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Oral and Lymphatic Delivery of Paclitaxel via Lipid Nanocapsules
Yakhak Hoeji 2021;65(5):375-385
Published online October 31, 2021
© 2021 The Pharmaceutical Society of Korea.

Phuong Tran*, Ji-Hun Jang*, Seung-Hyun Jeong*, and Yong-Bok Lee*,#

*College of Pharmacy, Chonnam National University
Correspondence to: Yong-Bok Lee, College of Pharmacy, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, Korea
Tel: +82-62-530-2931, Fax: +82-62-530-0106
E-mail: leeyb@chonnam.ac.kr
Received August 14, 2021; Revised October 18, 2021; Accepted October 22, 2021.
Abstract
Paclitaxel (Ptx) is a potent anticancer drug, especially in breast and ovarian cancers. However, orally administered Ptx presents a major therapeutic challenge because of its low bioavailability. In addition, an adjuvant consisting of Cremophor® EL and dehydrated alcohol is used in current clinical formulations of Ptx, which causes serious side effects. The side effects of Cremophor® EL include hypersensitivity reactions, nephrotoxicity, and neurotoxicity. Therefore, this study prepared lipid nanocapsules (LNCs) containing Ptx to reduce its toxicity and improve bioavailability. The LNCs were characterized using droplet size distribution, zeta potential, drug encapsulation efficiency, stability, differential scanning calorimetry, field emission-transmission electron microscopy, and in vitro release tests. The reference (Ptx) solution and LNCs containing Ptx were orally administered (12 mg/kg) to rats. The plasma and the mesenteric and axillary lymph nodes were obtained, and the concentrations of Ptx in these tissues were measured to compare and evaluate the pharmacokinetics and lymphatic delivery of Ptx. The mean droplet size, zeta potential, and encapsulation efficiency of the optimized Ptx-LNCs were approximately 87.6±6.2 nm, -4.0±0.4 mV, and 90.5±7.8%, respectively. The in vitro release profiles of the prepared Ptx-LNCs were affected by the pH of the dissolution media. Based on in vivo studies, the bioavailability of orally administered Ptx in LNCs was significantly (p<0.05) improved by approximately 2-fold compared to the reference solution. The prepared Ptx-LNCs also showed significantly (p<0.05) higher lymphatic targeting efficiencies than the reference solution. Based on these results, we conclude that the prepared Ptx-LNCs could be a good candidate as an effective oral formulation of Ptx.
Keywords : Paclitaxel, Lipid nanocapsule, Lymphatic delivery, Pharmacokinetics
Introduction

Paclitaxel (Ptx) is an anticancer drug with a diterpenoid pseudoalkaloid structure (Fig. 1).1-5) Ptx was isolated in the early 1960s from the bark of the Pacific Yew (Taxus brevifolia; family Taxaceae). Ptx was identified as the active substance in the bark in 1971.2,6) Ptx is therapeutically active against metastatic breast cancer and is currently being evaluated as an adjuvant and neoadjuvant treatment for early breast cancer.7,8) It has been approved by the US Food and Drug Administration to treat ovarian and breast cancers, Kaposi’s sarcoma, and a wide variety of carcinomas, including lung, colon, prostate, head and neck, cervical, and brain.9,10) Ptx induces a mitotic block by stabilizing the microtubules, blocking the late G2 and M phases of the tumor cell cycle, and decreasing the dynamic nature of these cytoskeletal structures.11) As a major component of the mitotic spindle, microtubules are essential for mitosis in all eukaryotic cells.



Fig. 1. Chemical structure of Ptx.

Furthermore, they are required to maintain cell structure, motility, and cytoplasmic motion within the cell. The synthesis of tubulin and the assembly of microtubules occur during the G2 phase and the prophase of mitosis, which are affected by Ptx, resulting in altered cell division and cell death. Ptx promotes microtubule assembly by binding to and stabilizing microtubules. Ptx binds to microtubules without competing with other microtubule-interacting agents such as colchicine, podophyllotoxin, or vinblastine, suggesting that Ptx binds to a different site on the tubulin molecule.12)

Orally administered Ptx presents a major therapeutic challenge because of its low bioavailability (less than 10%).13) This poor bioavailability results from limited aqueous solubility and dissolution, affinity for the intestinal and liver cytochrome P450 metabolic enzymes, and the multidrug efflux pump P-glycoprotein (P-gp), which is abundant in the gastrointestinal tract. When taken orally, Ptx is metabolized by enzymes or counter-transported by P-gp in the intestinal wall.13,14) To overcome its low bioavailability, Ptx, delivered through a parenteral route, is dissolved in a mixture of Cremophor® EL (Sigma-Aldrich, St. Louis, MO, USA) and ethanol (50:50, v/v) and requires dilution immediately before injection. However, Cremophor® EL has been associated with serious side effects, leading to hypersensitivity, nephrotoxicity, and neurotoxicity in many patients.3,15)

Various formulations to increase the bioavailability of Ptx and reduce the side effects of Cremophor® EL have been studied and developed. For example, liposomes,15,16) prodrugs,17) microspheres,18,19) self-emulsifying drug delivery systems,20) nanoparticles,21-24) nanosponges,13,25) and combination with P-gp inhibitors14,26) have been developed and evaluated. However, the attempts on clinical application of these formulations has not been carried out yet. Therefore, studies on other formulations for Ptx were needed.

Nanocapsules are nanoscale shells composed of non-toxic polymers. They are vesicular systems comprising a polymeric membrane that encapsulates an inner liquid core at the nanoscale level. Nanocapsules can be used in drug delivery, food enhancement, nutraceuticals, and the self-healing of various materials. Encapsulation protects materials against adverse environments and enables the controlled release and precision targeting of lipid nanocapsules (LNCs). LNCs are a new nanomedicine platform27) generated using original, solvent-free technology as drug nanocarriers to transport lipophilic drugs.28) Compared with other formulations, which are sensitive and metastable dispersed forms, LNCs are physically stable formulations that are easy to manufacture. LNCs have been found to be stable for one year (in suspenson ready condition).27) Thus, these systems may improve their rate and extent of absorption for lipophilic drug compounds, which are limited by their poor dissolution.

The advantages of LNC formulations are as follows:27,29) improved oral bioavailability and delivery by different routes, ease of manufacture and scale-up, dispersion stability >1 year, small particle size (adjustable between 20 and 150 nm), capacity to inhibit P-gp, stealth, cytostatic activity against glioma cells in vitro and in vivo, absence of organic solvents without the need for high energy, and simple preparation using generally recognized-as-safe excipients.

In this study, we prepared and characterized Ptx-LNCs via the phase-inversion temperature method with three cycles of heating and cooling.30-32) We also evaluated the pharmacokinetics and lymphatic targeting efficiency of this formulation in rats, and the results were compared with those of the reference Ptx solution for oral administration.

Methods

Materials and instruments

Ptx was obtained from the Korea Institute of Science and Technology (Seoul, Korea). Docetaxel was supplied by Sam Chun Dang Pharm. Co. Ltd. (Seoul, Korea). Normal saline solution and heparin sodium (25,000 IU/5 mL) were procured from JW Pharma. Co. (Seoul, Korea). Phosphate-buffered saline (PBS, pH 7.4), 70% ethanol, Lipoid® S100-3 (soybean lecithin about 100% phosphatidylcholine), Solutol® HS 15 (mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate), Captex® 8000 (tricaprylin), and tert-butyl methyl ether (TBME) were obtained from Sigma-Aldrich. Sodium chloride was purchased from Daejung Chemicals & Metal Co., Ltd. (Siheung-si, Gyeonggi-do, Korea). Methanol was obtained from Fisher Scientific Korea, Ltd. (Seoul, Korea). HPLC-grade water was obtained from a Milli-Q water purification system (Millipore Co., Milford, MA, USA) and used throughout the study. All other chemicals and solvents were of analytical grade or were of the highest quality available.

A chemical balance (EL204-IC, Mettler-Toledo, Greifensee, Switzerland), vortex mixer (G560, Scientific Industries Inc., Bohemia, NY, USA), and bath-type sonicator (Kodo Technical Research Co., Ltd, Hwaseong-si, Gyeonggi-do, Korea) were used for preparation of LNCs. A particle size analyzer (Scatteroscope-I, Qudix, Seoul, Korea), rotary vacuum evaporator (N-N series, EYELA, Tokyo, Japan), zeta potential analyzer (ELS-8000, OTSUKA Electronics, Osaka, Japan), field emission-transmission electron microscopy (FE-TEM, TECNAI-F20, Philips, Eindhoven, The Netherlands), shaking incubator (BS-11, JEIO TECH, Kimposi, Gyeonggi-do, Korea), and differential scanning calorimeter (DSC823e, Mettler-Toledo) were used to evaluate the prepared LNCs. The other instruments were a pH meter (SevenEasy, Mettler-Toledo), homogenizer (IKA-WERKE, KGD-79219, GmbH & Co., Staufen, Germany), centrifugal evaporator (Model CVE-200D, EYELA), and centrifuge (Model 5415C, Brinkman Instruments Inc., Westbury, NY, USA). Vacutainer® (K3 EDTA, 13 × 75 mm, Becton Dickinson, Meylan, UK) and polyethylene tubes (PE-10; Intramedic®, Clay Adams Co., Parsippany, NJ, USA) were used for the animal experiments. The LC-MS/MS system (Shimadzu Corp., Kyoto, Japan) used consisted of a system controller (CBM-20A), column oven (CTO-30A), autosampler, pump (LC-30AD 2 units), detector (SPD-M20A), and a MS detector (LCMS-8040).

Preparation method for LNCs

Blank LNCs were prepared according to an original method previously described.33) Briefly, Captex® 8000, Lipoid® S100-3, Solutol® HS 15, NaCl, and water (weight ratio, as shown in Table 1) were added and homogenized under magnetic stirring. Three cycles of progressive heating (for 10 min) and cooling (for 10 min) between 60 and 85°C (Step I) were performed, followed by an irreversible shock induced by the addition of cold water to the mixture at 78°C (Step II; for 5 min).33,34) Immediately, the suspension of LNCs was subjected to slow magnetic stirring for 5 min at room temperature (25°C).

Weight ratio (%) of components to prepared blank LNCs and their particle size

Solutol® HS 15 Lipoid® S100-3 Captex® 8000 NaCl Water Particle size (nm)
Blank LNCs 1 9.6 1.5 24.1 1.8 63.0 153.3±7.3
Blank LNCs 2 16.9 1.5 20.6 1.8 59.2 137.6±2.5
Blank LNCs 3 24.0 1.5 29.0 1.8 43.7 67.6±1.0


To prepare Ptx-LNCs, Ptx (29.3 mg) was dissolved in Captex® 8000 in the presence of methanol, and the solvent was evaporated at 85°C before use. The LNCs were prepared as described previously.33)

Measurement of droplet size, zeta potential, drug encapsulation efficiency, and stability

Droplet size and zeta potential

The mean particle sizes of the blank LNCs and Ptx-LNCs were analyzed via dynamic light scattering (DLS) at 25°C with a 90° scattering angle for optimum detection. Briefly, Ptx-LNCs were diluted in deionized water (DIW) and immediately sized at appropriate intervals (1, 2, 4, 8, and 12 h). The results (mean± standard deviation [SD]) represent three independent experiments.

The zeta potentials of the blank LNCs and Ptx-LNCs were measured with a zeta potential analyzer in triplicate among different batches to assess the surface charge and stability of the suspensions. One milliliter aliquots of each of the blank LNCs and Ptx-LNCs solutions were diluted with 10 mL of DIW in a cap vial. The mixture was gently shaken using a vortex mixer for 10 minutes, then resting the solution at 25°C. The resulting dispersion was measured after 1 h.

Drug encapsulation efficiency (EE)

Drug concentrations were determined using reversed-phase LCMS/ MS. Ptx-LNCs were mixed with methanol and vortexed vigorously for 1 min. The solution was diluted with a specific volume (1:1, v/v) of the mobile phase and injected into the LCMS/ MS system. The encapsulation efficiency (EE) was calculated as follows:

EE (%, w/w)=Weight of drug on Ptx-LNCsWeight of drug used in preperation of Ptx-LNCs×100

Stability tests in various media

Stability test in PBS (pH 7.4)

Stability tests in PBS (pH 7.4) at 4°C for an appropriate interval (1, 2, 3, 4, and 5 days) were performed by altering the size of the Ptx-LNCs. Briefly, 1 mL of Ptx-LNCs was added to 1 mL of PBS, and the mixture was vortexed vigorously for 1 min. Next, the solution was subjected to DLS. Each experiment was repeated at least three times.

Stability test in rat plasma and lymph nodes

The stabilities of Ptx-LNCs in rat plasma and lymph nodes were performed at 4°C for predetermined time intervals (1, 2, 3, 4, 5, 10, 15, and 23 days) and the size change of Ptx-LNCs was evaluated. The size change indicates an increased interaction of proteins with the lipid membrane of the Ptx-LNCs. Briefly, freshly prepared Ptx-LNCs (1 mL) were added to 1 mL of rat plasma and stored at 4°C. Next, the mixture was filtered with a polyvinylidene fluoride syringe filter (0.22 μm pore size; Millipore), and the filtrate was diluted with PBS to determine particle size. The size of each sample was measured using a particle size analyzer at 25°C. Each experiment was repeated at least three times. In these stability tests, the medium was maintained at a temperature of 4°C instead of 37°C to prevent the deterioration of the medium due to protein denaturation during the prolonged test period.

Differential scanning calorimetry

The endothermic melting temperatures of Ptx, a physical mixture of Ptx/blank LNCs, and Ptx-LNCs were determined via differential scanning calorimetry (DSC). The samples were scanned from −20 to 250°C at a rate of 10°C/min. An empty pan was used as a reference in all cases.

Morphological characterization

Ptx-LNCs were dropped on a carbon film coated on a copper grid and stained with 1% phosphotungstic acid neutralized to pH 7.0 with potassium hydroxide. After air-drying for 10 min, a drop was observed under FE-TEM at 200 kV using a TECNAI-F20 transmission electron microscopy system. The original magnification of the FE-TEM image was 50000×.

In vitro release studies

The in vitro release of Ptx from Ptx-LNCs at different pH levels (1.2, 6.8, and 7.4) was determined via dialysis. The prepared Ptx-LNCs were diluted with each medium, and the final concentration of Ptx was adjusted to 100 μg/mL. Next, each sample was transferred into a dialysis tube (MWCO: 12000) placed in a 50 mL screw-capped Falcon tube with 10 mL of each dissolution medium. The Falcon tubes were incubated in a shaking water bath at 37°C and shaken at 100 opm. Whole-media changes were used to prevent drug saturation in the drug release study. The whole medium (10 mL) was removed and replaced with an equivalent volume of fresh medium (10 mL) at predetermined time intervals (1, 2, 4, 6, 24, 48, 72, and 96 h) after incubation. The amount of released Ptx was measured using LC-MS/MS.

In vivo studies

Male Sprague-Dawley rats weighing 250±10 g were obtained from Dae Han Biolink (Eumseong-gun, Chungcheongbuk-do, Korea). The rats were fed tap water or food (Cheil Food and Chemical, Incheon, Korea). Animals were housed separately in a cage in a ventilated animal room at a controlled temperature (23±1°C) and humidity (50±5%), and maintained in a 12 h light/ dark cycle. After the acclimatization period, the weight of the rats just before drug administration was 292.4±13.6 g.

The femoral artery of the rats was cannulated with a polyethylene tube under light ether anesthesia. Cannulated rats were kept in restraining cages under normal conditions for 1-2 h until they recovered from anesthesia prior to the experiments. The rats were divided into two administration groups: (1) Ptx solution diluted with a 1:1 blend of Cremophor® EL and ethanol (6 mg/ mL), and (2) Ptx-LNCs. A single dose (12 mg/kg as Ptx) of each formulation was orally administered simultaneously to the rats. At predetermined intervals (0.5, 1, 2, 4, 6, 8, 12, and 24 h), whole blood samples (0.25 mL) were drawn via the femoral artery into a Vacutainer® tube with EDTA. The blood samples were immediately centrifuged (3000×g, 10 min), and the plasma samples were stored at −80°C until further analysis.

To compare and evaluate the lymphatic delivery effect of the prepared Ptx-LNCs, the rats were divided into two groups, as mentioned above. Whole blood was drawn from the abdominal aorta at 4, 8, and 24 h after oral administration, followed by the isolation and weighing of the mesenteric and axillary lymph nodes. These lymph node samples were homogenized for 1 min in PBS (pH 7.4) to achieve a final suspension concentration of 25 mg (lymph node)/mL and stored at −80°C until further analysis. This study was approved by the Chonnam National University Animal Experimental Ethics Committee, Gwangju, Korea (approval number: CNU IACUC-YB-2017-46).

Determination of Ptx concentration in rat plasma and lymph node suspensions

The Ptx concentrations in the rat plasma and lymph node (mesenteric and axillary) suspensions were determined using an LC-MS/MS assay from a previously reported method,35-40) with some modifications. The LC-MS/MS conditions for Ptx determination are listed in Table S1. A stock solution of 50 μg/mL Ptx was prepared in methanol and stored at −80°C. The internal standard (IS, docetaxel) solution (200 μg/mL) in methanol was prepared and stored at −80°C. Working standards of Ptx were prepared in methanol at concentrations ranging from 0.4 to 2000 ng/mL. A working IS solution (1.6 μg/mL) was prepared by diluting the docetaxel stock solution with methanol. We spiked 50 μL working solutions of Ptx (0.4, 2, 10, 50, 100, 200, 500, 1000, and 2000 ng/mL) and 40 μL working solutions of IS (1.6 μg/mL) into centrifuge tubes to prepare the calibration standards. After evaporating the solvent under a stream of nitrogen, 100 μL of blank plasma (or blank lymph node suspension) was added to each tube and vortexed. Final solutions of the resultant plasma (or lymph node suspension) containing 0.2, 1, 5, 25, 50, 100, 250, 500, and 1000 ng/mL of Ptx and 640 ng/mL of IS were used to evaluate the linearity of the calibration standards. Quality control samples of rat plasma were prepared similarly at concentrations of 5, 50, and 250 ng/mL within the range of the calibration standards and assayed in triplicate for three consecutive days for partial validation. Subsequently, the samples were mixed with 2 mL of TBME, vortexed, and centrifuged at 3000×g for 10 min. An aliquot (1.5 mL) of the upper organic layer was transferred to a new tube and evaporated to dryness under a stream of nitrogen. Each dried residue was reconstituted with 100 μL of a methanolwater solution (90:10, v/v) and vortexed for 30 s. The solutions were transferred to autosampler vials, and 5 μL of each sample was injected into the LC-MS/MS for analysis.

The LC-MS/MS system was fitted with a C18 column (1.7 μm, 50×2.1 mm; ACQUITY UPLC® BEH, Waters, Milford, MA, USA) using methanol and 0.1% formic acid (80:20, v/v) as the mobile phase with a total run time of 3 min. The flow rate was 0.2 mL/min, and the column temperature was maintained at 45°C. High-purity nitrogen was used as the nebulizing and curtain gas, and argon was used as the collision gas. Ptx levels in the plasma and lymph node samples were measured under the following parameters: ion spray voltage, 5900 V; capillary temperature, 215°C; collision energy, 30 V; source collision-induced disassociation, 12 V; and auxiliary gas pressure, 2 psi. Full-scan spectra were acquired over an m/z range of 200-1000 with a dwell time of 0.5 s. The peak-area ratios of Ptx to the IS were plotted versus the nominal concentration, and a least-squares linear regression model was used to generate a calibration curve, weighted by the reciprocal of the concentration.

Pharmacokinetic analysis and lymphatic delivery evaluation

The pharmacokinetic parameters associated with oral administration, such as the area under the plasma concentration-time curve (AUCt), time to maximum concentration (Tmax), and maximum concentration (Cmax) were estimated via non-compartmental methods using the WinNonlin program (Certara Inc., Princeton, NJ, USA). AUCt was calculated using a linear trapezoidal rule after oral administration. Cmax and Tmax were determined from the plasma concentration-time curve obtained for each rat. Clearance (CL/F) was calculated by dividing the oral dose of Ptx by the AUCt, where F represents the oral bioavailability. The targeting efficiency of Ptx in the lymphatic system was calculated as the ratio of Ptx concentration in the lymph nodes (mesenteric and axillary nodes) to the concentration in rat plasma at predetermined time intervals after oral administration of each formulation.

Statistical evaluation

All calculated values are expressed as the mean±SD. All data were analyzed for statistical significance using Student’s t-test (p<0.05).

Results and Discussion

Preparation of blank LNCs and Ptx-LNCs

Blank LNCs were prepared using different weight ratios (%) of Solutol® HS 15, Lipoid® S100-3, Captex® 8000, NaCl, and water (blank LNCs 1, 2, and 3) (Table 1). The components of the LNCs applied in this study were selected based on previous studies that reported similar formulations.33,34) Solutol® HS 15 and Captex® 8000 were used in this study as a co-surfactant and emulsifier, respectively. Lipoid® S100-3 was used to solubilize Ptx and the lipid core. NaCl and water were used as the salt and aqueous solvent components, respectively. Solutol® HS 15 is considered a key component in these formulations because it inhibits P-gp,27) which is crucial for increasing Ptx bioavailability.

Measurement of droplet size, zeta potential, drug EE and stability

Droplet size and zeta potential and drug EE

Table 1 shows the particle sizes of blank LNCs (Blank LNCs 1, 2, and 3) in DIW at 25°C, which were 153.3±7.3, 137.6±2.5, and 67.6±1.0 nm, respectively. These results showed that blank 3 was the best candidate with the highest weight ratio of Solutol® HS 15 (24%). This finding also confirms that a higher weight ratio of Solutol® HS 15 yields smaller particles. Therefore, we selected blank 3 as the basic formulation for this study. Fig. 2A depicts the size variation of the Ptx-LNCs in DIW for 12 h at room temperature. The polydispersity index of the prepared Ptx-LNCs was 0.21±0.03 (n=3). These results show that the particle size of Ptx-LNCs was stable in DIW for 12 h, and that the particle size of Ptx-LNCs measured using the DLS method was stable in DIW without significant size variation for 12 h.



Fig. 2. Particle size changes of Ptx-LNCs (A) suspended in DIW over a 12-h period at 25°C, (B) in PBS for 5 days at 4°C, (C) in rat plasma for 23 days at 4°C, and (D) in lymph node for 23 days at 4°C (n=3). *p<0.05 (Ptx-LNCs vs. days).

The zeta potential represents important physicochemical characteristics of the Ptx-LNCs surface. The zeta potential was −5.8±1.2 mV for the blank 3 LNCs and −4.0±0.4 mV for the Ptx-LNCs, ranging between +30 and -30 mV. This value indicates that the prepared Ptx-LNCs are relatively stable in colloidal dispersions.41)

The EE value of the optimized Ptx-LNCs was 90.5±7.8%. This result suggests that Ptx was sufficiently incorporated into LNCs, and thereby the particle size was significantly (p<0.05) increased from 67.6±1.0 nm (blank 3 LNCs) to 87.6±6.2 nm (Ptx-LNCs).

Stability tests in various media

In addition to DIW, tests in PBS, rat plasma, and lymph node suspensions were performed to evaluate the long-term stability of the Ptx-LNCs prepared in this study. Prior to in vivo pharmacokinetic evaluation, it was necessary to first evaluate the stability of Ptx-LNCs in in vivo matrices (especially in the plasma and lymph tissues). A test in PBS for five days was sufficient to evaluate the stability of Ptx-LNCs before administration to rats. The period for evaluating the stability of Ptx-LNCs was individually set arbitrarily (in PBS for 5 days and in rat plasma and lymph node suspension for 23 days) according to the purpose of the study.

Stability test in PBS (pH 7.4)

Fig. 2B shows the stability of Ptx-LNCs in PBS at 4°C with mean particle sizes ranging from 113.3 to 122.3 nm for 5 days. This result suggests that Ptx-LNCs were stable in PBS, even though the mean particle size of Ptx-LNCs in PBS was significantly (p<0.05) increased due to changes in pH and electrolytes compared with that of Ptx-LNCs in DIW.

Stability test in rat plasma and lymph node suspensions

When Ptx-LNCs enter the systemic circulation, some plasma proteins are adsorbed onto the surface of Ptx-LNCs. These plasma proteins act as “opsonins” or “dysopsonins.” Particle size measurement is a rapid and convenient method for measuring protein adsorption on particles. Increased particle sizes indicate binding via protein interactions with the lipid membrane of Ptx-LNCs. The size variation of Ptx-LNCs in rat plasma and lymph node suspensions is shown in Fig. 2C and 2D, respectively. The mean particle size of Ptx-LNCs at 4°C in rat plasma over 23 days ranged from 105.6 to 126.6 nm. When compared with the size variation of Ptx-LNCs in PBS at 4°C, the mean particle size of Ptx-LNCs in rat plasma was similar and not significantly different (p>0.05). The increased size of Ptx-LNCs fabricated from different components, including Solutol® HS 15, which carries a hydrophilic and lipophilic non-ionic surfactant corona, is attributed to hydrophilic PEGylation. The prepared Ptx-LNCs were resistant to protein binding in rat plasma due to PEGylation and were expected to be used in in vivo delivery systems for Ptx. Similar results were obtained for the prepared Ptx-LNCs in the lymph node suspensions.

DSC

DSC was conducted to characterize the drug inside the prepared LNCs and the melting point of each formulation. Figure 3 shows the DSC thermograms. Ptx has a melting point between 215 and 225°C. As shown in Fig. 3A, the endothermic peak of Ptx is at approximately 220°C. However, this peak was not observed in the physical mixture of Ptx/blank LNCs or the prepared Ptx-LNCs. These results suggest that Ptx is either molecularly dispersed in the LNCs or distributed in an amorphous or crystalline state at a small size.



Fig. 3. DSC thermogram of (A) Ptx, (B) physical mixture of Ptx and LNCs, and (C) prepared Ptx-LNCs.

Morphological characterization of Ptx-LNCs

Based on the FE-TEM observations (Fig. 4), the Ptx-LNCs prepared under the optimal conditions above were nearly 90 nm in size and had a relatively spherical and uniform shape with a smooth surface, suggesting the successful fabrication of Ptx-LNCs.



Fig. 4. FE-TEM image of Ptx-LNCs.

In vitro release studies

Figure 5 compares the release profiles of Ptx from the prepared Ptx-LNCs at various pH levels (pH 1.2, 6.8, and 7.4). The Ptx-LNC formulation exhibited a slow release for 96 h with an initial 6-h burst release, which can be explained by the dependence of the release rate of Ptx on the droplet size, which enables faster drug release into the aqueous phase and vice-versa. The release rate of Ptx in Ptx-LNCs was faster (more than 50%) in the dissolution medium at pH 1.2 than in other media. Therefore, Ptx-LNCs show a pH-dependent release with potential therapeutic applications in controlled drug release and delivery in the blood or intestinal fluid.



Fig. 5. In vitro release profiles of Ptx in the prepared Ptx-LNCs in various dissolution media (pH 1.2, 6.8 and 7.4). Each value represents the mean±SD (n=3).

Determination of Ptx levels in rat plasma and lymph node suspensions

Figure S1 shows the mass spectra of the parent ions of Ptx (A) (50 μg/mL in methanol) and docetaxel (B) (200 μg/mL in methanol) at a scan range of 200-1000 (m/z). Figure S2 shows the mass spectra of the daughter ions of Ptx (A) and docetaxel (B). Figure S3 shows chromatograms of Ptx in rat plasma (50.2 ng/mL) with a retention time of 0.913 min. The regression equations based on the calibration curves (in triplicate) for rat plasma and lymph node homogenates are Y=3.89701X+ 0.0475789 for rat plasma and Y=4.15434X+0.000805414 for lymph node homogenates (where Y=peak area ratio, X= concentration of Ptx), suggesting significant linearity (r2=0.998 and 0.998, respectively). The lower limit of quantitation in rat plasma was 0.2 ng/mL, with an accuracy ranging from 80% to 120%. The precision and accuracy of the assay for rat plasma were within acceptable limits (within 15%). The mean intraday and interday precision was less than 15%, and the accuracy was greater than 90% (Table S2).

Pharmacokinetic analysis and evaluation of lymphatic delivery of Ptx-LNCs

Figure 6 shows the mean plasma concentration-time profiles of Ptx after oral administration of Ptx-LNCs and the reference solution to rats. Table 2 lists the pharmacokinetic parameters obtained via non-compartmental methods. The AUCt, CL/F, and Cmax of Ptx-LNCs were 49.84±2.07 μg·h/mL, 240.97±100.66 mL/h, and 2.70±0.39 μg/mL, respectively, suggesting significant differences (p<0.05) compared with 26.93±4.80 μg·h/mL, 445.55±79.60 mL/h, and 1.57±0.01 μg/mL, respectively, in the reference solution (approximately 2-fold difference). These results indicated that more Ptx-LNCs were absorbed into the plasma than free Ptx. Interestingly, more Ptx-LNCs were absorbed into the systemic circulation faster than free Ptx when administered orally. According to previous reports,29,42,43) polymeric nanoparticles smaller than 200 nm can diffuse effectively through the mucosal barrier, and LNCs can be absorbed via fat absorption or be effectively transported due to P-gp inhibition.

Pharmacokinetic parameters of Ptx in rat plasma after oral administration of Ptx solution and Ptx-LNCs (12 mg/kg as Ptx) to rats

Parameters Oral administration

Paclitaxel solution Ptx-LNCs
AUCt (µg·h/mL) 26.93±4.80 49.84±2.07*
Tmax (h) 4.15±0.52 3.54±1.09
CL/F (mL/h) 445.55±79.60 240.97±100.66*
Cmax (µg/mL) 1.57±0.01 2.70±0.39*

*p<0.05 (n=3).




Fig. 6. Plasma concentration-time profiles of Ptx after oral administration of Ptx solution (●) and Ptx-LNCs (○) to rats (12 mg/kg as Ptx). Each value represents the mean±SD (n=3).

The concentrations of Ptx in the mesenteric and axillary lymph nodes at 4, 8, and 24 h after oral administration of Ptx solution and the prepared Ptx-LNCs are displayed in Fig. 7. Figure 7A shows that the concentrations of Ptx in mesenteric lymph nodes at 4, 8, and 24 h varied significantly compared with those of the reference solution (p<0.05). Fig. 7B shows that the concentration of Ptx in the axillary lymph nodes at 4 h was significantly different from that in the reference solution (p<0.05).



Fig. 7. Concentrations of Ptx in (A) mesenteric and (B) axillary lymph nodes at 4, 8 and 24 h after oral administration of Ptx solution (■) and Ptx-LNCs (▒) to rats (12 mg/kg as Ptx). Each value represents the mean±SD (n=3). *p<0.05.

The mesenteric and axillary lymphatic targeting efficiencies of Ptx, calculated as the ratio of the lymph node Ptx concentration to the plasma Ptx concentration, are shown in Fig. 8A and 8B, respectively. Figure 8 shows that the mesenteric and axillary lymphatic targeting efficiencies of Ptx-LNCs at 24 and 4 h were significantly higher than those of the reference solution, respectively (p<0.05). These results suggest that Ptx-LNCs transported Ptx more effectively into the lymphatic tissue, so we expect that this increased lymphatic delivery would enable the clinical use of lower doses of Ptx, leading to a decreased incidence of side effects.



Fig. 8. Targeting efficiencies of Ptx to (A) mesenteric and (B) axillary lymph nodes at 4, 8 and 24 h after oral administration of Ptx solution (■) and Ptx-LNCs (▒) to rats (12 mg/kg as Ptx). Each value represents the mean±SD (n=3). *p<0.05.
Conclusion

This study demonstrated the enhanced bioavailability and lymphatic delivery of Ptx after oral administration of Ptx-LNCs. Ptx, a poorly water-soluble drug, was easily and reproducibly incorporated into LNCs via three heating and cooling cycles using Solutol® HS 15 as a key component. The mean particle sizes of the blank LNCs and Ptx-LNCs under optimal conditions were 67.6±1.0 and 87.6±6.2 nm, respectively. Based on in vivo studies, the AUCt, CL/F, and Cmax of Ptx-LNCs after oral administration to rats varied significantly from those of the Ptx solution (p<0.05, approximately 2-fold change). The lymphatic targeting efficiency of Ptx-LNCs was significantly higher than that of the Ptx solution (p<0.05).

Therefore, the Ptx-LNCs prepared in this study can be a good candidate for an efficient Ptx delivery system as an oral formulation. It might also facilitate targeted delivery to tumor cells based on further experiments using LNCs.

Acknowledgment

This study was supported by a grant (2017RIDIAB04035667) from the Basic Science Research Program through the National Research Foundation funded by the Ministry of Education, Republic of Korea.

Conflict of Interest

All authors declare that they have no conflict of interest.

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