Development of a novel solid lipid nanoparticles-loaded dual-reverse thermosensitive nanomicelle for intramuscular administration with sustained release and reduced toxicity

Abstract

To develop a novel solid lipid nanoparticles (SLNs)-loaded dual-reverse thermosensitive nanomicelle (DRTN) for intramuscular administration of flurbiprofen with sustained release and reduced toxicity, the DRTN was prepared with flurbiprofen-loaded SLNs, poloxamer 407 (P 407), poloxamer 188 (P 188) and water. Its rheological characterization, release, stability, pharmacokinetics and morphology were evaluated after intramuscular administration to rats. These SLNs were solid at 25 °C and transformed into liquid form at physiological temperature due to their melting point of about 32 °C. Furthermore, the DRTN retained a liquid state at 25 °C and gelled inside the body owing to its gelation temperature of about 34.7 °C, leading to an opposite reversible property of the SLNs. When compared to the hydrogel, it significantly decreased the drug release and exhibited a reduced initial fast release. It sustained a high plasma concentration for 60 h, which was significantly higher when compared to the suspension, indicating enhanced bioavailability. However, it showed a lower plasma concentration, AUC, and C_max values than that found for the hydrogel, suggesting the retarded release and decreased side effects of the drug. Unlike the hydrogel, it induced no injury to rat muscle as a result of no direct contact of the drug. It was stable for four months. Therefore, this novel DRTN system could be a strong candidate for the intramuscular administration of flurbiprofen.

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1. Introduction

Hydrogel systems are the cross-linked networks composed of various polymers in aqueous solutions by means of intermolecular interactions, hydrogen, ionic bonding or hydrophobic forces.^1,2 In general, conventional thermosensitive hydrogels have been successfully developed using poloxamer solutions as a potential base.^3,4 They are easy to administer to the body and gel quickly, because they undergo a sol-to-gel conversion in reaction to temperature deviations. They are used as controlled release drug delivery systems for injectable,^5,6 ocular,⁷ and rectal administration.^8–10 Moreover, they show an enhanced bioavailability for the drug with appropriate safety in rats and human subjects.^2,8,11 However, in these conventional hydrogels, the drug is directly soluble or dispersed in an aqueous medium and thus may cause direct contact of the toxic drug to body tissue, leading to induced irritation or damage to the body tissue.¹² Furthermore, this may give an initial fast release and higher C_max value for the drug, resulting in increased intrinsic side effects for the drug.^8–10 Therefore, a product without an initial burst effect and direct contact of the toxic drug to body tissue must be developed.

In this study, to solve these serious problems, we have developed a novel dual-reverse thermosensitive nanomicelle (DRTN) system for intramuscular administration. The DRTN system is a dispersion of thermosensitive drug-loaded solid lipid nanoparticles (SLNs) in a thermosensitive poloxamer-based nanomicelle. The SLNs and nanomicelle in this DRTN system exist in a solid and liquid state at room temperature and reversely transform to a liquid and gel state at body temperature, respectively. Thus, the SLNs and nanomicelles in the DRTN system may prevent the contact of the drug with body tissue, leading to the reduced toxicity of the drug. Moreover, they regulate the release of the drug without a burst effect, which can reduce the initial fast release and C_max value of the drug, leading to decreased side effects. In addition, this system is administered easily to the body like a conventional injectable gel, as it exists in a liquid state at room temperature and changes to a gel state at body temperature. The SLN system has gained increasing interest as a colloidal drug carrier.^13,14 The nanoparticles were in the submicron size range (50–1000 nm) composed of biocompatible lipids. They were stabilized with non-toxic surfactants such as poloxamer and lecithin.¹⁵ The main advantages of SLNs include high absorption, appropriate tissue distribution, suitable pharmacokinetics, and biocompatibility.^14,16 Flurbiprofen, a widely used non-steroidal anti-inflammatory drug, was chosen as a model drug in this study. Because of its low aqueous solubility, flurbiprofen gave relatively weakened bioavailability and induces irritation to the mucous tissues of the human body.^17,18

2. Experimental section

2.1. Materials

Flurbiprofen was kindly donated by Kolon Life Science, (Gwacheon, South Korea). Poloxamer 407 (P 407) and poloxamer 188 (P 188) were purchased from BASF (Ludwigshafen, Germany). Tricaprin and triethanolamine were obtained from Tokyo Chem. Inc. (Tokyo, Japan). Semipermeable membrane tubs were obtained from Spectrum Laboratories, Inc. (Los Angeles, CA, USA). All the other chemicals were of reagent grade and were used without extra purification.

2.2. Animals

Male Sprague-Dawley rats (270 ± 20 g, 6–7 weeks old) were obtained from Nara Biotech (Seoul, South Korea) and had easy access to a usual standard laboratory diet and tap water. The animals were kept in a controlled environment at 23–26 °C and 50–55% humidity throughout the research. The procedures for the animal studies were performed with the National Institute of Health (NIH) Policy and Animal Welfare Act under the endorsement of the Institutional Animal Care and Use Committee (IACUC) at Hanyang University.

2.3. Preparation of flurbiprofen-loaded DRTN

Flurbiprofen (100 g) was added into 150 g of a lipid mixture of tricaprin and triethanolamine in a weight ratio of 8 : 2 at 45 °C. This drug loaded-lipid mixture was dispersed in 13 g of poloxamer solution (P 407, P 188, and water in a weight ratio of 15/25/60) at 45 °C using Ultra-Turrax (IKA, Guangzhou, China) and homogenized using a high pressure homogenizer (Emulsiflex B15, Avestin; Ottawa, ON, Canada) with a pressure drop of 500–1000 bar at 45 °C for three cycles.^19,20 Then, it was cooled down to room temperature to obtain the flurbiprofen-loaded SLNs dispersion.²¹ Subsequently, 15 g P 407 and 25 g P 188 were dissolved in 50 g of distilled water at 4 °C with mild stirring. Then, 10 g of the flurbiprofen-loaded SLNs dispersion was added to this poloxamer solution and kept overnight at 4 °C until a clear solution was obtained,⁸ leading to the development of the DRTN.

2.4. Measurement of gelation temperature

The gelation temperature was determined as previously reported.¹⁰ Briefly, 4 g of DRTN was placed in a 10 ml transparent glass vial with a magnetic bar (10 × 3 mm). The transparent glass vial was set in a low-temperature water bath and a digital thermometer (IKA ETS-D5; IKA, Guangzhou, China) was placed in the DRTN. The system was gradually heated at a constant rate of 1 °C min⁻¹ with a constant stirring of 50–70 rpm. The point at which the magnetic bar stopped rotation was noted as the gelation temperature.

2.5. Rheological behaviour of DRTN

A Brookfield Viscometer LVDV-II+P (Middleborough, MA, USA) was employed to determine the rheological behavior of the DRTN at 25 °C and 36.5 °C. A revolving water bath (WC-05G, Daejeon, South Korea) was used to control the temperature. Software (RHEOCALC; Lorch, Germany) was employed to determine the rheological parameters. The equipment was set up as explained previously.⁹

2.6. Transmission electron microscopy (TEM)

A transmission electron microscope (TEM) (Hitachi H-7600; Tokyo, Japan) was used at an accelerating voltage of 100 kV to observe the morphology of the DRTN. A drop of the DRTN was placed on a carbon-coated copper grid to adhere the particles to the carbon substrate. Negative staining was carried out using 2% phosphotungstic acid solution.

2.7. Release test

A release test was performed using the paddle method. It was executed at 36.5 °C and 100 rpm with 500 ml of phosphate buffer (pH 7.4) containing 0.3% Tween 80 as a dissolution medium in the dissolution tester (VISION-6 Classic, Chatsworth, CA, USA).^8,9,22 Each DRTN, hydrogel, and suspension containing 40 mg of flurbiprofen was introduced into a semipermeable membrane tube. DRTN was comprised of [SLN/P 407/P 188/H₂O (10/15/25/50%)], which enclosed 1% flurbiprofen. The hydrogel was composed of 1% drug, 15% P 407, 25% P 188, and 59% water. The suspension was composed of a 1% drug-loaded sodium carboxymethylcellulose (sodium CMC) aqueous solution. A clamp was used to tie both sides of the tube to prevent leakage, and then the tube was positioned in the dissolution tester. Two ml of the samples were withdrawn and filtered at 1 h intervals. The filtrate was examined using HPLC as stated below.

2.8. Pharmacokinetics

Eighteen rats were divided into three groups and were fasted for 24 h. These rats were intramuscularly administered with DRTN, hydrogel, and suspension at a dose of 12 mg kg⁻¹ in the gluteus maximus muscle. Sequential blood samples (300 μl) were obtained from the vein of the right or left ear at specific time periods and centrifuged at 9000 g for 10 min to obtain plasma using a centrifuge (Smart 15 micro centrifuge, Hanil Science Industrial Co.; Incheon, South Korea). Plasma was reserved at −18 °C prior to HPLC analysis. Plasma (150 μl) was mixed with acetonitrile (250 μl) and 10 μl of acetonitrile solution containing naproxen (40 μg ml⁻¹) as an internal standard. The mixture was centrifuged at 9000 g for 10 min, and the supernatant (10 μl) was injected into the inertsil ODS-4 C18 column (GL Science, 5 μm, 4.6 × 250 mm). The chromatograph was comprised of a high-performance liquid chromatograph (Agilent 1260 Infinity; Santa Clara, CA, USA) consisting of a variable wavelength detector (G1314B 1260) and a Quat Pump (G1311C 1260). The mobile phase was a mixture of 40% double distilled water adjusted to pH 2.6 with acetic acid and 60% acetonitrile. The effluent was observed at 245 nm at a flow rate of 1.0 ml min⁻¹.²³

2.9. Morphology characterization

The rats were intramuscularly administered with DRTN and hydrogel for 96 h. After 96 h of dosing, the muscle was entirely isolated, cleaned with a saline solution, fixed with 10% neutral carbonate-buffered formaldehyde, then embedded in paraffin, serially sectioned (3–4 μm), and stained with hematoxylin and eosin for overall characterization²⁴ or Masson's trichrome for collagen fibers.^25,26 The muscular tissue without dose administration was used as a control. Next, morphological evaluation of each sample was performed under a light microscope (E400, Nikon, Tokyo, Japan). Any damages to muscle tissues were observed based on degenerative lesions including muscle fiber necrosis, lysis and atrophy, inflammatory cell infiltrations, and fibrosis-collagen depositions.^24,27 To observe more detailed changes, the mean muscle fiber thicknesses (μm), mean number of muscle fibers (fibers per mm²), mononuclear cells (cells per mm²), and mean collagen-occupied regions (%mm⁻²) in muscle bundles around the hydrogel-applied regions were determined using a computer-based automated image analyzer (iSolution FL ver 9.1, IMT i-solution Inc.; Quebec, Canada).^28,29 Multiple comparison tests for different dose groups were conducted. The Levene test was performed to determine the variance of homogeneity.³⁰ If the Levene test failed to show a significant difference from the variance homogeneity, the data were assessed using a one-way ANOVA test and the least significant difference (LSD) multi-comparison test. In case of a significant deviation from the variance, homogeneity was observed in the Levene test, a non-parametric comparison test and Kruskal–Wallis H test. Statistical analysis was executed using SPSS for Windows (Release 14.0, SPSS Inc., USA).³¹

2.10. Stability

The 1% flurbiprofen-loaded DRTN was stored at 25 °C and 30 °C over a period of four months and analyzed for mean particle size and drug content.

3. Results and discussion

3.1. Preparation of DRTN

Generally, the SLN system was an aqueous colloidal dispersion containing solid lipids and surfactant.³² Similarly, the flurbiprofen-loaded SLN prepared in this study was an aqueous dispersion, in which the drug-loaded lipid mixture was dispersed in a poloxamer solution, resulting in a composition of the drug, lipid mixture, and surfactant in a weight ratio of 10/15/1.3. This SLN had an excellent entrapment efficiency of 91.4% ± 7.2% and nano size of 188.4 ± 4.0 nm (raw data not shown). In the preliminary study, this lipid mixture of tricaprin–triethanolamine at a weight ratio of 8 : 2 gave a melting point of 32.3 °C, suggesting that it was in solid form at 25 °C and changed to liquid form at body temperature. On the other hand, the DRTN system composed of 10% flurbiprofen-loaded SLNs, 15% P 407, 25% P 188, and 50% water was also an aqueous dispersion. This DRTN gave a gelation temperature, the temperature at which the liquid phase was changed to a gel phase,^8,9 of 34.7 ± 0.6 °C. It showed a free-flow characteristic at room temperature, but became a gel at 36.5 °C, suggesting that the hydrogel (poloxamer solution) in this DRTN gave the opposite thermosensitive property when compared to SLN. Thus, as shown in Fig. 1, in this DRTN system, the thermosensitive flurbiprofen-loaded SLNs and hydrogel retained a solid and liquid form at room temperature, respectively. Upon injection into muscle, they transformed oppositely to a liquid and gel form.

Fig. 1 Schematic for the dual-reverse thermosensitive nanomicelle (DRTN) system.

This SLNs-loaded DRTN exhibited a well-sustained round shape with a clear boundary between each particle (Fig. 2A). The flurbiprofen-loaded thermosensitive SLNs were a solid form with a nano size of about 160 nm (Fig. 2B), which were surrounded by a thermosensitive nanomicelle and poloxamer solution. The thermosensitive nanomicelles were formed owing to the gelling properties of the hydrogel, which were liquid at 25 °C. Therefore, such formation of a dually controlled thermosensitive system showed that the poloxamer solution formed a nanomicelle surrounding the thermosensitive SLNs.³³

Fig. 2 Transmission electron microscopy (10 000×) (A) and particle size (B) of DRTN.

In our preliminary study, the threshold of syringe-ability (viscosity at 25 °C) was determined by intramuscularly administering DRTN to rats using a syringe with a needle. It was easy to administer into muscle, beneath the threshold of syringe-ability, without the separation of the needle from the syringe. However, the needle separated from the syringe above the upper threshold value. In this study, the poloxamer-based DRTN had a viscosity of 275.9 ± 3.2 mPa s at 25 °C, which was beneath the threshold in syringe-ability, resulting in easy intramuscular administration of the DRTN to the body.³⁴ As shown in Fig. 3, at physiological temperature, the viscosity of the DRTN rapidly increased with the elapse of time and then maintained a constant intrinsic viscosity, gel strength. This could be due to the gelling effect of the nanomicelle, which was a liquid at room temperature; however, upon exposure to body temperature, it promptly converted into a gel.

Fig. 3 Rheological profile of DRTN at 36.5 °C. The arrow indicates the gel strength, which means the constant intrinsic viscosity of DRTH at 36.5 °C.

From these findings, the DRTN, composed of [SLN/P 407/P 188/H₂O (10/15/25/50%)], gave a suitable gelation temperature and good syringeability. Therefore, it was easy to administer intramuscularly and speedily formed a gel state in the body.

3.2. Release

The release profile of DRTN was compared with the suspension and hydrogel (Fig. 4A). DRTN was composed of [SLN/P 407/P 188/H₂O (10/15/25/50%)], which contained 1% drug. The hydrogel was composed of 1% drug, 15% P 407, 25% P 188, and 59% water. The suspension was composed of a 1% drug-loaded sodium CMC aqueous solution. The hydrogel was clear because the poloxamer mixture played the role of solubilising agent.^9,10 The DRTN significantly increased the release rate of the drug when compared to the suspension. However, this system significantly retarded the drug release when compared to the hydrogel. The SLNs and nanomicelles in this DRTN system, which exist in a solid and liquid state at room temperature, were reversely transformed into a liquid and a gel state at body temperature, respectively. Thus, this DRTN system could dually control the release of the drug by the SLNs and nanomicelles, resulting in the retardation of drug release.

Fig. 4 Release profile (A) and release kinetics (B) of the drug from DRTN, suspension and hydrogel.

To recognize the release mechanism of the drug from the DRTN and hydrogel, we designated the dissolution rate by the following equations: M_tM⁻¹ = ktⁿ, log[M_tM⁻¹] = log k + n log[t], where M_tM⁻¹ is the portion of dissolved drug at time t. The n and k values are an indication of the release mechanism and a characteristic constant of the DRTN, respectively. From the plot of log(M_tM⁻¹) versus log(t) (Fig. 4B), the kinetic parameters n and k were determined. Release becomes rapid as the k value increases.^35,36 The n value of 1 equals to zero-order dissolution kinetics, 0.5 < n < 1 corresponds to a non-Fickian dissolution model, and the n value of 0.5 specifies Fickian diffusion (Higuchi model).³⁵ Table 1 shows the n values were close to 0.5, both in the DRTN and hydrogel, suggesting that, like the hydrogel, the drug in this DRTN was released by Fickian diffusion through the extramicellar aqueous passages of the gel medium.^9,35 Their similar n values suggested that the presence of SLNs might hardly affect the release kinetics (mechanism) of the drug, because thermosensitive SLNs converted rapidly from solid to liquid form at 36.5 °C.^13,14 However, the lower k value indicated that flurbiprofen released more slowly from DRTN when compared to the hydrogel (Table 1). Thus, unlike the single control of hydrogel, the SLN and nanomicelle in the DRTN system could dually control the release of the drug, resulting in a reduced initial fast release and no burst effect.

Table 1 Release kinetic parameters^a

Formulations	Release exponent (n)	Kinetic constant (%h^−n)	Correlation coefficient (r^2)
a Each value represents the mean ± SD (n = 6).
DRTN	0.53	1.17	0.98
Hydrogel	0.50	1.20	0.99

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3.3. Pharmacokinetics

A pharmacokinetic study of DRTN after intramuscular administration at a dose of 12 mg kg⁻¹ drug to rats was carried out and compared to the hydrogel and suspension. Their mean plasma concentration–time profiles and pharmacokinetic parameters are given in Fig. 5 and Table 2, respectively. The DRTN was observed to attain a relatively high plasma concentration of about 18 μg ml⁻¹ at 2 h, followed by a maintained concentration of 18 μg ml⁻¹ for 60 h. Similarly, the hydrogel gave a constant high plasma concentration of about 25 μg ml⁻¹ from 1.75 to 60 h. In addition, the suspension showed a plasma concentration of about 4 μg ml⁻¹ over 48 h, and then gradually decreased to 0.87 μg ml⁻¹ at 96 h.

Fig. 5 Plasma concentration–time profiles for flurbiprofen after intramuscular administration of DRTN, hydrogel, and suspension to rats. Each value represents the mean ± S.D. (n = 6). All plasma concentrations in the DRTN and hydrogel at each time were significantly different from that found in the suspension (p < 0.05).

Table 2 Pharmacokinetic parameters^a

Parameters	Suspension	Hydrogel	DRTN
a Each value represents the mean ± SD (n = 6). *p < 0.05 compared to the suspension.
AUC (h μg ml^−1)	202.80 ± 27.23	1678.87 ± 575.90*	1196.37 ± 245.21*
tmax (h)	1.33 ± 0.57	1.75 ± 1.67	2.17 ± 1.47
Cmax (μg ml^−1)	3.54 ± 0.72	24.73 ± 6.91*	18.74 ± 3.22*
t1/2 (h)	0.03 ± 0.02	9.43 ± 1.70	10.09 ± 0.67
kel (h^−1)	30.99 ± 10.04	0.07 ± 0.02	0.06 ± 0.04

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All three formulations displayed a sustained plasma concentration, even if they gave significantly different plasma concentrations. At all times, the DRTN and hydrogel gave significantly higher plasma concentrations when compared to the suspension (p < 0.05). Furthermore, the DRTN and hydrogel showed a significantly higher AUC and C_max values when compared to the suspension (Table 2). In particular, these preparations improved about 5.3- and 7-fold bioavailability. The DRTN gave lower plasma concentration, AUC, and C_max values of flurbiprofen than that found for the hydrogel, even if they were not significantly different. In general, a high C_max of toxic drug may cause its intrinsic side effects as well as improve its therapeutic effect.^8–10 Thus, a relatively low C_max of flurbiprofen in the DRTN reduced its intrinsic side effects such as nausea, stomach pain, low fever and loss of appetite.³⁷ The drug in the hydrogel was fast and directly released from it and then absorbed in to the blood vessels of muscle, leading to an initial burst effect of the drug.^8–10 After injection to the muscle, the SLNs and nanomicelle in this DRTN system speedily and reversely changed to a liquid and a gel state, respectively. In the DRTN system, the drug encapsulated in SLNs may move out of the liquid SLNs phase and sequentially move out of gelled nanomicelle phase, followed by absorption into the blood vessels of muscle. Thus, unlike the hydrogel, the DRTN system composed of viscous SLNs and nanomicelles in the muscle could dually regulate the release of the drug without a burst effect, which could reduce the initial fast release and C_max value of the drug, leading to reduced intrinsic side effects of the drug.

3.4. Morphology analysis

The morphology analysis was performed to check any irritation or injury to the muscular tissues in the rats after the administration of DRTN when compared to the hydrogel. The normal muscular tissues without drug administration were used as a control. No abnormal damage or irritation signs were observed in the muscle tissues after intramuscular administration of DRTN, but noticeable focal muscle fiber necrosis and lysis were noticed with the hydrogel (Fig. 6). It was also demonstrated that no meaningful or significant changes in the histological damage scores, mean muscle fiber thicknesses and numbers, mononuclear cell numbers, and mean collagen occupied regions in the muscle bundles were demonstrated in the DRTN when compared with the control (Table 3). However, significantly (p < 0.01) increased histological damage scores and significantly (p < 0.001) decreased mean muscle fiber thicknesses and numbers were observed with the hydrogel when compared with the control (Table 3).

Fig. 6 Morphology of rat muscle: (A) control, (B) after the application of DRTN, (C) after the application of hydrogel. The muscles were isolated, rinsed with a saline solution, fixed in 10% neutral carbonate-buffered formaldehyde, entrenched in paraffin successively sectioned (3–4 μm), and stained with hematoxylin and eosin. Each muscle was observed under a light microscope. MB = muscle bundle; MF = muscle fiber; IS = interstitial. Scale bars = 120 μm. The muscular tissues without administration were used as a control.

Table 3 Morphological analysis of DRTN and hydrogel applied to muscle^a

Morphology	Control	Hydrogel	DRTN
a Each histological value represents the mean ± SD (n = 9). *p < 0.01 and ^#p < 0.001 compared with the control.
Muscle bundles	0.00 ± 0.00	1.89 ± 0.78^#	0.00 ± 0.00
Muscle fiber thickness (μm)	46.30 ± 5.21	30.53 ± 6.55*	46.36 ± 5.05
Muscle numbers (fibers per mm^2)	148.22 ± 13.35	85.89 ± 28.34*	148.89 ± 18.20
Mononuclear cell numbers (cells per mm^2)	46.78 ± 14.31	58.67 ± 18.81	47.22 ± 11.17
Collagen percentage (%mm^−2)	7.24 ± 3.80	7.53 ± 2.98	7.13 ± 3.34

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These findings are viewed as direct confirmation that the DRTN did not cause any local irritation or injury to the applied muscle, but the hydrogel could cause noticeable local irritable signs, focal muscle fiber necrosis, and lysis. In general, flurbiprofen, a NSAID drug, causes local irritation or injury to body tissue.^17,18 Thus, the drug in the hydrogel came into direct contact with the muscle tissue, leading to irritation or injury. However, because the SLNs encapsulated the drug and were dispersed in the nanomicelle, the DRTN system prevented direct contact of the drug with the body tissue, leading to no induced injury or damage to the muscle tissue.

3.5. Stability

The stability of the DRTN was evaluated by the drug content, mean particle size, and physical appearance over four months at 25 and 30 °C (Table 4). There was no noticeable change in the physical appearance of the DRTN during this period. Moreover, no significant and meaningful difference was found in the drug content. The content of flurbiprofen decreased to less than 3%, even at 30 °C, suggesting that the DRTN was stable for at least four months.

Table 4 Stability of DRTN^a

	Particle size (nm)			Drug content (%)
Period (months)	0	2	4	0	2	4
a Each value represents the mean ± SD (n = 3).
25 °C	168.7 ± 7.7	169.9 ± 8.5	169.9 ± 6.9	100	98.03 ± 0.2	97.30 ± 0.4
30 °C	168.5 ± 7.5	169.8 ± 9.3	170.0 ± 5.2	100	97.53 ± 0.8	97.32 ± 0.9

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4. Conclusions

A novel DRTN system composed of SLN and nanomicelles was easy to administer intramuscularly. It retarded the sustained release of the drug when compared to the hydrogel and improved the drug bioavailability when compared to the suspension without a burst effect. Furthermore, it caused no injury or irritation to the muscle, unlike the hydrogel, and remained physically and chemically stable over a period of four months. Thus, this DRTN system with sustained release, reduced toxicity, and excellent stability could be a strong candidate for the intramuscular administration of flurbiprofen.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. 2012R1A2A2A01045658).

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