Materials
Polyvinyl alcohol (PVA, MW = 9000-10000, 80% hydrolyzed), D (+)-Sucrose (MW = 342.29, purity ≥ 99.9%), Sulforhodamine B (SRB, MW = 580.65), and Citric acid buffer (0.1 M, pH = 4.5) were purchased from Sigma-Aldrich. Streptozotocin (STZ) was obtained from AbMole Bioscience. Insulin (28 IU/mg) extracted from pig pancreas was purchased from Xuzhou Wanbang Biochemical Pharmaceutical Co., Ltd. Polydimethylsiloxane (PDMS, Sylgard 184), consisting of Base Part A and Curing Agent Part B was purchased from Dow Corning. Other chemicals used were analytical grade without further purification. Porcine cadaver skin was purchased from a local slaughterhouse (Beijing, China).
Methods
Fabrication of PDMS molds
PDMS molds with uniform cavities and different dimensions were initially fabricated using a laser-based and micromolding techniques as our previous study proposed (38). PDMS prepolymer solution and curing agent were first mixed in a mold at a ratio of 10:1 (v/v), then carried it out for 30 min under vacuum to remove air bubbles generated by stirring, next cured at 60 °C for 5 h to form PDMS sheet. Subsequently, a laser engraving machine (VLS3.50, 50 W, Universal Laser System, USA) was used to prepare a series of conical cavities on the PDMS sheets to form PDMS molds.
Preparation of PVA solution
PVA powder was firstly dissolved in deionized (DI) water and heated with a magnetic stirring at 90 ℃ for 6 h to obtain 32% (w/w) homogeneous solution. Following sucrose (acts as a stabilizer) was added into the solution (sucrose to PVA weight is 3:4) and kept stirring the mixture at 90 ℃ for 1 h and then cooled it down to room temperature to form 25.8% PVA mixed solution.
Fabrication and characterization of DMNRs
DMNRs were fabricated based on a casting process. Briefly, 200 μl of prepared drug solution (insulin solution or 2 mg/ml SRB) was first applied to the PDMS mold under vacuum for 15 minutes. Then, the residual drug solution was removed from the mold surface and the vacuum was continued to concentrate the drugs on the tips of the cavities of PDMS mold. Third, 25.8% PVA/sucrose solution (about 200 mg) was cast into the PDMS mold under vacuum for 30 min at room temperature to make sure PVA/sucrose solution filled with the MN cavities. After that, the mold was dried at room temperature in a fume hood. Subsequently, 3M double-sided tape with a thickness of 0.2 mm was attached to the outer circumferential surface of a hollow cylindrical plastic roller with a length of 5 cm, an inner diameter of 2 mm, and an outer diameter of 4 mm. DMNRs were finally formed by demolding the drug-loaded MNs from the PDMS mold using the prepared roller with double-side tape. SRB was used as a model drug, and insulin solution was used as a therapeutic drug for diabetes. Detailed characteristics of DMNRs were observed using fluorescence microscope (SZX7, Olympus, Japan) and scanning electron microscope (SEM, Hitachi S-4000, Japan).
Demolding time study of DMNR
To ensure that the DMNs can be demolded from the PDMS mold without causing any MNs damage, the demolding time of DMNR was first optimized. Briefly, DMNs were demold from PDMS mold after drying 2, 4, and 8 hours. Each DMNR demolded at different times was then weighed immediately. The relative water content of DMNR demolded at different times was determined by comparing the weight change after immediate demolding and after drying under vacuum for 48 hours.
Mechanical property tests of DMNRs
Mechanical properties of DMNRs were determined by a displacement-force test station with a mechanical sensor (Mark-10, Force Gauge Model, USA). Both axial force and shear forces of SRB-loaded DMNs with different heights (400, 600, and 700 μm) that mounted on the rollers were tested. Briefly, on the one hand, to access the axial compression performance, a DMNR with only one row containing five DMNs was attached to a flat stainless-steel baseplate of the sensor probe while the tips of MNs pointing downwards, and the sensor probe was then lowered in the vertical direction at a speed of 0.1 mm/s. The data of displacement and force measurements were recorded when the tips of the MNs first touched the smooth stainless-steel platform and continued until the force reached 5 N. On the other hand, to test the shear force of DMNRs, the roller with a single MN was fixed on a vertically placed stainless steel plate, and the tips of MNs faced horizontal direction. The sensor probe was lowered in the vertical direction at a speed of 0.1 mm/s while a shear force was then given at 250 μm from the tips of the DMNs. Displacement and force measurements were recorded when the sensor probe was first touched the MNs and continued until the shear force was zero. Subsequently, the ultimate shear force was obtained when the MN was fractured under shear force. These experiments were repeated five times to determine the mechanical property of DMNR. After each test, the sensor probe was moved upwards at a speed of 10 mm/min, and DMNs were then visualized using fluorescence microscope.
Insertion studies of DMNRs on porcine cadaver skin
To assess the in vitro insertion abilities of DMNRs with DMN heights of 400, 600, and 700 μm, porcine cadaver skin without subcutaneous tissues was used as a human skin model. Before experiments, skin was separated into small pieces of 5 × 5 cm and stored in a refrigerator at -20 °C. Prior to insertion test, the skin was first immersed in PBS and thawed for 30 minutes, and excess moisture on the skin surface was absorbed using commercial tissue papers, and then the hair of the pig skin was carefully removed by a disposable razor. SRB-loaded DMNRs were installed on a homemade applicator and then rolled forward once row by row across the skin. To control the force applied on the DMNRs, we referred to the method of previous study (30) that placed the skin on a balance which DMNRs were applied. The skin surface was imaged by fluorescence microscope after DMNR insertion. To clearly evaluate the insertion depth of DMNRs with different height DMNs, the skin samples were sliced from the middle of the DMNs insertion position points and viewed from the side using fluorescence microscope.
Insertion studies of DMNRs on Parafilm M®
To further observe the holes created of DMNR visually, Parafilm M®, a validated skin model (39), was used as an artificial skin simulants. The Parafilm M® sheet was folded to get an eight-layer film (appromiximately127 μm thickness per layer). Following, SRB-loaded DMNR (375 DMNs, 600 μm) was installed on a homemade applicator and rolled forward once across the eight-layer film row by row. After insertion, the sheet was unfolded and observed using a handheld microscope (AD7013MZT, Dino-lite, Taiwan, China). The number of holes created in each layer by DMNRs were then calculated under microscope after washing off the fractured MNs in the sheet.
Rapid drug delivery performance tests of DMNRs
The in vitro drug delivery performance of DMNRs were investigated by controlling the insertion time and insertion ways. Porcine cadaver skin described above was used as a skin model. For easy observation, DMNPs and DMNRs with only one row (5 MNs/row, 600 μm height) were used to insert the skin, respectively. The experiments were divided into two groups: (1) DMNP with 5 MNs/row was vertically inserted into the skin and then removed vertically at 1 second; (2) DMNR was inserted into the skin by rolling it once across the skin and removed within 1 second. Immediately, top views and side views of the skin after insertion were imaged using fluorescence microscope. DMNs were visualized using fluorescence microscope and SEM before and after insertion. The DMNs for each group had the same geometry. Each group repeated five times. Moreover, the model drug does not diffuse immediately in a short time after being transported into the skin by DMNs, so the insertion situation can be evaluated by observing the number of red spots left after the drug was delivered into the skin.
Insertion rate and drug delivery efficiency tests of DMNRs in vivo
To assess and compare the in vivo insertion and drug delivery performances of DMNRs and traditional DMNPs, the insertion rate and drug delivery efficiency were measured. UV-sterilized DMNRs contains different number of SRB-loaded DMNs (225, 375, 525) were separately applied to the dorsal skin (with a certain curvature) of female SD rats, and the DMNPs with the same geometry and same number of DMNs to DMNRs were fabricated as control group. Prior to the experiment, the dorsal region hair of rats was first removed using electric clippers and depilatory cream under anesthesia, and then cleaned by 75% ethanol. Notably, DMNRs were rolled once across the dorsal skin and removed immediately, while DMNPs were inserted into the skin and removed after 2 minutes. Subsequently, the skin of insertion sites was immediately observed by handheld microscope, and the amounts of red spots created by SRB-loaded DMNRs or DMNPs on the insertion sites were counted to calculate the insertion rate by the following equation:
At the same time, collecting the drug residues remained on the rollers and patches respectively, as well as residual drug on the insertion sites administrated by DMNRs and DMNPs, to measure the fluorescence intensity using a fluorescence microplate reader (Fluoroskan Ascent 374, Thermo Scientific), and then the amounts of residual drug were calculated. Drug loading of per DMNR or DMNP was also determined using fluorescence microplate reader Therefore, drug delivery efficacy was calculated according to the following equation:
In vivo visualization of drug delivery of DMNR using IVIS imaging tool
To visualize cutaneous permeation of model drug in live SD rats, the live anesthetized rats were first treated with the same size model drug-loaded DMNRs and DMNPs (600 μm height), respectively. Then imaging of the insertion sites of the rat skin at different times (0, 2, 4, 6, and 8 h) after treatment of DMNRs or DMNPs using a non-invasive analytical in vivo imaging system (IVIS, Xenogen 200, Caliper Life Sciences, Hopkinton, MA) to visualize cutaneous permeation of drug in live rats.
Rat model of Type I diabetes
Sprague-Dawley (SD) rats (6-8 weeks, 200-230 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Rats were housed in laminar flow cages, provided with ad libitum access to food and water, and maintained in a specific environment with temperature of 22 ± 1 ℃, relative humidity of 50 ± 10%, and a 12 h light-dark cycle. All rats were allowed to acclimatize the laboratory environment for 1 week before experiments. After overnight fasting, Type 1 diabetic models were induced in female SD rats by single intraperitoneal injection with 75 mg/kg streptozotocin in 200 μl sodium citrate buffer (0.1 M, pH 4.5). Rats with fasting blood glucose levels exceeding 300 mg/dl were confirmed as Type 1 diabetic rats. All procedures of animal studies were conducted in accordance with the guidelines of China-Japan Friendship Hospital for care and use of laboratory.
Blood glucose control studies in type 1 diabetic rats
Prior to the experiment, type 1 diabetic rats were fasted for 6 h but received water ad libitum, and the back hair of each group was shaved using an electric shaver under anesthesia. Then all animals were casually divided into the following 3 groups (n=4 for each group): (1) control group: DMNRs without insulin loaded were applied to the dorsal skin of diabetic rats; (2) subcutaneous (SC) group: diabetic rats were injected 0.3 IU insulin solution subcutaneously by disposable insulin syringes on the dorsal skin of diabetic rats; (3) DMNRs group: insulin-loaded DMNRs (0.3 IU per roller) were applied to the dorsal skin of diabetic rats. The blood glucose levels of rats in each group were measured using One Touch glucometer before and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, and 6 h after insulin-loaded DMNRs administration.