An Integrated Microcurrent Delivery System Facilitates Human Parathyroid Hormone Delivery for Enhancing Osteoanabolic Effect

Abstract

Human parathyroid hormone (1-34) (PTH) exhibits osteoanabolic and osteocatabolic effects, with shorter plasma exposure times favoring bone formation. Subcutaneous injection (SCI) is the conventional delivery route for PTH but faces low delivery efficiency due to limited passive diffusion and the obstruction of the vascular endothelial barrier, leading to prolonged drug exposure times and reduced osteoanabolic effects. In this work, a microcurrent delivery system (MDS) based on multimicrochannel microneedle arrays (MMAs) is proposed, achieving high efficiency and safety for PTH transdermal delivery. The internal microchannels of the MMAs are fabricated using high-precision 3D printing technology, providing a concentrated and safe electric field that not only accelerates the movement of PTH but also reversibly increases vascular endothelial permeability by regulating the actin cytoskeleton and interendothelial junctions through Ca²⁺-dependent cAMP signaling, ultimately promoting PTH absorption and shortening exposure times. The MDS enhances the osteoanabolic effect of PTH in an osteoporosis model by inhibiting osteoclast differentiation on the bone surface compared to SCI. Moreover, histopathological analysis of the skin and organs demonstrated the good safety of PTH delivered by MDS in vivo. In addition to PTH, the MDS shows broad prospects for the high-efficiency transdermal delivery of macromolecular drugs.

Keywords: PTH; microchannels; microcurrent; microneedle; osteoanabolic effect.

Figure 1

Schematic diagram of MDS improving the osteoanabolic effect of PTH compared to subcutaneous injection and the Ca²⁺‐dependent cAMP signaling mechanism.

Figure 2

Design and characterization of the MDS. A) Components of the MDS. The counter electrode MNs and the pusher head of the syringe act as the negative and the positive electrode, respectively. The negative electrode, the positive electrode and the MMAs are loaded into the injection cap together to assemble the MDS. B,C) Optical micrographs showing the top view (B) and the bottom view (C) of the injection cap. The red arrows and the blue circle represent the rectangular groove for loading counter electrode MNs and the circular groove for loading MMAs, respectively. The red triangle represents the drug reservoir. Scale bar, 1000 µm. D,E) The silver layer and the silver wire (yellow arrows) at the end of the syringe pusher as the positive electrode, and the head of syringe can be fitted into the injection cap. F) Optical image of the counter electrode MNs. Scale bar, 100 µm. G) Schematic diagram of the 3D‐print process of the MMAs (top), and the layer‐by‐layer structure of the MMAs shows the internal microchannels through the base and the inside of MNs (bottom). H,I) Representative side view and top view of MMAs. Scale bar, 100 µm. J) Representative image of a single MN. Scale bar, 100 µm. K) Mechanical curve of MMAs with microchannels of 100 µm diameter under compressive force. L) Rhodamine B staining of MMAs penetration wounds in porcine skin. Scale bar, 200 µm.

Figure 3

The endothelial permeability increased reversibly triggered by MDS in vitro. A,B) The transendothelial electrical resistance (TEER) and the permeability to 5 kDa FITC–dextran of HUVEC monolayers were measured following MDS exposure with different microcurrent densities. The data are presented as mean ± SD (n = 9). P values were analyzed by one‐way ANOVA. C,D) The TEER and the permeability to 5 kDa FITC–dextran of HUVEC monolayers as function of post‐exposure incubation time, for both control and MDS stimulated (7 min at 1 mA cm⁻²). The data are presented as mean ± SD (n = 9). P values were analyzed by unpaired Student's t‐test. E–I) Representative confocal microscopy images of HUVEC monolayers 0.5 h after MDS stimulation under varying microcurrent densities for 7 min. Scale bar, 50 µm. The middle column showing a magnification of the region indicated in the VE‐cadherin channel. The white arrows point to the cytoplasm with reduced SFs. The percentage of intracellular actin (F), the relative VE‐cadherin enriched in the junctional regions (G), the cell size (H) measured by the area enclosed by VE‐cadherin, and the linearity index (I) defined as the ratio of the actual junction length to the linear junction length of VE‐cadherin concerning that of untreated cells (Control). The data are presented as mean ± SD (n = 30). P values were analyzed by one‐way ANOVA. J–N) Representative confocal microscopy images of HUVEC monolayers as function of incubation time after MDS stimulation at 1 mA cm⁻² for 7 min. Scale bar, 50 µm. The middle column showing a magnification of the region indicated in the VE‐cadherin channel. The white arrows point to the cytoplasm with reduced SFs. Scale bar, 50 µm. The intracellular actin (K), the VE‐cadherin enrichment (L), the cell size (M), and the linearity index (N) are described above. The data are presented as mean ± SD (n = 30). P values were analyzed by one‐way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Ca²⁺‐dependent cAMP mediates reversible increase of endothelial permeability via ROCK activation and inhibition. A) Intracellular Ca²⁺ in HUVEC monolayers as function of post‐stimulus time for both control (untreated) and MDS stimulation (1 mA cm⁻² for 7 min) cells. The data are presented as mean ± SD (n = 3). P values were analyzed by unpaired Student's t‐test. B–E) Western blot analyses of insoluble VE‐cadherin, soluble VE‐cadherin (total), ROCK and GAPDH protein samples in HUVEC monolayers triggered by MDS (1 mA cm⁻² for 7 min) at different post‐stimulus time (B,D). Quantitative analyses of insoluble VE‐cadherin (C) and ROCK protein expression (E) relative to soluble VE‐cadherin and GAPDH, respectively. Data are presented as mean ± SD (n = 6). P values were analyzed by one‐way ANOVA. F) Relative RhoA activity in HUVEC monolayers triggered by MDS (1 mA cm⁻² for 7 min) at different post‐stimulus times with respect to that of control cells (untreated). Data are presented as mean ± SD (n = 6). P values were analyzed by one‐way ANOVA. G) Intracellular cAMP levels in HUVEC monolayers as function of post‐stimulus time for both control (untreated) and MDS stimulation (1 mA cm⁻² for 7 min) cells. The data are presented as mean ± SD (n = 3). P values were analyzed by unpaired Student's t‐test. H) Relative PKA activity in HUVEC monolayers triggered by MDS (7 min at 1 mA cm⁻²) at different post‐stimulus times with respect to that of control cells (untreated). Data are presented as mean ± SD (n = 6). P values were analyzed by one‐way ANOVA. I, J) The effect of MDS stimulation (7 min at 1 mA cm⁻²) and extracellular Ca²⁺ on intracellular cAMP levels in HUVEC monolayers with and without intracellular Ca²⁺, respectively. Intracellular Ca²⁺ were chelated by BAPTA‐AM. The data are presented as mean ± SD (n = 3). K) Mechanism diagram of spontaneous recovery of endothelial permeability. *p < 0.05 and ***p < 0.001.

Figure 5

MDS‐mediated PTH efficient delivery and osteogenic enhancement in vivo. A) PTH delivery efficiency of MDS at different microcurrent densities in vitro. Data are presented as mean ± SD (n = 9). P values were analyzed by one‐way ANOVA. B) Plasma concentration‐time profiles of PTH using three different delivery methods. Data are presented as mean ± SD (n = 3). C) 3D reconstruction of distal femurs from sham‐operated and OVX rats treated with PBS or PTH by different methods for 4 weeks. D–H) Quantitative results of micro‐CT: (D) bone mineral density, (E) bone volume fraction, (F) trabecular number, (G) trabecular thickness, and (H) trabecular separation. Data are presented as mean ± SD (n = 5). P values were analyzed by one‐way ANOVA. I) Hematoxylin and eosin (H&E) staining of distal femurs. Scale bar, 200 µm. J) Masson staining of distal femurs from sham‐operated and OVX rats. Blue marks new bone collagen fibers, while red marks mature bone. Scale bar, 200 µm. Enlarged scale bar, 50 µm. K,L) Plasma concentration of PINP and CTX‐I. Data are presented as mean ± SD (n = 5). P values were analyzed by one‐way ANOVA. M,N) Immunohistochemistry analyses for osteoblastic makers Col1 (M) and OCN (N). Col⁺ and OCN⁺ osteoblasts on the bone surface are marked by red arrows. Scale bar, 200 µm. Enlarged scale bar, 50 µm. O,P) The number of Col⁺ and OCN⁺ osteoblasts per bone surface. Data are presented as mean ± SD (n = 5). P values were analyzed by one‐way ANOVA. Q) Immunohistochemistry analyses for osteoclastic makers Ctsk. Ctsk⁺ osteoclasts on the bone surface are marked by red arrows. Scale bar, 200 µm. Enlarged scale bar, 50 µm. R) Trap staining for assessment of osteoclast activity. Red arrows indicate mature osteoclasts. Scale bar, 200 µm. Enlarged scale bar, 50 µm. S,T) The number of Ctsk⁺ and Trap⁺ osteoclasts per bone surface. Data are presented as mean ± SD (n = 5). P values were analyzed by one‐way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6

MDS for safe PTH delivery in vivo. A) Representative images of skin from the back of hairless rats after the treatment of MMAs (MDS without microcurrent) or MDS (1 mA cm⁻²) for 7 min. Compared to the untreated sites (control), the H & E staining showed more scattered immune cells (black arrows) at 4 h after treatments, which alleviated within 24 h. Scale bar, 100 µm. B) Representative photographs of the heart, lung, liver, kidney, and spleen at the end of the experiment. C) Statistical analysis of the final weight of organs. Data are presented as mean ± SD (n = 3). P values were analyzed by one‐way ANOVA. D) H&E staining of the heart, lung, liver, kidney, and spleen of rats at the end of the experiment. Scale bar, 100 µm.