Painless Skin Electroporation as a Novel Way for Insulin Delivery

Abstract

Background:

Rigorous research efforts have been undertaken worldwide to develop a needle-free insulin delivery for many decades with limited success. This translational study aims to deliver insulin through skin with painless electroporation.

Methods:

A recently designed microelectrode array was used to deliver insulin in mice with diabetes under electroporation conditions that are painless and harmless on human skin.

Results:

Under such condition, a therapeutic amount of insulin was delivered successfully through mouse skin. Electroporation alone increased insulin transport around 100-fold compared with passive diffusion. Increased skin temperature to 40°C for 20 min augmented insulin transport to 237-fold more than the control value. Repeated electroporation showed no harm on human skin.

Conclusion:

The results indicate the potential of painless delivery of insulin through human skin in future clinical practice.

Introduction

D iabetes mellitus currently affects 285 million people worldwide, a figure likely to reach 438 million at the 2030 horizon.¹ Of the approximately 16 million subjects with diabetes in the United States, about 1.5 million suffer from type 1 diabetes, afflicting predominantly young individuals who need daily subcutaneous insulin injections.² To achieve and maintain long-term optimal blood glucose control remains a major challenge to both physicians and patients. Most patients are reluctant to undergo lifelong multiple daily insulin injections. Alternative ways to deliver insulin without injections may improve patients' adherence to insulin treatments and improve their quality of life.³ Among alternative routes⁴ of insulin delivery are dermal, nasal, oral, buccal, and pulmonary; only pulmonary delivery has gained wide acceptance.⁵ Exubera™ (Pfizer, New York, NY), an inhaled powder form of recombinant human insulin for the treatment of adult patients with type 1 and type 2 diabetes, was approved by the U.S. Food and Drug Administration in January 2006. However, Pfizer suspended sales in October 2007 because of low sales of the product. This might probably relate to its high price, bulky inhaler, and lingering doubts over long-term safety. In April 2008, the company announced that six users out of 4,740 had developed lung cancer.⁶ Although there were too few cases to determine whether the incidence is related to the drug, other companies also suspended their inhalable insulin plans.

Skin is the largest and most accessible organ of human body. Transdermal drug delivery to the systemic circulation has been studied as an alternative method for noninvasive drug administration.⁷ It offers several advantages over oral route and subcutaneous injections: (1) avoidance of degradation in digestive tract and first pass of the liver, (2) improvement of patient compliance because of a user-friendly approach, and (3) potential for steady and time-varying controlled release.⁸ However, only liphophilic molecules that have a molecular size less than 500 Da can be passively transported across the stratum corneum (SC), at low dosage (5–10 mg daily).⁸ The SC is the outermost skin layer, consisting of dead tissue composed of flattened keratinocytes filled with cross-linked keratin and an extracellular matrix made up of lipids arranged largely in bilayers and is impermeable to most hydrophilic substances. The SC varies in thickness (10–20 μm), depending on the body location.⁹ Once the drug has passed through the SC, subsequent layers are easier to cross to reach the circulation system.¹⁰

Transdermal insulin delivery has had limited success. Using iontophoresis to deliver monomeric insulin through skin is only possible after significant disruption of the barrier with pretreatment of the skin by alcohol¹¹ or by depilatory cream.¹² The iontophoresis process takes 2–3 h and is cumbersome to carry out in the clinic. Methotrexate (MTX), a folic acid antagonist with antineoplastic activity, poses the same challenge in transdermal delivery. Transport of MTX through isolated porcine skin could be detected only after 10 h of continuous iontophoresis.¹³ Recently, Wong et al.¹⁴ have successfully transdermally delivered a relevant amount of MTX (molecular mass of 0.45 kDa) to mice using electroporation (EP) with newly designed microarray electrodes under conditions that are painless to human skin. The monomeric size of insulin (∼6 kDa) is around 10 times larger than MTX. Whether the same technology can be applied for transdermal insulin delivery is unknown.

EP involves the creation of transient aqueous pathways across lipid bilayer membranes by applying a short, high-voltage pulse.¹⁵ EP of the SC was first demonstrated in 1993.¹⁵ Upon application of high-voltage pulses, large increases in transdermal transport of molecules, mainly due to electrophoretic movement and diffusion through newly created aqueous pathways, were observed ex vivo with human skin and in vivo in hairless rats.¹⁵ The voltage required to break down 20 layers of eight to 10 lipid bilayers each in the SC is around 75–100 V. Microchannels or so-called “local transport regions” are created through the breakdown sites of the SC.^15,16 These microchannels remain open for minutes after electric pulses, and more drugs are transported during the post-pulse period than during the pulse application period.¹⁷ The amount and the molecular size limits of drugs transported by EP (up to 10 kDa)¹⁸ have been reported to exceed by far the transport by diffusion and iontophoresis.^15,19,20 Sen et al.²¹ have successfully delivered insulin transcutaneously by EP with anionic lipid enhancers in isolated porcine epidermis in a short treatment time (minutes). This raised the question of clinical application of transdermal delivery of commercial insulin by EP. However, most, if not all, electrodes available today used for EP induce painful sensation on human skin.²² Many new electrodes had been designed aiming at reducing the pain sensation while maintaining the enhanced delivery efficacy.^14,23,24

In this preclinical study, we evaluate the efficacy of transdermal insulin delivery into mice with diabetes by using a painless and harmless EP condition on human skin.

Materials and Methods

Animals

Female C57BL/6J mice, 6–8 weeks old, were obtained from the University Laboratory Animal Center (Tainan, Taiwan). All procedures were approved by the University Animal Care Committee and followed the guidelines for care and use of laboratory animals of the U.S. National Institutes of Health. Diabetes was induced by intraperitoneal injection of 50 mg/kg streptozotocin (Sigma, St. Louis, MO) dissolved in 0.05 M sodium citrate buffer (pH 4.5) for 5 consecutive days.²⁵ After a week, the blood glucose level was determined using a glucometer (Accu-Chek^® Advantage II, Roche Diagnostics Corp., Indianapolis, IN). Animals with a blood glucose level higher than 300 mg/dL were considered to have diabetes.

EP procedures

Mice were anesthetized by subcutaneous injection of chloral hydrate (400 mg/kg). To confirm that EP created electropores successfully, transepidermal water loss (TEWL) of mouse abdominal skin, a sensitive and reliable parameter of the intactness of the SC,²⁶ was measured with a Tewameter (model TM 300, Courage and Khazaka, Köln, Germany) before and after EP. The hair was removed at least 3 days in advance to avoid skin barrier disruption. Mouse skin with a pre-EP TEWL >5/g²/h was excluded. A 2-×2-cm² microelectrode array block¹⁴ connected to a pulse generator (Gene Pulser Xcell, Bio-Rad, Hercules, CA) was used to deliver multiple unipolar square pulses. Skin was pulsed with 150 V, 60–120 pulses with a pulse length of 0.2 ms at 1 Hz, a condition that is sufficient to create electropores painlessly on the human skin.^14,15

Determination of optimal insulin occlusion time after EP

A 2-×2-cm² gauze saturated with 200 μL of fluorescein isothiocyanate (FITC)–insulin solution (200 μg/mL) (Sigma) was applied on the mouse abdomen, and occlusion was induced for different time periods (2, 5, 10, 15, or 30 min) with adhesive tape and aluminum foil successively to avoid evaporation. Immediately after occlusion, residual insulin on the skin was removed by a 1-min washing with tap water. The skin was cut and fixed on a wooden board with either epidermis or dermis side facing upward.²⁷ FITC fluorescence was examined under a fluorescence microscope. The time point with maximal fluorescence was selected for subsequent experiments. An intermediate-acting insulin (biphasic insulin aspart, 30% soluble and 70% protamine-bound insulin aspart) (NovoMix^® 30, 100 U/mL, Novo Nordisk Pharma, Bagsvaerd, Denmark) was used in subsequent pharmacokinetics experiments. Biphasic insulin aspart generally provides significantly better postprandial glucose control and fewer side effects in patients with type 1 or 2 diabetes mellitus.²⁸ This insulin has similar bioactivity in mice.

Uptake of insulin in skin of mice with diabetes

Skin insulin level was measured using a solid-phase two-site enzyme-linked immunosorbent assay (ultrasensitive insulin enzyme-linked immunosorbent assay, Mercodia, Uppsala, Sweden).²⁹ After treatment, a 2-×2-cm piece of washed mouse skin under the electrode was cut into small pieces and grounded into powder in liquid nitrogen. The powder was dissolved and homogenized with a Polytron™ homogenizer (model PT3100, Kinematica, Lucerne, Switzerland) in 1 mL of radioimmunoprecipitation solution containing protease inhibitors.²⁰ The supernatant was collected after centrifugation for 15 min at 14,488 g and stored at −20°C until analysis.

Hyperthermia

Mild hyperthermia at 40°C has been shown to enhance transdermal delivery of macromolecules³⁰ and MTX.²⁰ The warm porcelain surface (2×2 cm²) of a thermoelectric cooler (Kryotherm Thermoelectrics, St. Petersburg, Russia) was attached on the skin to provide constant heating at 40±0.5°C 5 min before pulsing and during 15 min of insulin application post-pulsing. A thermosensor (National Instruments Co., Austin, TX) was attached to the mouse skin to monitor skin temperature continuously.

Oral glucose tolerance test

The oral glucose tolerance test (OGTT)³¹ was used to examine insulin actions in mice with diabetes at different time points before and after EP. Following overnight fasting (16–18 h), mice were subjected to either electric or sham EP and then to insulin occlusion for 15 min, an interval we determined from previous experiments. At 3 h after EP/insulin treatment, the time of peak hypoglycemia after treatment in mice that was determined in separate experiments (data not shown), 1.5 mg of glucose/g (around 100 μL) was administered orally through a gavage tube.³² After receiving the oral glucose solution, mice were put back to cages and received water ad libitum. Seven hours later, mice were allowed to access food freely. Blood glucose was measured before and hourly for the first 6 h after EP and then every 2–4 h until 34 h.

Acute effects and safety of EP on human skin

The institutional review board approved this study, and all subjects gave written informed consent before any study-related activities. All studies followed the Declaration of Helsinki protocols. Healthy volunteers without diabetes, seven men and two women 19–48 (range, 29±9) years old, were tested for the safety of the EP protocol. Volunteers with heart problem were excluded. EP was done with the conditions mentioned above on forearm skin after a 12-h fast. They graded their pain sensation with a 0–10 pain score during EP and compared it with needle injection.¹⁴ A 2-×2-cm² gauze saturated with 60 μL (6 U) of human insulin (Novomix) was applied, and occlusion was applied for 15 min with Parafilm (Pechiney Plastic Packaging Co., Chicago, IL) and a self-adhesive polyurethane dressing (Tagaderm^™, 3M, St. Paul, MN) in succession immediately after EP. Blood glucose was measured by fingertip blood sampling before and 1.5 h (peak action of Novomix) after insulin occlusion. TEWL was measured before and every hour after treatment until values returned to the baseline level. Digital images of the skin after treatment were taken for further analysis by a dermatologist who was blinded to the study. Clinical scores of skin damage were graded as follows: 0 for no visible change, 1 for minimal erythema, 2 for moderate erythema, 3 for marked erythema, and 4 for chapping and oozing. Three of the volunteers were treated daily for 3 consecutive days.

Statistics

One-way analysis of variance was performed to determine whether there were significant differences between the different test conditions. The Student–Newman–Keuls method was applied for multiple pairwise comparisons among individual groups. Data were analyzed using SigmaStat^™ (Systat Software Inc., San Jose, CA) version 3.11. A value of P<0.05 was considered significant. Data were calculated from two or three separate independent experiments.

Results

EP enhances insulin delivery into mouse skin

FITC-labeled insulin was delivered transdermally through skin of mice with diabetes after EP (Fig. 1). Immediately after insulin occlusion, vivid green fluorescence was detected on both the epidermis and dermis of mouse skin. The intensity of fluorescence concentrated within the electrode–skin contact areas. Figure 2 shows the insulin level in mouse skin samples after EP and hyperthermia. Each group contained three mice, and at least two independent experiments had been done in all groups. EP alone enhanced insulin delivery into mouse skin 93-fold compared with the control: 10,727±1,927 mU/L and 115±78 mU/L, respectively (P=0.004) (Fig. 2).

FIG. 1.

Enhanced transdermal delivery of fluorescein isothiocyanate–labeled insulin with electroporation in skin from mice with diabetes. Electric pulsing with a microelectrode array at 150 V, 0.2 ms (pulse length), 1 Hz for a total of 60 pulses was then followed by 15 min of insulin occlusion. Bright green fluorescence was detected on both the epidermis and dermis of pulsed mouse skin immediately. The intensity of fluorescence was concentrated within the electrode–skin contact areas. Images were representative data from one of three separate independent experiments.

FIG. 2.

Cumulative transport of insulin in mouse skin, calculated from the excised samples, is plotted for each protocol: Control, insulin occlusion only; Hyp, hyperthermia at 40°C for 5 min before and during the 15-min insulin occlusion; EP, electroporation. Data are mean±SE values (n=5 in the control, n=3 in the other groups), pooled from two separate repeated experiments. **P<0.01 by one-way analysis of variance.

Mild hyperthermia enhanced insulin influx synergistically with EP. Maintenance of the mouse skin at 40±0.5°C 5 min before and during insulin occlusion (in total, 20 min) provided a 69-fold higher insulin transport (7,907±2,399 mU/L, P=0.004) (Fig. 2) into mouse skin than the control. When EP was performed in addition to hyperthermia, the transport of insulin was enhanced by 237-fold (27,210±9,378 mU/L, P=0.004) (Fig. 2) more than the control.

Reduction of blood glucose in diabetic mice by transdermal delivery of insulin with EP and hyperthermia

The pharmacodynamics of insulin were monitored with OGTT. Oral administration of glucose produced a peak in blood glucose in both pulsed and control mice 4 h after electric pulsing (Fig. 3). Mild hyperthermia alone did not alter blood glucose kinetics compared with the control except at 4 h after EP (no peak of glucose, Fig. 3). EP actuated an effective transdermal insulin delivery as revealed by the significant hypoglycemic effect immediately after pulsing that continued up to 10 h. The peak glucose level in pulsed mice was still below the prepulse level. When EP was applied in conjunction with hyperthermia (Fig. 3), a marked reduction of blood glucose was observed at all tested time points compared with the other groups. The glucose peak in OGTT was again absent.

FIG. 3.

Hyperthermia (Hyp) and electroporation (EP) provide synergistic hypoglycemic effects in mice with diabetes. EP (▾) (150 V, 120 pulses at 0.2 ms, 1 Hz, n=6) on mouse skin and 15 min of post-pulsing insulin occlusion reduced blood glucose significantly. Hyp + EP (▵) (40±0.5°C 5 min before EP and during 15-min post-pulsing, n=9) initiated a rapid and profound hypoglycemic effect. Mice receiving Hyp alone (○) (n=11) showed a trend but insignificant changes in blood glucose except at 4 h post-EP (P<0.05) compared with the control (•) (insulin application only, n=9). Oral glucose solution was administered (oral glucose tolerance test [OGTT]) to each mouse 3 h after EP, and all had free access to food 10 h later. Blood glucose values at each time point were normalized relative to pretreatment values. Data are mean±SE values, pooled from two separate repeated experiments. *P<0.05, **P<0.01, ***P<0.001 by Student–Newman–Keuls method.

Repeated EP is safe and painless on human skin

Figure 4a shows that the skin barrier is temporarily disrupted with EP with the present protocol. TEWL increased around twofold immediately after 15 min post-pulse insulin occlusion and returned to the basal level in 90 min. The blood glucose in EP treated subjects declined slightly but not significantly (around 10 mg/dL, P>0.05) (Fig. 4b). There was no change of blood glucose in the non-EP group (Fig. 4b). Skin damage scores were 0 before and after treatments in both groups. All volunteers felt almost no pain (0.6±1.0 in pain scores) during electric pulsing, whereas they felt obvious pain during needle injection (5.0±1.4, P<0.001) (Fig. 4c).

FIG. 4.

Electroporation (EP) with the microelectrode array is safe on human skin. Nine healthy volunteers without diabetes were occluded with 6 U of human insulin for 15 min after EP or sham EP (control). (a) The skin barrier was disrupted temporarily as revealed by a twofold increase of transepidermal water loss (TEWL) immediately after insulin occlusion and electric pulsing, and values returned to baseline in 90 min. (b) EP induced a slight but not significant (P>0.05) decrease of blood glucose. (c) All volunteers felt almost no pain during electric pulsing, whereas they felt obvious pain (***P<0.001) during needle injection.

Discussion

In this study, a therapeutic level of insulin was transdermally delivered into mouse skin successfully under a painless EP condition in humans. To adapt this EP technology for insulin delivery to patients, two critical concerns must be addressed: (1) Can it deliver a sufficient amount of drug for human insulin therapy? (2) Is this procedure safe on human skin? There are two possible ways to further increase the quantity of drug delivery: one is to increase the electrode–skin contact area and time of application, and another is to use adjunct physical and chemical enhancers with EP. It is known that a larger electrode–skin contact area produces more electropores for drug delivery after electric pulsing.^14,18 A flexible electrode may be more comfortable for the patient. A larger and flexible microelectrode array with the same principle for painless EP is currently being designed and fabricated.

Raising the skin epidermis temperature from 25°C to 40°C, the first transition temperature of SC lipids, was found to reduce the electric resistance at least twofold.³⁰ In the present study, the transport of insulin into the skin was enhanced by 69-fold over passive diffusion with mild hyperthermia. Elevated temperature may induce the SC to swell and incorporate more drug solution, as well as increase the uptake of the hydrophobic portion of insulin in Novomix into the SC. The insulin in SC might diffuse steadily into the blood circulation as the OGTT curve (Fig. 3) is relatively flat. The results agree with those of Sen and co-workers;^21,33 they found that without electric pulsing, most of the insulin remained in the SC domain of the skin treated with mild hyperthermia and lipid enhancer. The transport of a chemotherapeutic agent, MTX, through isolated porcine skin was increased 11-fold by EP concurrent with anionic lipid enhancer and iontophoresis at 40°C hyperthermia, whereas the increase was 4.4-fold at 25°C compared with passive diffusion.²⁰ The enhanced transport is most likely due to the prolonged postpulse permeable state of the skin.³⁰ In the present study, when EP was performed before insulin application, increased skin permeability enhanced synergistically the total transport of insulin to 237-fold.

The elevation of local skin temperature to 40°C for 20 min can be easily achieved clinically and is well tolerated by the patient.³⁴ The insulin concentration delivered is equivalent to 0.0066 U/cm² (0.23 μg/cm² [i.e., 0.13% of the applied insulin]), which is sufficient to cover the daily basal insulin requirements (the basal rate for a continuous insulin pump is 0.22 U/kg/day).³⁵ The daily requirement of a 30-kg child, for example, can be satisfied by 40 2-min EP treatments with a 10-×10-cm² sized microelectrode array following a 10–20-min insulin occlusion with our protocol. Although the present protocol may be inconvenient, it is possible to deliver a preprogrammable amount of insulin via skin EP to maintain physiological blood glucose, which may also increase the psychological well-being in the patient for being free of injections.

The present EP protocol with insulin is safe and painless on human skin. Prolonged repeated disruption of the skin barrier may increase the risk of skin infection or dermatitis. We found that repeated treatments on the same area for 3 consecutive days showed no gross changes on skin surface and TEWL. Apparently the complete recovery in 90 min after EP and occlusion reduced such risk. As expected, glucose levels in healthy volunteers remained normal after EP and insulin occlusion with only a slight trend of reduction because of a tight regulation of blood glucose in the healthy human body.³⁶ The future challenge is to design a portable device that can be used by the patient him- or herself at home. This can be done by miniaturizing the electrode array and powering by battery.

In conclusion, we have demonstrated the success of transdermal delivery of insulin to mice using a new electrodes array with an EP condition that is painless and harmless to humans. The study gives hope to those type 1 and type 2 diabetes patients who have to endure lifelong insulin injections, especially young children who are likely to refuse multiple injections.

Footnotes

Acknowledgments

This study is supported by the National Science Council (grants NSC952314B006040MY2 and NSC982314B006043), a New Faculty Grant from National Cheng Kung University, and National Cheng Kung University Hospital (grant NCKUH94023) of Taiwan. We thank Ms. Shu-Fen Ko and Cindy H.J. Lin for their excellent technical assistance.

Author Disclosure Statement

None of the authors has a conflict of interest to declare.

Portions of this work have been submitted in abstract form to the conference of the 22^nd World Congress of Dermatology in Korea, May 22, 2011.

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