Transdermal delivery of gentamicin using dissolving microneedle arrays for potential treatment of neonatal sepsis

Abstract

Neonatal infections are a leading cause of childhood mortality in low-resource settings. World Health Organization guidelines for outpatient treatment of possible serious bacterial infection (PSBI) in neonates and young infants when referral for hospital treatment is not feasible include intramuscular gentamicin (GEN) and oral amoxicillin. GEN is supplied as an aqueous solution of gentamicin sulphate in vials or ampoules and requires health care workers to be trained in dose calculation or selection of an appropriate dose based on the patient's weight band and to have access to safe injection supplies and appropriate sharps disposal. A simplified formulation, packaging, and delivery method to treat PSBI in low-resource settings could decrease user error and expand access to lifesaving outpatient antibiotic treatment for infants with severe infection during the neonatal period. We developed dissolving polymeric microneedles (MN) arrays to deliver GEN transdermally. MN arrays were produced from aqueous blends containing 30% (w/w) of GEN and two polymers approved by the US Food and Drug Administration: sodium hyaluronate and poly(vinylpyrrolidone). The arrays (19×19 needles and 500μm height) were mechanically strong and were able to penetrate a skin simulant to a depth of 378μm. The MN arrays were tested in vitro using a Franz Cell setup delivering approximately 4.45mg of GEN over 6h. Finally, three different doses (low, medium, and high) of GEN delivered by MN arrays were tested in an animal model. Maximum plasma levels of GEN were dose-dependent and ranged between 2 and 5μg/mL. The time required to reach these levels post-MN array application ranged between 1 and 6h. This work demonstrated the potential of dissolving MN arrays to deliver GEN transdermally at therapeutic levels in vivo.

Keywords: Gentamicin; Microneedle; Neonatal sepsis.

Graphical abstract

Fig. 1

Diagrammatic representation of MN fabrication process in (A) one-step and (B) two-step.

Fig. 2

Schematic representation showing the four treatment groups investigated in the in vivo experiment. (A) IM administration of GEN, 7.5 mg/kg. Transdermal application of (B) one, (C) two and (D) four MN arrays to the hairless back of the animals and the setup used to ensure the MNs were kept in place.

Fig. 3

(A) Comparison of percentage of MN height reduction of dissolving MN formulations tested, observed following the application of a force of 32 N/array (means + S.D., n = 4). (B) Percentage of holes created in each Parafilm M® layer following insertion of dissolving MN formulations investigated. MN arrays used were 19 × 19 with a needle height of 500 μm, base width of 300 μm and a base interspacing of 50 μm (means + S.D., n = 4).

Fig. 4

Digital pictures showing two-layered MN array taken with (A) the Leica EZ4D digital microscope and (B) the Keyence VHX-700F digital microscope (Keyence, Milton Keynes, UK). (C) Comparison of percentage recovery of GEN from dissolving MNs prepared from F1 and F6 in one-step and two-step (means ± S.D., n = 3). (D) Representative OCT images showing in vitro dissolution kinetics of a 19 × 19 MN array prepared using F1 and inserted manually in neonatal porcine skin. False colours were applied to the skin and MNs. The original OCT images can be found on the Supplementary content (Fig. S1). The white scale bar at top right represents a length of 1 mm. Digital pictures showing two-layered MN array taken with (A) the Leica EZ4D digital microscope and (B) the Keyence VHX-700F digital microscope (Keyence, Milton Keynes, UK). (C) Comparison of percentage recovery of GEN from dissolving MNs prepared from F1 and F6 in one-step and two-step (means ± S.D., n = 3). (D) Representative OCT images showing in vitro dissolution kinetics of a 19 × 19 MN array prepared using F1 and inserted manually in neonatal porcine skin. False colours were applied to the skin and MNs. The original OCT images can be found on the Supplementary content (Fig. S1). The white scale bar at top right represents a length of 1 mm.

Fig. 5

In vitro permeation profile of GEN across 350 μm neonatal porcine skin when delivered from dissolving MNs made of formulation F1 over a 24 h period. Means ± S.D., n = 4.

Fig. 6

(A) In vivo plasma profile of GEN following IM injection of gentamicin sulphate dissolved in sterilised water for injections (dose was 7.5 mg/kg). Means ± S.D., n = 5 at 1 h, 2 h, 4 h and 6 h; n = 10 at 24 h. (B) Transdermal delivery by using one dissolving MN (low dose). Means ± S.D., n = 5 at 1 h, 2 h and 6 h; n = 4 at 4 h; n = 10 at 24 h. (C) Transdermal delivery by using two dissolving MNs (medium dose). Means ± S.D., n = 4 at 1 h, 2 h, 4 h and 6 h; n = 9 at 24 h. (D) Transdermal delivery by using four dissolving MNs (high dose). Means ± S.D., n = 4 at 1 h; n = 5 at 2 h, 4 h and 6 h; n = 10 at 24 h. Dashed lines represent limit of quantification for GEN (LoQ = 0.30 μg/mL).

Fig. 7

Comparison of mean peak plasma concentrations of GEN (Cmax) following IM injection and transdermal delivery of GEN at increasing doses. Means ± S.D., n = 5 for the control group and the low dose and n = 4 for medium and high dose. (ns) indicates p values > 0.05, (*) indicates p values ≤ 0.05 and (***) indicates p values ≤ 0.001.