Transdermal delivery of estradiol-loaded PLGA nanoparticles using iontophoresis for treatment of osteoporosis

Abstract

Background:

Estradiol is one of the therapeutic agents for osteoporosis. We have reported transdermal permeability of estradiol-loaded nanoparticles, and permeability effect of estradiol was enhanced by using nanoparticle system and iontophoresis [Colloids and Surfaces B: Biointerfaces 97 (2012), 84–89].

Objective:

This study was conducted in vivo to evaluate therapeutic efficacy of the estradiol-loaded PLGA nanoparticles for osteoporosis.

Methods:

Prior to the in vivo study, we have determined the surface charge density of the particles and found they have negatively charged polyelectrolyte layers on the surfaces. Ovariectomized female Sprague–Dawley rats were used as an animal model of osteoporosis. They were separated into three groups by administration route of estradiol-loaded PLGA nanoparticles, passive diffusion group, iontophoresis group and control. After treatment, we have measured bone mineral density of spine using an X-ray computed tomography system.

Results:

Bone mineral density after iontophoresis was significantly higher than that of passive diffusion and control group. By usage of iontophoresis, the nanoparticles were permeated through follicles and migrated into capillary vessel around follicles, and the loaded drug reached effective blood concentration in plasma of rat.

Conclusions:

From this study, we found that the combination with charged nanoparticle system and iontophoresis is useful to osteoporosis treatment.

Keywords

Estradiol transdermal administration iontophoresis osteoporosis nanoparticle therapeutic efficacy

1. Introduction

Postmenopausal osteoporosis is a major public health problem, which occurs by ovarian hormone deficiency [2,3]. The risk of fractures increases when bone mineral density is reduced. Prevention of osteoporosis is important, especially in countries in which society is aging [4]. 17 β-estradiol (E2) is used to prevent and treat this disease as a drug of hormone replacement therapy (HRT). Oral administration is one of the most major route of drug administration. This drug is significantly affected by first-pass hepatic metabolism, and high dose administration must be needed to obtain drug efficacy [5]. Intravenous administration of estradiol is also difficult because it causes rapid increase of drug level in blood followed by rapid elimination. These administration routes have possibility of inducing adverse effect such as thrombosis, endometriosis and uterus carcinoma. Therefore, alternative administration route is needed to keep suitable drug level in blood. We have focused on the transdermal drug delivery system and studied estradiol-loaded poly (lactide-co-glycolide) (PLGA) nanoparticles using iontophoresis [1,6–9]. Transdermal delivery can avoid the effect of first-pass hepatic metabolism, and deliver therapeutic agent as systemic or local administration for long period of time [10]. The major barrier to transdermal delivery is stratum corneum of skin. This superficial layer provides reduction of the percutaneous absorption, and metabolic activity of the skin assists this function. Enhancing skin permeability is required at transdermal administration of many candidate agents. To enhance the percutaneous absorption of drugs, chemical penetration enhancer has been studied. These chemical compounds interact with stratum corneum after partition, and reduce the resistance of skin to drug diffusion [11,12]. However, chemical enhancers have the risks of inducing irritation and reducing the skin barrier function in the case of using long-term administration drugs. In transdermal administration, advantages of the PLGA nanospheres were demonstrated. It improves permeability of medical agents, protects unstable agents in the particles, controls release ratio of active agent from the carrier and can use iontophoresis technique [1,6,13,14]. Iontophoresis, which promotes skin permeability by using electropotential energy [15–17], is one of the physical penetration enhancers. Electroporation by producing small pore on the surface of stratum corneum by adding pulse voltage [18,19], and sonophoresis by using the effect of cavitation of ultrasonic wave [20,21] have also been studied. Recently, to bypass the stratum corneum, microneedle treatment has been studied as a new physical penetration enhancement method composed many materials (for example, silicon, glass, stainless steel and biodegradable polymers) [22,23]. We have reported combination use of nanoparticle system and iontophoresis promoted skin permeability of indomethacin [6,7] and estradiol [1].

In this report, therapeutic efficacy of estradiol-loaded PLGA nanoparticles for osteoporosis was studied. Average diameter of the selected nanoparticles was 160 nm, and they were stable in physiological saline at 32°C as we have reported [1]. We have measured electrophoretic mobility of the particles at nine different ionic strengths and analyzed them to evaluate the surface charge density of the particles. An animal model for osteoporosis was produced from female rat because its pathophysiological response of skeleton is similar to that of human [24]. To evaluate the therapeutic efficacy, we have measured bone mineral density of spine using computed tomography (CT).

2. Materials and methods

2.1. Materials

PLGA (Mw: 10,000, monomer composition of lactic acid/glycolic acid = 75/25), polyvinyl alcohol (PVA, degree of polymerization: 500), trehalose dihydrate (C₁₂H₂₂O₁₁·2H₂O, purity > 97%), indomethacin (C₁₉H₁₆ClNO₄, purity > 98%), dichloromethane (CH₂Cl₂, purity > 99.5%), toluene (C₆H₅CH₃, purity > 99.5%), heparin sodium, potassium dihydrogen phosphate (KH₂PO₄, purity > 99.5%), disodium hydrogenphosphate 12-water (NaHPO₄·12H₂O, purity > 99%), trisodium citrate dihydrate (C₆H₅Na₃O₇·2H₂O, purity > 99%) and citric acid monohydrate (C₆H₈O₇·H₂O, purity > 99.5%) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). 17 β-estradiol (C₁₈H₂₄O₂, purity > 97%) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Acetonitrile (CH₃CN, JP, USP/NF, EP) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Pentobarbital sodium salt (C₁₁H₁₇N₂NaO₃, purity > 95%) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Physiological saline (Otsuka normal saline, JP) was purchased from Otsuka Pharmaceutical Co., Ltd (Tokyo, Japan).

2.2. Preparation of 17 β-estradiol-loaded PLGA nanoparticles

17 β-estradiol-loaded PLGA nanoparticles were prepared by using emulsion solvent evaporation method [1]. Briefly, 490 mg of PLGA and 10 mg of 17 β-estradiol were dissolved in 20 mL of dichloromethane. The solution was added to 100 mL of 1.0 (w/v%) PVA aqueous solution, and was emulsified using a probe sonicator (Digital Sonifier S-250D, Branson Ultrasonics Co., Danbury, CT) at 100 W of energy output for 1 min on ice bath. Prepared O/W emulsion was stirred during three hours on a magnetic stir plate at room temperature to evaporate dichloromethane. Obtained nanoparticle suspension was ultracentrifugated at 20,000 rpm for 20 min (Himac 80WX, Hitachi Koki Co. Ltd, Tokyo, Japan). After centrifugation, the precipitated nanoparticles were washed with distilled water in ultrasonic bath sonicator to remove residual PVA. This centrifugation and wash handling were repeated three times. The precipitation of nanoparticles were redispersed in distilled water, and trehalose dihydrate with equal weight of precipitated nanoparticles was added. After freezing at −30°C, this suspension was dried using freeze dryer (FD-1000, Tokyo Rikakikai Co., Ltd, Tokyo, Japan).

2.3. Evaluation of the estradiol-loaded PLGA nanoparticles

Surface properties of nanoparticles were observed using scanning electron microscope (SEM, JSM-6060LA, JEOL Ltd, Akishima, Japan). The mean volume diameter, the size distribution and the electrophoretic mobility of the prepared estradiol-loaded PLGA nanoparticles were measured using a zeta-potential and particle size analyzer (ELSZ-2, Otsuka Electronics Co., Ltd, Osaka, Japan). The electrophoretic mobility of the particles were measured in phosphate-buffered solutions (PBS, pH 7.4) with nine different ionic strengths ( $I = 0.005, 0.01, 0.02, 0.04, 0.06, 0.075, 0.0.1, 0.125, 0.154 M$ ), at 32°C, skin surface temperature [25]. The loading ratio of 17 β-estradiol in the prepared particles were measured using high-performance liquid chromatography (HPLC, SIL-20A prominence, SPD-20A prominence, LC-20AD prominence, CTO-10ASvp, DGU-20A₃ prominence, SHIMADZU Co., Kyoto, Japan) at 281 nm with an ODS column (Develosil ODS-HG-5, size: 4.6 mm–150 mm, Nomura Chemical Co. Ltd, Seto, Japan). To prepare mobile phase, 0.1 M of acetic acid solution was mixed with equivalent amount of acetonitrile. 2 μg/mL indomethacin solution, made of indomethacin powder and mobile phase, was used as an internal standard substance solution. 15 mg of prepared estradiol-loaded PLGA nanoparticles were dissolved in 5 mL of mobile phase. Then, 0.1 mL of this solution and 0.1 mL of internal standard substance solution were added to 1.8 mL of mobile phase, and 17 β-estradiol concentration was measured at 40°C (flow rate of HPLC: 1.5 mL/min). All measurements were repeated three times.

2.4. Animal experiments

Female Sprague-Dawley rats, approximately 7 weeks old and averaging 300 g in weight, were purchased from Japan SLC Inc. (Tokyo Japan) and animal care was conducted under the Guidelines for Animal Experimentation of Tokyo University of Science, which are based on the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science. They were ovariectomized and then housed in stainless steel cages under standard environmental conditions ( $23 \pm 1 ° C$ , $55 \pm 5 %$ humidity and a 12/12 h light/dark cycle) and maintained with free access to water and a low-calcium diet (0.02%, PMI Nutrition International., St. Louis, MO) for 50 days. After 3 days from ovariectomy, rats were separated into three groups by administration route of estradiol-loaded PLGA nanoparticles, passive diffusion group (PD), iontophoresis group (IP) and control. Rats of the PD and IP groups were subcutaneously injected 64.8 mg/kg rat weight of pentobarbital sodium salt, which is dissolved in physiological saline, and shaved their abdominal skins. In the PD group, square gauze sheet (2 cm × 2 cm) with 12.0 mg of prepared nanoparticles which were redispersed in 1.0 mL of PBS (pH 7.4, $I = 5 mM$ ) was placed on the skin and diffused passively for 6 hours. In the IP group, two square gauze sheets (2 cm × 2 cm) were placed on the skin. The samples of 12.0 mg were redispersed in 1.0 mL of PBS (pH 7.4, $I = 5 mM$ ), and then it was added on the cathode-side gauze (donor side) and 1.0 mL of physiological saline was added on the anode-side gauze (receptor side). Ag/AgCl electrodes were used and iontophoresis was performed at constant voltages of 3 V/cm for 6 hours. Both administrations were carried out twice a week.

2.5. Evaluation of therapeutic efficacy of estradiol-loaded PLGA nanoparticles for osteoporosis

Therapeutic efficacy was evaluated to measure bone mineral density of spine using an X-ray CT system (LaTheta LCT-200, Hitachi Aloka Medical Ltd, Mitaka, Japan). The bone mineral density was calculated from 10 CT images which were obtained by scanning at 160 μm intervals.

Fig. 1.

SEM image of prepared estradiol-loaded PLGA nanoparticles.

Fig. 2.

Electrophoretic mobility of prepared estradiol-loaded PLGA nanoparticles. Solid line is theoretical results calculated with $z N = - 3.59 \times 10^{- 3} M$ and $1 / λ = 1.82 nm$ ( $n = 3$ ).

3. Results and discussion

3.1. Characterization of prepared 17 β-estradiol-loaded PLGA nanoparticles

We have prepared estradiol-loaded PLGA nanoparticles with mean volume diameter of $165.0 \pm 13.1 nm$ . Figure 1 shows SEM image of the particles. We have observed spherical dispersed particles. The yield and the estradiol content of the particles were $63.4 \pm 3.09 %$ and $1.66 \pm 0.18 w/w%$ , respectively. Electrophoretic mobility μ was also measured and the results were shown in Fig. 2. The data were analyzed by using Ohshima’s electrokinetic theory for soft particles [26]. At a colloidal particle covered with a layer of polyelectrolyte chains moving in a liquid containing a symmetrical electrolyte of valency v and bulk concentration (number density) n (m⁻³), ionized groups of valency z are uniformly distributed at a number density of N (m⁻³). Therefore, it is expressed as $\begin{matrix} (1) & μ = \frac{ε_{r} ε_{0}}{η} \frac{ψ_{0} / κ_{m} + ψ_{DON} / λ}{1 / κ_{m} + 1 / λ} f (\frac{d}{a}) + \frac{z e N}{η λ^{2}} \end{matrix}$ with $\begin{array}{l} (2) & f (\frac{d}{a}) = \frac{2}{3} [1 + \frac{1}{2 {(1 + (d / a))}^{3}}], \\ (3) & ψ_{DON} = \frac{k T}{v e} ln [\frac{z N}{2 v n} + {{(\frac{z N}{2 v n})}^{2} + 1}^{1 / 2}], \\ (4) & ψ_{0} = \frac{k T}{v e} (ln [\frac{z N}{2 v n} + {{(\frac{z N}{2 v n})}^{2} + 1}^{1 / 2}] + \frac{2 v n}{z N} [1 - {{(\frac{z N}{2 v n})}^{2} + 1}^{1 / 2}]), \\ (5) & λ = {(γ / η)}^{1 / 2}, \\ (6) & κ_{m} = κ {[1 + {(\frac{z N}{2 v n})}^{2}]}^{1 / 4}, \\ (7) & κ = {(\frac{2 n e^{2} v^{2}}{ε_{r} ε_{0} k T})}^{1 / 2}, \end{array}$ where a is the particle diameter, d is the thickness of the ionpenetrable surface layer, η is the viscosity, λ is the frictional coefficient of the surface layer, $ε_{r}$ is the relative permittivity of the solution, $ε_{0}$ is the permittivity of a vacuum, $Ψ_{DON}$ is the Donnan potential of the surface layer, $Ψ_{0}$ is the potential at the boundary between the surface layer (it means the surface potential of the polyelectrolyte-coated particle) and the surrounding solution and k is the Boltzmann constant. The parameter κ is the Debye–Hückel parameter, and $κ_{m}$ shows the Debye–Hückel parameter in the polyelectrolyte layer. We have determined the density of fixed-charges $z N$ and the reciprocal of λ (λ: the degree of friction exerted on the liquid flow in the polyelectrolyte layer of the particles). $1 / λ$ has the dimension of length, and shows the permeability of the polyelectrolyte layer. Therefore, it can be considered as the parameter of “softness” of particle surface [26–28]. In previous report, curve-fitting procedure was used to determine $z N$ and $1 / λ$ [29,30]. We have applied it to our measurement result of electrophoretic mobility of estradiol-loaded PLGA nanoparticles (Fig. 2). From the result of the procedure (shown as a solid line in Fig. 2), $z N = - 3.59 \times 10^{- 3} M$ and $1 / λ = 1.82 nm$ were obtained. It was found that prepared particles were “soft particles” and have negative charged polyelectrolyte layer [31,32].

Table 1
Bone mineral densities in rats (mean ± S.D., $n = 3$ )

Bone mineral density of total bone (mg/cm³) Bone mineral density of cortical bone (mg/cm³) Bone mineral density of cancellous bone (mg/cm³)

Control 203.2 ± 19.5 314.1 ± 21.0 151.5 ± 14.2

PD 214.5 ± 8.6 313.2 ± 3.0 157.5 ± 9.7

IP 247.8 ± 17.7 372.4 ± 25.9 175.7 ± 6.1

	Bone mineral density of total bone (mg/cm³)	Bone mineral density of cortical bone (mg/cm³)	Bone mineral density of cancellous bone (mg/cm³)
Control	203.2 ± 19.5	314.1 ± 21.0	151.5 ± 14.2
PD	214.5 ± 8.6	313.2 ± 3.0	157.5 ± 9.7
IP	247.8 ± 17.7	372.4 ± 25.9	175.7 ± 6.1

Fig. 3.

Bone mineral densities in rats. The data were analyzed by Dunnett’s multiple comparison test. ^∗ $P < 0.05$ ( $n = 3$ ).

3.2. Therapeutic efficacy of the estradiol-loaded PLGA nanoparticles

As shown in Table 1 and Fig. 3, bone mineral density in the rats of IP group was significantly different from that of control group ( $P < 0.05$ ). This therapeutic efficacies were observed in total bone and cortical bone. In IP group, it is suggested that transdermal permeability of estradiol-loaded PLGA nanoparticles was enhanced by the application of iontophoresis and loaded estradiol inhibited bone resorption of ovariectomized rats. The possible pathways of drug in the stratum corneum are transcellular pathway, paracellular pathway and transaccessory pathway. Nanoparticles penetrate into skin through transaccessory pathway [1]. Therefore, we assume that the nanoparticles were permeated through follicles and migrated into capillary vessel around follicles, and the loaded drug was confirmed in plasma of rat. In cancellous bone, significant difference has not been observed. This result is considered to be related to the greater loss of cancellous than cortical bone in ovariectomized rats [33]. Efficient transdermal penetration is required, and a method of analysis of particle surface condition using Ohshima’s electrokinetic theory for soft particles became an effective tool to develop more effective transdermal delivery of estradiol.

4. Conclusion

17 β-estradiol-loaded PLGA nanoparticles with negative charged polyelectrolyte layer show therapeutic efficacy for ovariectomized rats by usage of iontophoresis. From this result, we found that the combination with charged nanoparticle system and iontophoresis is useful to osteoporosis treatment. This technique will contribute development of effective transdermal therapy.

Footnotes

Acknowledgement

This work was supported by Program for Development of Strategic Research Center in Private Universities supported by MEXT (2010-2014, Grant Number: S1001019).

Conflict of interest

The authors have no conflict of interest to report

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