Iontophoresis-driven penetration of nanovesicles through microneedle-induced skin microchannels for enhancing transdermal delivery of insulin

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Iontophoresis-driven penetration of nanovesicles through microneedle-induced skin microchannels for enhancing transdermal delivery of insulin Huabing Chen a,1, Hongda Zhu a,1, Jingnan Zheng a, Dongsheng Mou a, Jiangling Wan a, Junyong Zhang b, Tielin Shi b, Yingjun Zhao b, Huibi Xu a, Xiangliang Yang a,⁎ a b

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China College of Mechanical Science and Engneering, Huazhong University of Science and Technology, Wuhan 430074, China

a r t i c l e

i n f o

Article history: Received 9 February 2009 Accepted 25 May 2009 Available online 28 May 2009 Keywords: Nanovesicles Microneedles Iontophoresis Insulin Transdermal delivery

a b s t r a c t The transdsermal delivery of insulin remains a significant challenge due to low permeation rates at therapeutically useful rates. We report unilamellar nanovesicles with membrane thickness of 3–5 nm and entrapment efficiency of 89.05 ± 0.91%, which can be driven by iontophoresis for enhancing transdermal delivery of insulin through microneedle-induced skin microchannels. The permeation rates of insulin from positive nanovesicles driven by iontophoresis through skins with microneedle-induced microchannels were 713.3 times higher than that of its passive diffusion. The in vivo studies show that the blood glucose levels of diabetic rats induced by the positive nanovesicles driven by iontophoresis through skins with microneedleinduced microchannels are 33.3% and 28.3% of the initial levels at 4 and 6 h, which are comparable to those induced by subcutaneous injection of insulin. The fluorescence imaging validated the penetration of insulin from the nanovesicles driven by iontophoresis through skins with microchannels. The nanovesicles with charges show significant permeation ability with the assistance of physical devices including microneedles and iontophoresis. This approach offers a new strategy for non-invasive delivery of peptides with large molecular weights using nanovesicles. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The non-invasive delivery of insulin including the transdermal, pulmonary, nasal, and buccal delivery remains a great challenge due to low permeation rates at therapeutically useful rates [1–4]. The transdermal delivery of insulin for avoiding barriers and increasing patient acceptance and compliance is an increasingly attractive alternative [5,6]. Many attempts have been made to enhance the permeation rates of insulin through skin. The chemical and physical permeation enhancing methods and drug carriers have been explored for enhancing transdermal delivery of peptides through intact skins [7–11]. Some chemical permeation enhancer such as fatty acids, iodine, short synthetic peptide (ACSSSPSKHCG) and trypsin can facilitate effective transdermal delivery of insulin through intact skin and decrease the glucose level [12–14]. Iontophoresis for charged molecules shows many advantages for transdermal delivery of peptides [15–17]. The microneedles were constructed to deliver insulin cross the skins by the painless microinfusion of insulin solution [18,19]. Drug carriers such as transfersomes and nanoparticles were used to break through the skins barrier for enhancing the transdermal delivery of insulin [20,21]. The combinations of ionto⁎ Corresponding author. Tel.: +86 2787792147; fax: +86 2787794517. E-mail address: [email protected] (X. Yang). 1 These authors contributed equally to this work. 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.05.031

phoresis with electroporation, chemical enhancers, poloxamer gels were also found to enhance penetration ability of insulin through skins than iontophoresis alone [22–24]. However, it is still an unresolved attracting focus in field of transdermal therapy of diabetes due to formidable barrier of stratum corneum and the poor penetration of insulin [25]. The vesicles including flexible liposomes, ethosomes and niosomes as drug carriers have mainly been used to enhance transdermal delivery of hydrophilic drugs [26]. It is necessary to further enhance transdermal delivery of drugs with large molecular weights, due to powerful skin barriers, even though vesicles have been used to enhance permeation rates of some peptides such as insulin and cyclosporine A through skins [27–29]. The physical enhancing methods such as microneedles, iontophoresis and electrophoresis were found to have good potential to enhance transdermal delivery of drugs with large molecular weights through skins [1,30–33]. For example, the vesicles were found to enhance the permeation rates of cyclosporin A and ascorbyl palmitate when combined with iontophoresis [34,35]. Our previous study showed that the nanovesicles could significantly enhance the penetration of insulin through mucous membrane of oral cavity [36]. The nanovesicles combined with physical enhancing methods might be promising for transdermal delivery of insulin. In this work, we report a novel active strategy for enhancing transdermal delivery of insulin using insulin-loaded nanovesicles with various charges driven by iontophoresis through skins containing

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microneedle-induced microchannels (Fig. 1). The nanovesicles are expected to provide a significant synergetic effect of the penetration of insulin through skins for reducing blood glucose level when combined with microneedles and iontophoresis. This study aims to understand the potential biological effect of nanovesicles when combined with the mechanical and electric devices, and construct a promising general route for the non-invasive delivery of peptides with large molecular weights.

loaded nanovesicles were prepared according to the composition of ILV1 formulation. Soybean lecithin and propylene glycol were mixed with chloroform solution of QDs/ZnS (4 nm). Then the solution was evaporated in round-bottom flask and the lipid membrane was obtained. The bulk dispersion was obtained by dispersing the lipid in distilled water under moderate stirring. The QDs-loaded nanovesicles were obtained by homogenizing the dispersion using ultrasonication. The blank nanovesicles without QDs were also prepared according to this process. All the samples were stored at low temperature (4 °C).

2. Materials and methods 2.3. Size and zeta potentials measurement 2.1. Materials Insulin was purchased from Jiangsu Wanbang Pharm. Company (Xuzhou, China). Soybean lecithin was obtained from Jinban food company (Shanghai, China). Cetyltrimethyl ammonium bromide (CTAB) was obtained from Shanghai Bio Life Sci. & Tech. Co. Ltd. Sodium dodecyl sulfate (SDS) and propylene glycol were purchased from Shanghai Shiyi Chemicals Reagent Co. Ltd (China). FITC-labeled insulin (FITC-INS) was a gift from Bioekon Company (Beijing, China). Streptozotocin was purchased from Sigma. Urethane was obtained from Shanpu Chemical Co. Ltd. All the other materials were of pharmaceutical and analytical grades. 2.2. Preparation of insulin-loaded nanovesicles Insulin (27.9 IU/mg) was dissolved in PBS solution (pH = 7.4) to form the insulin stock solution. Soybean lecithin was dissolved in propylene glycol, followed by mixing with the weighed stock solution containing insulin. Then coarse mixture dispersion containing 40 IU/ ml of insulin was obtained by adding PBS solution (pH = 7.4). Then, the nanovesicles were obtained by homogenizing the mixture dispersion using homogenization (120 MPa, 8 cycles) or ultrasound method (1200 W, 1:1). In addition, 0.4% cetyltrimethyl ammonium bromide (CTAB), 0.4% and 0.03% sodium dodecyl sulfate (SDS) were also added to the above nanovesicles for constructing the positive and negative nanovesicles, respectively. Additionally, FITC-labeled insulin (FITC-INS) was incorporated into the nanovesicles for constructing FITC-INS-loaded nanovesicles with 0.4% CTAB using the above method. The control solution containing insulin (40 IU/ml) was prepared by dissolving insulin into PBS solution (pH 7.4). The QDs-

The average diameters and zeta potentials of samples were measured by photon correlation spectroscopy (PCS) (Nano ZS90, Malvern Instruments, U.K.) at 633 nm. The measurements were performed at 25 °C using a He–Ne laser. Prior to measurement, the samples were diluted for avoiding the multi-scattering phenomenon. The results are expressed as the z-average diameter, which is obtained the Stockes– Einstein equation (D = kT/3ηπd). 2.4. Microstructure characterization Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) were used to probe the morphology of nanovesicles. The nanovesicles were placed on a carbon-coated copper grid and dried in air. Then dried samples were observed using Tecnai G2 20 TEM (FEI Corp., Netherlands) at 180 kV. In order to probe the microstructure of insulin-loaded nanovesicles, the samples were placed on a carboncoated copper grid and dried in air. Then the solution of 1% phosphotungstic acid was covered on samples and the superfluous phosphotungstic acid on sample was wiped off by filter paper, followed by being dried in air. The dried samples were observed using JEM-2100F HR-TEM (JEOL, Japan). The point resolution and lattice resolution of HR-TEM are 0.23 nm and 0.10 nm, respectively. 2.5. Entrapment efficiency The entrapment efficiency of the nanovesicles was measured using FITC-INS-loaded nanovesicles. A F-4500 Spectrofluorophotometer was used to analyze FITC-INS and the assay was linear in the concentration range of 0.05–1.0 µg/ml. The nanovesicles were separated

Fig. 1. The sketch of the transdermal delivery of insulin by utilizing iontophoresis-driven penetration of nanovesicles through microneedle-induced microchannels of skins.

using the Sephadex-G25 column (15.0 cm × 1.5 cm) and the eluent of 0.12 mol/l NaCl solution. The entrapped and free FITC-INS was respectively collected at continuous volume intervals of 2.0 ml and the collected samples were analyzed. The entrapment efficiency of nanovesicles was calculated by dividing the entrapped insulin in the nanovesicles using the total amounts of insulin. 2.6. Microneedles The solid stainless microneedle arrays were fabricated by photochemical etching stainless steel sheets according to our previous studies. Briefly, the structure of the arrays was drafted using a CAD software and the pattern was transferred onto steel sheets by photoetching. Then, the matrix of microneedles was obtained by chemical etching. Finally, the microneedles were punched from the plane by a die (Fig. 2). The microneedles array was arranged in rotundities with 8 needles around each cycle. Each array with an area of 2.0 cm2 contains 296 needles. The needles of array have a triangular shape with a length of 800 μm, a maximal width of 260 μm and a thickness of 80 μm.

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were continuously stirred at 300 rpm. The nanovesicle systems (2.5 ml) were gently placed in the donor chamber. At 1, 2, 3, 4 and 5 h, 0.5 ml of the solution in the receptor chambers were removed for HPLC determination and replaced immediately with an equal volume of fresh physiological saline. In addition, for the application of iontophoresis, the current of 0.2 mA/cm2 with an on/off ration of 1:1 and frequency of 100 Hz was applied continuously for 5 h through Ag/AgCl electrodes using a four-channel power supply unit. The anodes were placed in the donor chambers and cathodes were set at the receptor chambers when the electropositive ILV4 was applied into the donor chamber. When electronegative nanovesicles were applied, cathodes were placed in the donor chamber. After the permeation studies were ended, the nanovesicles in cells showed no significant changes in physical stability. Each sample was performed three times. Cumulative corrections were made to obtain the total amount of insulin permeated at each time interval. The cumulative amounts of insulin permeated through mouse skins were plotted as a function of time. The permeation rates of insulin at a steady-state through skins were calculated from the slope of linear portion of the cumulative amounts permeated through the skins per unit area versus time plot.

2.7. In vitro permeation studies [35] 2.8. The HPLC analysis The male guinea pigs (400–600 g) were obtained from the Experimental Animal Center, Hubei College of Traditional Chinese Medicine (Hubei, China). The animals were sacrificed by excessive urethane injection and hair was removed from the dorsal portions using an animal hair clipper and a depilatory. After the skins were excised, the subcutaneous fat was carefully removed, and then the skins were washed and examined for the integrity. In order to create pathways for transdermal transport, the microneedle arrays were employed to pierce into the integrated skins under a quantitative force of 9.0 N for 2 min and then were withdrawn from the skins [37]. The skins with microchannels induced by microneedle arrays were applied in the following in vitro permeation experiments. The permeation experiments were performed using a diffusion instrument (TK-12A, Shanghai Kaikai Corp., China) with a re-circulating water bath and 12 diffusion cells. The integrated and microneedlepretreated skins were respectively clamped between the donor and receptor chambers of vertical diffusion cells with an effective diffusion area of 2.8 cm2 and a volume of 7.0 ml. The receptor chambers were filled with freshly prepared physiological saline. The receptor chambers were set at 37 °C and the solutions in the receptor chambers

Fig. 2. The configuration of microneedle array fabricated by photochemical etching, followed by being punched from the plane of stainless steel sheet. (a) side elevation, (b) planform, and (c) micrograph. Each array with an area of 2 cm2 contains 296 needles, which have a triangular shape with a length of 800 μm, a maximal width of 260 μm, and a thickness of 80 μm.

Insulin was detected using reversed phase HPLC using Agilent 1100 series. The column was a Hypersil ODS2 C18 column (5 μm, 4.6 mm ID × 25 cm). The mobile phase was a mixture of acetonitrile and sodium dihydrogen phosphate (pH 3.0, 30:70 v/v) with a flow rate of 1.0 ml/min and the detection wavelength was set at 214 nm. The assay was linear in the concentration range of 0.36–36.0 μg/ml (A = 0.6828 C–0.07259, r2 = 0.9999) with a lowest detection limit of 0.18 μg/ml and the precision of RSD = 1.03%. The percentage recoveries ranged from 99.0 to 101.5%. No interference from the other formulation components was observed. All samples filtered through an aqueous membrane filter (0.45 μm pore size) before injection. 2.9. In vivo studies [22] The male Sprague–Dawley rats (180–200 g) (the Experimental Animal Center, Huazhong University of Science & Technology, China) were administrated by intraperitoneal (i.p.) injections of streptozotocin (65.0 mg /kg body weight). The blood glucose levels (BGL) were measured using Freestyle Glucometer (Therasense, USA) after 48 h, and the BGL were allowed to be stable for one week. Only the rats with blood glucose levels over 16.7 mmol/l were considered as the diabetic rats and used in this study. The diabetic rats were randomly assigned to treatment groups with five rats per group. The animals were fasted for 12 h before the experiment and only water was supplied. The rats were anesthetized by an i.p. injection of urethane (25.0% aqueous solution, 4.0 ml/kg) before an experimental procedure. To the control groups, a subcutaneous (s.c) injection of insulin (1.0 IU/kg) was administered. In the case of transdermal treatment groups, the rats were fixed supinely and abdominal hair was carefully shaved using electrical clippers, and skin surface was washed using cotton soaked in 70% ethanol. Two acrylic cells were attached to the skins (1.5 × 2 cm i. d.) using polyacrylate glue and the distance between two cells was 10 mm (Supporting information). The animals were kept at repose for 1 h and allowed the glucose levels to be stabilized. The microneedles were employed to pierce into the exposed abdominal skins under a quantitative force of 9.0 N for 2 min and then were withdrawn from the skins. The nanovesicles (ILV4) (2.0 ml, 40.0 IU/ml) was applied to one of the acrylic cells and an equal volume of fresh physiological saline was applied in the other cells. In addition, for the application of iontophoresis, the current of 0.2 mA/cm2 with an on/off ration of 1:1 and frequency of 100 Hz was applied continuously for 3 h through Ag/ AgCl electrodes using a four-channel power supply unit. The Ag/AgCl

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Table 1 The insulin-loaded nanovesicles with various average diameters and zeta potentials.

3.2. Characterization of nanovesicles

Nanovesicles

Zeta potentials (mV)

Average diameters (nm)

PDI

ILV1 ILV2 ILV3 ILV4 ILV5 ILV6

− 16.2 − 19.1 − 19.2 + 27.8 − 50.5 − 25.3

91.0 134.0 175.9 107.4 98.4 103.6

0.213 0.231 0.254 0.294 0.278 0.255

Transmission electric microscopy (TEM) imaging showed that ILV1 had a spherical morphology and their average diameters ranged from 60 nm to 110 nm (Fig. 3a). However, this image did not reveal the microstructure of nanovesicles. It is difficult for the conventional TEM imaging to provide the microstructure of organic nanoparticles with small diameters [39]. So far only a few studies were performed to directly observe the microstructure of nanovesicles, even though some methods such as small-angle neutron scattering have been used to validate the membrane structure of nanovesicles [40]. We employed HR-TEM imaging was used to probe the structure of the insulin-loaded nanovesicles [41]. The HR-TEM images of insulinloaded nanovesicles (ILV4) and control solution are shown in Fig. 3b and c. The insulin-loaded nanovesicles had the spherical shape and

electrodes were immersed in samples in the acrylic cells and the current was applied using a constant power supply. The anodes were placed in the cell with electropositive vesicles, while the cathodes were placed in the other cell. One treatment control group received 2.0 ml PBS (pH 7.4) solution containing 80 IU insulin and the negative control groups two cells received 2.0 ml fresh physiological saline. In the treatment group, the nanovesicles (ILV4) (2.0 ml, 40 IU/ml) were respectively applied using the microneedles, iontophoresis, microneedles/iontophoresis, or without any physical permeation enhancing method. At various time points after treatment (0, 1, 2, 3, 4 and 6 h), blood was drawn from the tails and assayed for blood glucose as described above. 2.10. Fluorescence imaging Insulin-FITC was incorporated into the nanovesicles (ILV4, 40 IU/ ml) or was diluted in PBS (40 IU/ml) for transdermal administration. Insulin-FITC nanovesicles or PBS solution were applied to the exposed abdominal skin of rats using the iontophoresis and microneedles. After 3 h the skins were carefully cleaned with 70% alcohol, harvested and frozen at −20 °C. A freezing microtome (LEICA CM 1900) was used to make the horizontal and vertical sections with the thickness of 8 µm. Fluorescence photomicrographs of the sections were obtained using OLYMPUS IX-70 microscope with the excitation and emission wavelengths of 490 nm and 525 nm, respectively [38]. The same procedure was performed for the skin penetration of PBS solution containing free FITC-insulin. 2.11. Statistical analysis The statistical significance of the differences between groups about in vitro skin permeation and in vivo animal experiments was evaluated by variance analysis, followed by Student's t-test. P b 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Preparation of nanovesicles The insulin-loaded (40 IU/ml insulin) nanovesicles with various diameters and zeta potentials were prepared using high pressure homogenization and ultrasound method (Table 1) [38]. The coarse mixture dispersion consisting of 1.5% soybean lecithin and 3.0% propylene glycol were homogenized using high pressure homogenization (ILV1 obtained at 120 MPa and 8 cycles) and ultrasound method (ILV2 for 6 min and ILV3 for 4 min) in order to obtain the nanovesicles with various average diameters. The nanovesicles with similar zeta potentials (ILV1, ILV2 and ILV3) had an average diameter of 91.0 nm, 134.0 nm and 175.9 nm. The nanovesicles with various charges were obtained by incorporating 0.4% cetyltrimethyl ammonium bromide (CTAB), 0.4% sodium dodecyl sulfate (SDS) and 0.03% SDS into the nanovesicles from ultrasound method. The nanovesicles with similar average diameters (ILV4, ILV5 and ILV6) had zeta potentials of + 27.8 mV, − 50.5 mV and −25.3 mV, respectively. Particle size of these insulin-loaded nanovesicles showed no significant change in 3 months at 4 °C (Supporting information).

Fig. 3. The TEM and HR-TEM images of insulin-loaded nanovesicles. (a) ILV1 consisting of 1.5% soybean lecithin, 3.0% propylene glycol and 40 IU/ml insulin with the average diameter of 91.0 nm and zeta potential of − 16.2 mV, (b) ILV4 at the magnification times of 150,000, (c) the control solution containing free insulin.

particle size of about 60 nm, which were smaller that that obtained from PCS. The HR-TEM images of the insulin-loaded nanovesicles differed from their TEM image. The images showed that the nanovesicles were unilamellar vesicles with membrane thickness of 3∼5 nm. According to the composition and their particle size of nanovesicles (Fig. 3b and c), the small particles might be the insulin hexamers in Fig. 3b [42]. FITC-INS was used to replace insulin in ILV4 for measuring the entrapment efficiency of the nanovesicles by utilizing its fluorescence property. The nanovesicles had a high entrapment efficiency of 89.05 ± 0.91%. The direct homogenization of the mixture of lecithin, propylene glycol and insulin resulted in a high entrapment efficiency of the nanovesicles. But when insulin was added to the blank nanovesicles, only a low entrapment efficiency could be obtained. The order of the addition of the ingredients and insulin play an important role for the encapsulation of insulin. In order to trace the microstructure of nanovesicles and the integrity of lipid bilayers, we encapsulated QDs (Fig. 4a) into nanovesicles and observed the fluorescence imaging of QDs-loaded nanovesicles (Fig. 4b). The fluorescence emission spectrums of QDs-loaded nanovesicles after one day and 30 days at room temperature are shown in Fig. 4c and d. It shows that QDs in nanovesicles still had good fluorescent intensity, which provided a stable hydrophobic environment for lipophilic QDs in the lipid region. The TEM images of QDs-loaded nanovesicles and blank nanovesicles at various magnifications are shown in Fig. 5, respectively. Fig. 5a showed that the QDs-loaded nanovesicles had a spherical or near-spherical shape, which was similar to the TEM image of insulinloaded nanovesicles (ILV1). When the TEM imaging was set at high magnifications, the QDs-loaded nanovesicles showed the clear unilamellar microstructure (Fig. 5b and c). Additionally, QDs also showed uniform arrangement in lipid layers. However, the TEM imaging showed that the blank nanovesicles had no clear microstructure of vesicles (Fig. 5d−f). It shows that QDs as a microstructure

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probe can trace the microstructure of nanovesicles in the TEM imaging and enhance the clarity of microstructure of organic lecithin. The lipid bilayers had a good integrity and showed no significant structure defects of lipid arrangement. It is estimated that the thickness of the lipid membranes of the unilamellar nanovesicles was about 3–5 nm (Fig. 5c), which was similar to the above results and those obtained from small-angle neutron scattering [40]. The stable fluorescence intensity of QDs in nanovesicles implied that the lipid bilayers of 3– 5 nm might be very potential for providing a good encapsulation of aqueous insulin into the caves of nanovesicles. The integrity of lipid bilayers would be valuable for maintaining good drug-loading ability and stability of nanovesicles in the iontophoresis-driven penetration through skins. 3.3. Permeation studies of nanovesicles The in vitro permeation studies were performed for evaluating the permeation rates of insulin through skins. The passive diffusions of insulin from the various nanovesicle formulations and control solution are shown in Fig. 6a. The nanovesicles with various diameters and zeta potentials could penetrate through skins, but the control solution could not penetrate through skins in 5 h. Soybean lecithin as an enhancer can disturb the lipid structure of skins, which might contribute to the decrease of the barrier of stratum corneum. ILV4 with positive zeta potential and small average diameter had the highest permeation rate of 0.19 ± 0.01 μg·cm− 2·h− 1. The permeation rates of ILV5 with high negative zeta potentials was 0.14 ± 0.01 μg·cm− 2·h− 1. However, all the nanovesicles lacked enough ability to deliver insulin through the integrated skin in point of clinical potentials. 3.4. Permeation studies of nanovesicles driven by iontophoresis The permeation rates of insulin from various nanovesicles and control solution through the integrated skins with the facilitation of iontophoresis are showed in Fig. 6b. The permeation rates of insulin

Fig. 4. (a) the TEM image of QDs, (b) Fluorescence imaging of QDs-loaded nanovesicles, (c) Fluorescence emission spectrums of QDs-loaded nanovesicles after one day and (d) Fluorescence emission spectrums of QDs-loaded nanovesicles after 30 days at room temperature.

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Fig. 5. TEM images of QDs-loaded nanovesicles at various magnifications (a, b and c) and TEM images of the blank nanovesicles without QDs at various magnifications (d, e and f).

from nanovesicles with the facilitation of iontophoresis were increased 3.3–5.3 times when compared with those without the facilitation of iontophoresis (Fig. 6a). The combination of nanovesicles and iontophoresis resulted in more powerful ability to enhance the permeation rates of insulin. ILV4 had the highest permeation rate of 0.97 ± 0.05 μg·cm− 2·h− 1. The nanovesicles (ILV4) with smaller diameter and positive zeta potentials had higher permeation rates of insulin under the electrical forces from iontophoresis. The iontophoresis were advantageous to enhance the penetration of positive nanovesicles

Fig. 6. (a) Permeation rates of insulin-loaded nanovesicles and control solution, (b) Iontophoretic permeation rates of insulin from nanovesicles and control solution through integrity skins.

due to the active electroosmosis and electrostatic interaction between skins and nanovesicles [35]. When the negative nanovesicles were driven by the cathode of iontophoresis, the reverse electroosmosis was disadvantageous to the penetration ability of insulin. All the nanovesicles also had higher permeation rates of insulin than control solution when iontophoresis was applied on skins. The iontophoresis did not change the permeation behavior of insulin through skins and still showed a Fick's first law of diffusion, which is described as the equation of J = DKC / h = PC (J is the permeation rate, D is the diffusion coefficient of drug in stratum corneum, C is the concentration of drug in formulations, K is the partition coefficient of drug between skin and formulation, P is the penetration coefficient and h is the thickness of skin). The iontophoresis might enhance the diffusion coefficient of insulin and the nanovesicles could improve the diffusion coefficient of drug. Then, the permeation rates of insulin were enhanced due to the combination of nanovesicles and iontophoresis. It shows that the nanovesicles play an important role in the iontophoretic penetration of insulin, in which lecithin might enhance the lipid fluidity, permeability of skin and electroosmosis [43]. In addition, peptide candidates for iontophoretic delivery should generally have either a pI b 4 or pI N 8, because there is a pH gradient in skin which goes from a weakly acidic ∼5 at the surface to a physiologic 7.4 in the interior, then it is clear that the ionization state of insulin will change as it passes through the skins because of insulin has a pI of ∼5.4. So, the nanovesicles with high entrapment efficiency encapsulated insulin and reduce the charge reversal of insulin in the penetration [44]. Even though the iontophoresis significantly enhanced the permeation ability of nanovesicles, it only induced a limited increase of permeation rate of insulin. Generally, the iontophoresis could only drive the movement of nanovesicles with charges along the skin appendages such as follicular and eccrine routes. The limited space of the integrated skin appendages restricted the enhancing effect of penetration induced by iontophoresis. The Nernst–Plank equation has been used to describe the permeation of ionic drugs with low molecular weights, which is controlled by the electric conditions such as electric force. So, the permeation rates of drug can be optimized by enhancing electric conditions. But, only this optimization might not be able to conquer the insufficient permeation rates of insulin with large

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molecular weight. The penetration abilities of drugs with large molecular weights are also influenced by many other factors. Then, it necessary to explored more effective strategy for enhancing the permeation ability of insulin [7]. 3.5. Permeation studies of nanovesicles combined with microneedles The permeation rates of insulin from various nanovesicles and control solution through the skins with microchannels pretreated by microneedles are shown in Fig. 7. The permeation rates of insulin from these nanovesicles with the facilitation of microneedles were increased 86.1–166.7 times when compared with those without the facilitation of microneedles (Fig. 6 and Fig. 7). The combination of nanovesicles and microneedles resulted in more powerful abilities to enhance the permeation rates of insulin, which might attribute to the skin microchannels pretreated by microneedles. The permeation profiles of insulin from all the nanovesicles through skins accorded with Fick's first law of diffusion (Supporting information) and their permeation time lag ranged from 0.9 h to 1.2 h. The microchannels effectively reduced the skin barrier of stratum corneum and contributed to the increase of diffusion coefficient of insulin, which resulted in the significant increase of the permeation rates. ILV4 with smallest average diameter and positive zeta potentials had the highest permeation rate of 31.68 ± 0.79 μg·cm− 2·h− 1. However, there is no linear correlation between their average diameters and permeation rates. ILV1 had higher permeation rates when compared with ILV5 and ILV6. It implies that the addition of SDS to nanovesicles might weaken the permeation ability of nanovesicles. ILV5 with high negative zeta potentials had the lowest permeation rates of 12.91 ± 0.48 μg·cm− 2·h− 1. Additionally, all the nanovesicles also had higher permeation rates of insulin than control solution when the microneedles were used to pretreat the skins. It shows that the nanovesicles still had significant influence on the penetration of insulin in the penetration of insulin through skin microchannels. According to Figs. 6 and 7, the microneedles had much more powerful ability to enhance the penetration of insulin from nanovesicles through skins than iontophoresis. The microneedles provided a large amount of microchannels, which could contribute to the increase of permeability coefficient of drugs with large molecular weights [31]. In order to obtain higher permeation rates, the skin microchannels induced by microneedles might provide the sufficient channels for iontophoretic transport of nanovesicles in addition to the skin appendages, so it is possible to obtain the synergetic penetration of nanovesicles with facilitation of both iontophoresis and microneedles [45].

Fig. 8. Iontophoretic permeation rates of insulin from nanovesicles and control solution through microneedle-pretreated skins.

of insulin. Fig. 8 shows the permeation rates of the nanovesicles and control solution driven by iontophoresis through the skins with microchannels pretreated by microneedles. All formulations showed the significant increases of penetration abilities in presence of the iontophoresis and microneedles. The permeation rates of ILV1, ILV2 and ILV3 were 76.13 ±5.10 μg·cm− 2·h− 1, 61.13 ±4.25 μg·cm− 2·h− 1 and 60.23± 2.29 μg·cm− 2·h− 1, respectively. ILV1 with smallest diameters had high permeation rates. ILV4, ILV5 and ILV6 had the permeation rates of 106.99 ±2.78 μg·cm− 2·h− 1, 99.86 ± 4.29 μg·cm− 2·h− 1 and 70.44 ±4.58 μg·cm− 2·h− 1, respectively. The positive ILV4 had the highest permeation rate, which is higher 7 times over that from control solution because of the anodic transport of positive nanovesicles. The penetration of ILV4 driven by iontophoresis through skins with microchannels increases the permeation rates of insulin by 3.4–7.1, 92.5–134.9 and 359.6–713.3 times, respectively, when compared with those from nanovesicles combined with microneedles alone, nanovesicles combined with iontophoresis alone and nanovesicles at passive diffusion. There is a smaller difference between the permeation rates of insulin from ILV4 and ILV5 in Fig. 8 when compared with that in Fig. 7. It might attribute to the powerful iontophoretic penetration of insulin-loaded nanovesicles with charges and avoidance of negative electroosmotic flow in skin microchannels. All the permeation profiles followed with Fick's first law of diffusion and the permeation time lag ranged from 1.0 h to 1.3 h (Supporting information). According to Figs. 6–8, iontophoresis, microneedles and nanovesicles showed a significant synergetic effect on the penetration of insulin through skins, which resulted in the high in vitro permeation rates of insulin [45]. The microneedles might have the significant contribution in the synergetic effect, which contribute to the enhancement of diffusion coefficient. The microchannels provided enough space for the iontophoretic penetration of nanovesicles and insulin, which play an

3.6. Permeation studies of nanovesicles combined with iontophoresis and microneedles Both the iontophoresis and microneedles were jointly used to realize the iontophoresis-driven penetration of nanovesicles through microneedle-induced skin microchannels for enhancing transdermal delivery

Fig. 7. Permeation rates of insulin from nanovesicles and control solution through microneedle-pretreated skins.

Fig. 9. The blood glucose levels of various treatment groups (n = 5). (A) Negative control, (B) Free insulin administrated by subcutaneous injection (1.0 IU/kg), (C) ILV4 combined with iontophoresis and microneedles, (D) Free Insulin solution combined with iontophoresis and microneedles, (E) ILV4 combined only with microneedles, (F) ILV4 combined only with Iontophoresis, (G) ILV4 based on passive penetration. Glucose levels of streptozotocin-induced diabetic rats were normalized against the initial (0 h) value.

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important role in the penetration routes of nanovesicles. The nanovesicles also acted as the effective carrier of insulin and permeation enhancer, which resulted in the significant increase of permeation coefficient (Supporting information). The encapsulation of insulin in nanovesicles with high entrapment efficiency could avoid the charge reversal of iontophoretic penetration of insulin through skins. It implies that this strategy is promising for breaking through the limit of pI value of peptides for iontophoresis. The enrichment of nanovesicles consisting of lecithin and propylene glycol in skins could also reduce the skin barrier, which might reduce the partition coefficient and diffusion coefficient. Then, a large amount of insulin-loaded nanovesicles with the drive of iontophoresis could easily penetrate through skins along both the microchannels and stratum corneum routes. Certainly, the electrical force from iontophoresis as the propelling force resulted in the active transport of nanovesicles and insulin through microchannels, which contributed to the enhancement of diffusion coefficient [31]. In addition, the microneedles and iontophoresis resulted in the lowest increase of the penetration of insulin from control solution through skins, even though its permeation rate was only 15.37 ± 0.66 μg·cm− 2·h− 1. They were jointly used to enhance transdermal delivery of drugs with small molecular weights such as oligonucleotide and methotrexate [46,47]. Wu et al reported that both could jointly enhance the in vitro penetration of dextran with various molecular weights through skins and also result in the enrichment of dextran in dermal layers [48]. 3.7. In vivo studies To demonstrate the advantage of this strategy based on the iontophoresis-driven nanovesicles through the skin microchannels, the in vivo studies were performed to validate the change of blood glucose of diabetic rats. The blood glucose levels (BGL) of diabetic rats are shown in Fig. 9. The BGL at various times were normalized against the initial glucose level [49]. According to Fig. 9, ILV4 combined only with microneedles or iontophoresis also resulted in the decrease of BGL at different time and their BGL were 65.6% and 66.5% of their initial values at 6 h, respectively, which is also significantly lower than that of control group (P b 0.05) [7,50]. But their BGL were significantly higher than that of ILV4 driven by iontophoresis through microneedle-induced skin microchannels. It means that the nanovesicles might lack enough ability to penetrate through skins, even though the nanovesicles or insulin molecules released from the nanovesicles can penetrate though skins with the assistance of microchannels or iontophoresis. The free insulin solution combined with iontophoresis and microneedles showed a higher BGL compared to the other treatment groups, whose BGL were decreased to 70.3% and 68% of its initial in 4 h and 6 h (P b 0.05). It implies that the iontophoresis showed a poor ability to drive the free insulin because of its low charges and only the passive diffusion of insulin in skins was insufficient for transdermal delivery, even though a combination of microneedle pretreatment and iontophoresis was reported to enhance the transdermal delivery of free dextrans [45]. The nanovesicles play an important role in the in vivo transport of insulin of the synergetic penetration. ILV4 only with passive penetration also revealed similar BGL to those of ILV4 combined only with iontophoresis or microneedles at 3 and 6 h. Only the weak permeation enhancement of soybean lecithin contributed to the passive penetration of ILV4. As shown in Fig. 9, the various groups show different decreases of BGL. The subcutaneous injection of insulin resulted in a rapid decrease Fig. 10. The fluorescence images of penetration of free FITC-INS and FITC-INS-loaded nanovesicles combination with iontophoresis through skins containing microchannles. (a) the vertical section of skin treated by nanovesicles, (b) the vertical skin section from free FITC-INS, (c) Horizontal section of stratum corneum treated by the nanovesicles, (d) Horizontal section of stratum corneum treated by free FITC-INS, (e) Horizontal section of skin at the depth of about 600 µm treated by the nanovesicles, (f) Horizontal section of skin at the depth of about 600 µm treated by free FITC-INS. The right images are the original microscopy image of the left fluorescence images.

of BGL. The BGL at 2 h was only about 28% of its initial value and the low BGL lasted for at least 6 h (28.3–39.6%, P b 0.001 compared to the negative control group). ILV4 driven by iontophoresis through

microneedle-induced skin microchannels also resulted in a significant decrease of BLG, when compared with the other treatment groups (P b 0.05). The BGL of this group was only 33.3% and 28.3% of initial levels at 4 h and 6 h (P b 0.001 compared to negative control group), which is no significance when compared with those of subcutaneous injection group (P N 0.05). Even though the iontophoresis and microneedles for transdermal delivery of drugs with small molecular weights have been found to be clinically effective, it was still unconquered for transdermal delivery of insulin [7,32,51]. Martanto et al. reported that the free solution containing 100 IU/ml insulin could also result in the BGL decrease of 65% at 4 h by pretreating skins using microneedles [52]. The insulinloaded microneedles were also found to result in a significant decrease of BGL [30,53–55]. The iontophoresis also found to reduce the BGL of rats when combined with electrophoresis and the low BGL could last for 2– 3 h [22]. Our strategy resulted in the significant synergetic effect on the decrease of BGL, which is comparable to the subcutaneous injection of insulin. It shows that this strategy has a powerful penetration for transdermal delivery of insulin and implies a promising potential for clinical therapy of diabetes by transdermal administration. There was also a significant difference of time lag between different groups. The subcutaneous injection of insulin and ILV4 combined with iontophoresis and microneedles showed a rapid decrease of BGL. The other groups showed a slow decrease of BGL compared to the above. It reveals that the nanovesicles combined with pretreatment of microneedles and iontophoresis might had higher in vivo permeation rates of insulin through skins and implies this strategy can decrease the time lag, which can usually delay the therapy in transdermal delivery. Additionally, the profile of group C in Fig. 9 showed a steady decrease, which might be more advantageous to the therapy of diabetes and avoidance of adverse side effects when compared with subcutaneous injection of insulin. The in vitro permeation studies showed that the microneedles resulted in the most significant role when compared with the nanovesicles and iontophoresis. However, the in vivo studies showed similar effects on the decrease of BGL of diabetic rats between microneedles, iontophoresis and nanovesicles. Both the in vitro and in vivo studies showed that the penetration of the insulin-loaded nanovesicles driven by iontophoresis could result in the synergetic effect for transdermal delivery of insulin. The nanovesicles may carry a large amount of insulin molecules within them and the microneedleinduced microchannels open a routeway for the movement of nanovesicles in skin. The drive of iontophoresis leads to the active penetration of nanovesicles along the routeway. The comparable effects on BGL between nanovesicles and subcutaneous injection imply that this strategy provides a valuable alternative for the future clinical consideration. Furthermore, this strategy opens a general active route for transdermal delivery of peptides.

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it also have a significant enrichment of fluorescence at the surface of skins or along the microchannels. It validates that the nanovesicles play an important role and mainly carry insulin to the deep layers of skin. Horizontal sections (sectioning parallel to the skin surface) at various skin depths (Fig. 10c–f) showed FITC-INS from both nanovesicles and free solution can permeate into the epidermis and dermis. The fluorescence images of stratum corneum (Fig. 10c and d) from nanovesicles and free FITC-INS revealed that nonuniform fluorescence distribution, respectively. The fluorescence might derive from the penetration of nanovesicles and free FITC-INS along microchannels and also hair follicles. Fig. 10e and f showed the fluorescence of sections from the nanovesicles and free FITC-INS at the depth of about 600 µm in skins, respectively. The nanovesicles resulted in stronger fluorescence intensity (Fig. 10e) than free FITCINS (Fig. 10e). Both fluorescence images showed a uniform distribution, which are different from their images at stratum corneum. The in vivo and in vitro results validates that the nanovesicles with the assistance of iontophoresis can penetrate into the deep layers of skin along the microchannels and then insulin might be released from nanovesicles in skins. The insulin molecules further diffuse through skins and also uniform the distribution in the deep layers of skins. This strategy shows an active delivery for non-invasive delivery of insulin via the multimodal permeation enhancing routes, which is expected to conquer formidable barrier of skins. 4. Conclusions In summary, the insulin-loaded nanovesicles with the high entrapment efficiency of 89.05 ± 0.91% were constructed and characterization. The microneedle array was used to induce the skin microchannels, which could enhance the penetration of insulin through skin significantly. The positive zeta potential and small diameters of nanovesicles are significantly advantageous to the penetration of the insulin-loaded nanovesicles when combined with iontophoresis and microneedles. Both the in vitro and in vivo studies showed that the penetration of the insulin-loaded nanovesicles driven by iontophoresis could result in the synergetic effect for transdermal delivery of insulin, due to the increase of diffusion coefficient and partition coefficient. The nanovesicles with the facilitation of iontophoresis can penetrate into the deep layers of skin along the microchannels and then insulin might be released from nanovesicles in skins, followed by the penetration through skins. This strategy provides a general active route for transdermal delivery of noncharge peptides by establishing iontophoresis-driven nanoparticles with charges through skins containing microchannels. This work also provides an encouraging support for the future clinical development of novel therapy strategy of diabetes and shows a conceptual advance in the field of transdermal delivery of peptides. Acknowledgments

3.8. Fluorescence imaging The fluorescent observation of the skin sections were used to trace the penetration of free FITC-insulin and FITC-INS-loaded nanovesicles driven by iontophoresis through skins with microneedle-induced skin microchannels in order to investigate the mechanism of penetration [38,49]. The fluorescence imaging is shown in Fig. 10. The vertical skin sections (sectioning perpendicular to the skin surface, Fig. 10a) from FITC-INS-loaded nanovesicles showed high fluorescence intensity of FITC along microchannels of skins. FITC-INSloaded nanovesicles (Fig. 10a) might deeply penetrate into epidermis and dermis. Then, the more amounts of insulin molecules from nanovesicles can penetrate through skins. The vertical section from the free FITC-INS solution (Fig. 10b) showed weak fluorescence intensity and reveals a poor penetration of free FITC-INS when compared with that from nanovesicles. Additionally, a low fluorescence distribution outside microchannels at the deep layers of skins was also observed, even though

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