”) to measure differences before and after the reaction and isolation. Gelation temperature was measured on a rheometer to analyze effects on gelation from fluorescently labeling the poloxamer. The gelation temperature of the P407 formulations was determined by measuring the storage modulus (G’) as temperature was increased by 1°C/min from 15 to 40°C. Gelation temperature was defined as the temperature at which G’ is halfway between the G’ of the liquid and G’ of the gel. Migration of P407 into the micropores was analyzed using confocal microscopy. Excised porcine skin was dermatomed to 1 mm and treated with one MN array consisting of 56 projections with a length of 600 µm. The formulations (5-DTAF solution in HEPES buffer, and 5-DTAF-labaled P407 in HEPES buffer as a liquid or a gel) were applied for ten minutes to intact skin and skin treated with MN. Excess formulation was then removed, and the skin samples were rinsed in HEPES buffer before being placed on microscope slides. Skin samples were analyzed using a z-stack on a confocal microscope using an excitation wavelength of 492nm and detected at a wavelength of 516nm.

Results: P407 content did not change drastically (17.1% removed) after dialysis suggesting that the polymer was large enough to be contained within the dialysis tubing while the drastic change (46.9% removed) in 5-DTAF suggests that unreacted 5-DTAF was removed. The final product concentrations show that 21.3% of the P407 terminal groups were labeled. Gradient HPLC showed different peaks based on the unreacted components and reaction products. 5-DTAF solution and mixed reactants produced a large and small peak of the same size at seven and five minutes, respectively, suggesting that the presence of P407 in solution does not change the column interaction of 5-DTAF. The peak at 7 minutes decreased by 31.4% after the reaction proceeded, while the peak at 5 minutes increased 3.2-fold. This change suggests that the 5-DTAF is exiting the column at a different time due to bonding with P407. The peak at seven minutes decreased by 3.6-fold when comparing the solutions before and after dialysis, while the area under the peak at five minutes stayed constant. This further supports that unreacted 5-DTAF was effectively removed via dialysis. Gelation temperature of P407 and labeled P407 solution was measured using rheology. Storage modulus of labeled P407 increased over the tested temperature range with a measured gelation temperature of 33.5°C. The P407 solution did not gel before 40°C. This demonstrates that covalently bonding 5-DTAF to P407 decreases gelation temperature. Confocal microscopy was used to image the migration of 5-DTAF (in solution or as a label on P407) into intact and MN-treated skin. On intact skin, 5-DTAF solution and labeled P407 both do not penetrate the skin more than 38.3 µm. 5-DTAF in solution can freely move into the micropores and permeate the deeper layers of the skin. Both liquid- and gel-form labeled P407 show the same migration pattern in the skin suggesting that gelation does not prevent labeled P407 from entering the micropores. Within the micropores, 5-DTAF was present as deep as 127.7µm in the skin.

Conclusion: 5-DTAF can be used to fluorescently label P407, and unreacted 5-DTAF can be efficiently separated from reacted products using dialysis. Gelation temperature of labeled P407 formulations is lower than blank P407 formulations of the same concentration. Using confocal microscopy to visualize MN-treated skin, labeled P407 migrated into micropores in excised porcine skin.

Chromatographs of P407 solution, 5-DTAF solution, immediately after mixing the components (“Reactants mixed”), before dialysis (“RXN undialyzed”), and after dialysis (“RXN dialyzed”) (n=1; chromatograms are offset for clarity).



Temperature ramp of blank P407 and fluorescently labeled P407.



Cross sections of confocal z stack of 5-DTAF solution and Labeled P407 as gel or liquid on intact or MN treated skin. Green shows 5-DTAF.