Targeted solid lipid nanoparticles with peptide ligand for oral delivery of atorvastatin calcium

Abstract

Feasible and effective peptide ligand-modified solid lipid nanoparticles (SLNs) have been designed to improve the oral bioavailability of atorvastatin calcium (ATC). In the present work, the peptide ligand-modified SLNs loaded with ATC, namely ATC CSK-SLNs, were prepared by coupling the peptide ligand CSKSSDYQC (CSK), which showed affinity with goblet cells, to stearic acid. The physicochemical properties of the SLNs were characterized by TEM, DSC and FT-IR, which unravelled the transformation of ATC to an amorphous or molecular state from the native crystalline form. Compared with unmodified SLNs, the CSK-SLNs exhibited a more efficient cellular uptake across the Caco-2/HT29 co-cultured cell monolayer as evidenced by confocal laser microscopy. Following absorption, the mechanisms were studied using a modified in situ perfusion method in rats, which showed the segment-dependent absorption characteristics of ATC, ATC SLNs as well as ATC CSK-SLNs. The K_a (0.076 ± 0.23 min⁻¹) and P_app (0.011 ± 0.63 cm min⁻¹) values of the ATC CSK-SLNs were raised 2.97-fold and 2.99-fold in comparison with those of the ATC solution, implying that CSK peptide modification enhances the permeation of drugs across the epithelium. In conclusion, our results demonstrated that CSK-modified SLNs could be potential carriers for the transport of drugs across intestinal barriers.

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1. Introduction

Oral administration is most preferred because of its various advantages over other routes of drug delivery, including patient convenience, cost effectiveness, and ease of dosage control.¹ However, the development of oral drug delivery systems is hindered by low drug solubility, poor gastrointestinal (GI) absorption, metabolism-related issues, continuous fluctuation of drug plasma levels and variability due to food effects.² Solid lipid nanoparticles (SLNs), first reported as an oral drug carrier in the middle 1990s, have attracted lots of interest due to their good tolerability, biodegradability, physical stability, possibility of large-scale production, sustained release, efficient incorporation of hydrophobic drugs, and reduction of toxic and side effects simultaneously.^3,4 Moreover, SLNs have the potential for targeted drug delivery, which could increase mucus adhesion and retention in the GI tract, and bypass the first metabolism.^5–7

Coating nanoparticles with peptide ligands was intended to enhance the binding specificity and decrease the elimination rate.⁶ Recently, two categories of peptide ligands were applied for oral drug delivery systems: epithelium-targeting peptides and permeation-enhancing peptides. A CSKSSDYQC (CSK) peptide identified from a random phage-peptide library through an in vivo phage display technique was found to have affinity with goblet cells which are important intestinal epithelium cells.⁸ Previous studies demonstrated that CSK peptide-modified trimethyl chitosan chloride possessed an improved hypoglycemic effect and higher relative bioavailability compared with the unmodified versions.^9,10 Therefore, the CSK peptide ligand could be available as a leading peptide for the carrier-drug conjugate strategy with molecular therapeutics to facilitate efficient transport of drugs across the intestinal mucosal barrier. Furthermore, liposomes modified with the CSK peptide exhibited greatly improved cellular internalization via different mechanisms.¹¹

Atorvastatin calcium (ATC) is an important and efficacious member of a class statins in treating dyslipidemia and coronary heart disease. It works by inhibiting 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase which reduces hepatocyte cholesterol levels which in turn causes up-regulation of low-density lipoprotein (LDL) receptors and increases clearance of LDL-cholesterol (LDL-C) from the plasma.¹² Previous studies indicated that ATC had low systemic availability because of its instability, incomplete intestinal absorption and/or extensive gut wall extraction.¹³ The poor solubility, low oral bioavailability (12%), and high first pass metabolism of ATC¹⁴ make it a suitable agent for SLN formulation.

In the present work, therefore, based on the above-mentioned research, the peptide ligand-modified, ATC-loaded SLNs were designed and established by conjugating the peptide ligand CSK to stearic acid (CSK-SLNs), with unmodified SLNs as the control, as schematically depicted in Fig. 1. The physicochemical characteristics of the SLNs were investigated. The influence of mucus and the targeting effect in vitro were investigated using a mucus-secreting Caco-2/HT29 co-culture cell monolayer model, which was applied as they closely mimic the mucus layer and the epithelium of the intestine.^15–17 The chemical conjugates of octadecylamine and fluorescein isothiocyanate (ODA-FITC) were synthesized, and encapsulated into the SLN formulation as a fluorescence probe to investigate the cellular uptake. Finally, the absorption of the drug and drug-loaded SLNs in the GI tract were studied using the in situ single-pass perfusion method in rats.

Fig. 1 Schematic of the preparation of ATC SLNs and ATC CSK-SLNs.

2. Materials and methods

2.1. Materials

Atorvastatin calcium (ATC) was provided by Shanghai Ecust Biomedicine Co., Ltd. (Shanghai, China). CSKSSDYQC (CSK) was chemically synthesized by ChinaPeptides Co., Ltd. (Shanghai, China). Stearic acid (SA), glycerol tripalmitate (GTP), palmitic acid, poloxamer188, sodium deoxycholate, octadecylamine (ODA) and fluorescein isothiocyanate (FITC) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) was gained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). N-Hydroxy-succinimide (NHS) was supplied by Aladdin Chemistry Co., Ltd. (Shanghai, China). Sodium laurylsulphate was obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). All the other reagents and solvents were of analytical grade or chromatographic grade.

HT29 cells were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). The Caco-2 cell line was a kind gift from Dr Liu (ECUST, Shanghai, China). Male Sprague-Dawley rats weighing 250 ± 20 g were supplied by the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (Shanghai, China). The rats were housed at a room temperature of 22 ± 2 °C and a relative humidity of 50 ± 10%.

2.2. Synthesis of SA-CSK

The amine group of the CSK peptide was conjugated with the carboxylic group of SA using a previously reported method.¹⁸ SA (45 mg) was dissolved in a 6 mL mixed solvent of DMF and water (2 : 1, v/v), followed by the addition of excess EDC·HCl (30 mg) and NHS (20 mg). After adjusting the pH to 8.0, the solution was stirred at room temperature for 1 h in the dark. CSK (15 mg) was added and the reaction was conducted at ambient temperature for 24 h. The resultant SA-CSK was purified by 3 days of dialysis (spectra/pormolecularporous membrane tubing cut-off M_w 500–1000) against very frequently changed deionized water, lyophilized and stored at 4 °C. The obtained SA-CSK was identified by ¹H NMR (BrukerBiospin, Germany), and Fourier transform infrared (FT-IR) spectroscopy (Thermo Fisher Scientific Inc., NY, USA). The content of the conjugated peptide was determined through high performance liquid chromatography (HPLC, Shimadzu).

2.3. Preparation of SLNs

SLNs were prepared using a modified emulsion/solvent evaporation method.¹⁹ Briefly, GTP (118 mg) and palmitic acid (32 mg) were dissolved in a 2 mL mixture of dichloromethane and ethanol (1 : 1, v/v), and the mixture was sonicated and warmed to obtain a clear melting organic phase. Then the organic phase was added to 10 mL aqueous solution containing 1.25% poloxamr188 and 0.25% sodium deoxycholate in a glass vial. The mixture was sonicated using an ultrasonicator at 0.5 cycles and 60% amplitude for 10 min. The pre-emulsion formed was then submitted to high-speed Ultra-Turrax homogenization (IKA T25) at 24 000 rpm for 7 min. After this step, the organic solvent was completely evaporated under reduced pressure using a rotary evaporator. The resultant SLN suspension was concentrated to 1 mL by ultrafiltration using an Amicon device (Millipore Inc., 30 000 MWCO) (30 min, 4 °C). Finally, the SLNs were briefly sonicated to eliminate aggregates due to the concentrating process.

The CSK-SLNs were prepared by adding SA-CSK (1 mg mL⁻¹) to the aqueous solution containing poloxamer188 and sodium deoxycholate.⁹ After that, a 2 mL lipid solution was added dropwise to the CSK-SA solution, with magnetic stirring. The emulsion (O/W) was finally formed by the aforementioned method. For the observation and evaluation of SLNs in the following tests, ODA was labelled with FITC (FITC-ODA) according to previous reports,²⁰ and then encapsulated into the SLNs to prepare fluorescence-labelled nanoparticles. In addition, the SLN dispersion was lyophilized using trehalose as a cryoprotectant, and then stored at 4 °C for future use.

2.4. Drug loading and release

ATC-loaded SLNs or CSK-SLNs were prepared by adding the drug to the solid lipid solution before sonication. The other procedures were similar to those used for the SLNs. The amount of ATC was 4% (w/w) with respect to the solid lipid matrix. For SLNs that were used in the animal study, the ultrafiltration period was extended to 60 min to further concentrate the suspension.

To evaluate the entrapment efficiency of ATC, the free drug in the ATC SLN suspension was separated by the ultrafiltration method using a filter membrane with a weight cut-off (MWCO) of 10 kDa (Millipore, USA) at 3500 rpm for 20 min. The SLN dispersion was destroyed by adding 100-fold DMSO in an 80 °C water bath for 10 min. This solution was cooled down to room temperature and centrifuged for 15 min at 12 000 rpm. The amount of ATC in the supernatant and SLNs was determined by the reversed-phase HPLC (Shimadzu, LC-M20AT) method. Separation was achieved on a Diamonsil C₈ column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile–ammonium acetate buffer (60 : 40, the pH was adjusted to about 4 with acetic acid). The flow rate was 0.8 mL min⁻¹, and the detection wavelength was set at 246 nm. The drug entrapment (EE, %) and drug loading (DL, %) could be achieved from the following equations:

where C_T is the total added ATC (mg mL⁻¹) concentration, C_AP is the ATC (mg mL⁻¹) concentration in the aqueous phase, W_DL is the weight of ATC loaded in the nanoparticles (mg), and W_L is the weight of lipid (mg).

The in vitro drug release behaviours from the ATC SLNs and ATC CSK-SLNs were measured using a treated dialysis membrane (M_w = 8000 Da).²¹ The dialysis bag was immersed in the dissolution medium that is the phosphate buffer (pH 6.8) containing 0.8% sodium laurylsulphate (SLS). The freshly prepared 5 mL of atorvastatin calcium suspension, SLNs and CSK-SLN dispersion containing 2.5 mg of the drug were placed in a treated dialysis bag which was immersed in 100 mL of freshly prepared dissolution medium (phosphate buffer + 0.8% SLS) at 37 ± 0.5 °C. The rotation speed of the magnetic stirrer was adjusted to 50 rpm. At pre-determined time intervals, 1 mL of the dissolution medium was withdrawn. Then the same amount of media was replaced to maintain the sink condition. The drug concentration was analyzed by the HPLC method as described above. The error bars were obtained from triplicate samples.

2.5. In vitro cellular studies

2.5.1. Cell culture. The human colon adenocarcinoma cells, Caco-2 cells and HT29 cells were cultivated separately in culture dishes using DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, penicillin (100 UI per mL) and streptomycin (100 μg mL⁻¹). Both cultures were maintained at 37 °C, with 95% relative humidity and 5% CO₂. Prior to the test, cells were digested with 0.25% trypsin containing 0.05 mM ethylene diamine tetraacetic acid (EDTA) and then diluted with fresh DMEM to a density of 1 × 10⁵ cells per mL. For the transport experiments, co-cultures of Caco-2 cells and HT29 cells were re-suspended in a 1 : 1 ratio and seeded onto 96-well plates with a density of 3 × 10⁴ cells per well, into which DMEM was added. The cells were allowed to grow and differentiate for 21 days before use. The integrity of the monolayer was evaluated by measuring the transepithelial electric resistance (TEER).²²

2.5.2. In vitro cytotoxicity study. The cytotoxicity of SLNs and CSK-SLNs were assessed using MTT assay with Caco-2 cells and HT29 cells, respectively. In brief, the culture medium in the 96-well plates was discarded. Subsequently, the cells were washed with PBS and incubated with SLN suspensions (blank SLNs, blank CSK-SLNs, ATC SLNs or ATC CSK-SLNs) at concentrations of SLNs varying from 0.2 to 1 mg mL⁻¹ for 4 h at 37 °C. After incubation, the test solution was aspirated from the wells and the cell viability was tested by MTT assay. Tests were performed in triplicate for each sample.

2.5.3. Cellular uptake study. To further investigate the transport of the CSK peptide-modified SLNs across the epithelial cells and the influence of mucus on the targeting recognition, the cellular uptake study was performed using Caco-2/HT29 co-cultured cells as model cells. For this assay, co-cultured cells were incubated with fluorescence-labelled SLNs (at a concentration of 200 μg mL⁻¹) for various times (1 h, 2 h, 4 h) at 37 °C. After incubation, the medium was removed, and the cells were washed three times with PBS (0.01 M, pH 7.4) at room temperature. Then the cells were fixed by 70% ethanol for 20 min. The cells were further washed thrice by PBS and the nuclei were then counter-stained by Hoechst 33342 for 30 min. The fixed cell monolayer was finally washed thrice by PBS and observed by confocal laser scanning microscopy (Leica Microsystems, Weltzer, Germany).

2.6. In situ single-pass intestinal perfusion study

The in situ single-pass perfusion study was performed according to the previously published methods.^23,24 Sprague-Dawley rats weighing 200–250 g were fasted overnight but had free access to water. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg kg⁻¹). The intestines were exposed by a midline abdominal incision. The proximal and distal ends of the duodenum, jejunum and ileum were identified and cannulated with silicone tubes. Krebs Ringer’s buffer was gently injected through the inlet tube to rinse the intestinal contents. Perfusates were prepared into Krebs Ringer’s buffer by diluting the stock solution of ATC solution, ATC SLNs or ATC CSK-SLNs with an ATC concentration of 50 μg mL⁻¹. After perfusion for 30 min, the samples of perfusate were collected in 15 min intervals up to 120 min. At the end of the experiment, the length and radius of the infused intestinal segments were measured accurately. The net water flux in the perfusion experiment was calibrated by weight. All the perfusate solutions collected were weighed and stored at −20 °C until further analysis using a validated HPLC method. The absorption rate constant (K_a) and apparent permeability coefficients (P_app) were calculated using the following equations:

where C_in is the concentration (μg mL⁻¹) of ATC in the entering solution, and C_out is the ATC concentration (μg mL⁻¹) in the receptor tube, Q_in is the inlet volume and Q_out is the outlet volume (mL), r is the intestinal radius (cm), l is the length of the segment (cm), and v is the perfusion flow rate (mL min⁻¹).

2.7. Sample characterization

The particle size and zeta potential of the SLNs were characterized by Malvern Zetasizer Nano ZS90 (Malvern instrument Ltd., Malvern, UK). All measurements were taken in triplicate.

The SLNs were morphologically characterized by transmission electron microscopy (TEM, JEOL, JEM-1230, Japan). Samples were prepared by depositing a drop on the surface of copper grids coated with carbon film and negatively stained with a 2% (w/v) solution of phosphotungstic acid.

The thermal behaviors of ATC, GTP, the physical mixture and lyophilized SLN preparation were examined by differential scanning calorimetry (DSC, Thermo Fisher Scientific Inc., NY, USA). Samples were accurately weighed (5–6 mg), sealed in aluminum crimp cells, and analyzed while heating from 30 °C to 200 °C at a heating rate of 10 °C per minute.

The intermolecular interactions of the native lipid, pure drug and lyophilized SLN formulation were analyzed using a FT-IR spectrometer. The spectra were acquired from the wavelength of 500 to 4000 cm⁻¹ and the resolution was 4 cm⁻¹.

3. Results and discussion

3.1. Synthesis of polymers

The CSK peptide was conjugated to stearic acid following a previously reported method with slight modification. The carboxylic group of SA was conjugated to the amine group of the CSK peptide. The molar ratio of SA and the CSK peptide was 3 : 1. ¹H NMR and FT-IR spectra are shown in Fig. 2. According to the results of the ¹H NMR spectra, the characteristic peaks of the SA-CSK peptide were at 6.74 and 7.02 ppm and were attributable to the two protons of the benzene ring of tyrosine in the CSK peptide sequence, respectively. The other proton peaks of the CSK peptide ranging from 0.9 to 4.7 ppm are undistinguished due to the peaks of SA.²⁵ And in the FT-IR spectra, the peak at 1704 cm⁻¹ corresponds to the C=O bond of –COOH on SA, while the spectrum of SA-CSK exhibits obvious N–H bending, C–N stretching from amide II (∼1567 cm⁻¹), and C=O stretching from amide I (∼1651 cm⁻¹), confirming the presence of amide bonds.²⁶ Besides, the characteristic peak of the benzene ring at 798 cm⁻¹ for SA-CSK was observed, again indicating the conjugation of the CSK peptide with SA. Then, according to the result of HPLC detection, the content of the CSK peptide in the SA-CSK polymer was calculated to be about 98%.

Fig. 2 FT-IR spectra of SA (A) and SA-CSK (B). ¹H NMR spectra of CSK (C) and SA-CSK (D). The test solvent is D₂O and field frequency is 500 MHz.

3.2. Preparation of SLNs

SLNs were prepared by the modified emulsion/solvent evaporation method. The size measurement results of the blank and drug-loaded SLNs (including the FITC-labelled SLNs and FITC-labelled CSK-SLNs) are summarized in Table 1. It was clear that the prepared SLNs loaded with ATC had a negative charge with about a −33.5 mV zeta potential, and were nano-sized. In addition, all the blank SLNs and drug-loaded SLNs prepared by SA-CSK exhibited a larger size and lower zeta potential in comparison with the unmodified SLNs. This might be due to the introduction of CSK, a peptide with a molecular weight (MW) of 1020 Da and a negative charge.⁹ Otherwise, when it comes to the fluorescence-labelled batches, the drug-loaded SLNs and CSK-SLNs possessed both a larger particle size and lower zeta potential compared with that of the blank batches, due to the entrapment of the negatively charged ODA-FITC.

Table 1 Characterization of the prepared SLNs^a

Material	Size (nm)	Zeta potential (mV)	PDI	EE%	DL%
a EE%: entrapment efficiency; DL%: loading content.
SLNs	152.4 ± 5.39	−32.4 ± 1.62	0.124
CSK-SLNs	162.5 ± 6.35	−35.2 ± 1.85	0.080
FITC SLNs	171.6 ± 8.20	−38.1 ± 2.17	0.194
FITC CSK-SLNs	178.5 ± 8.64	−40.9 ± 1.80	0.286
ATC SLNs	158.3 ± 4.23	−33.5 ± 1.35	0.135	79.8 ± 0.45	3.09 ± 0.04
ATC CSK-SLNs	168.5 ± 6.11	−36.0 ± 2.06	0.210	81.2 ± 0.63	3.15 ± 0.07

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TEM has been widely applied as a powerful tool for detecting the size and surface morphology of nanoparticles. Fig. 3C and D present TEM of the ATC-loaded SLNs and CSK-SLNs, indicating that all of the nanoparticles had spherical shapes, with average sizes similar to those obtained by dynamic light scattering. The digital photos and turbidity of the SLN products are shown in Fig. 3 which generally agreed with the particle size results.

Fig. 3 Particle size intensity of ATC SLNs and ATC CSK-SLNs (A); digital photos and turbidity of ATC SLNs (left) and ATC CSK-SLNs (right) (B); and TEM photographs of ATC SLNs (C) and ATC CSK-SLNs (D).

DSC thermal curves for the drug and pure excipients are shown in Fig. 4 along with the thermal curves for the SLNs loaded with ATC and the DSC curve for a physical mixture of the lipid and drug. All the samples showed one or more endothermic phase transitions. An endotherm starting from 60 °C with a peak at 67 °C was observed for pure lipid. ATC exhibited a melting peak at 159.2 °C. No significant change in the position of the endothermic peaks was observed after running the physical mixture of the drug and solid lipid (Fig. 4c). Thus, physical incompatibility between the components was discarded. A small endothermic peak was observed at around 50 °C in both the ATC SLNs and ATC CSK-SLNs. This peak indicated the presence of poloxamer188 either in the form of a coating surrounding the SLNs or as a residue after ultrafiltration and lyophilization.²⁷ In accordance with the literature, the phase transition temperature and enthalpy of the SLN formulation is lower than the melting temperature of the bulk material. The melting points of the SLNs were distinctly decreased by about 3–8 °C.²⁸ The ATC melting peak was not recorded upon running the SLN formulation attributed to the solubility of the drug within the solid lipid matrix.

Fig. 4 DSC curves of ATC (a), GTP (b), a physical mixture of lipid and ATC (c), ATC SLNs (d), and ATC CSK-SLNs (e).

The FT-IR spectra of the SLNs and the pure ingredients are shown in Fig. 5. The SLN control exhibited several characteristic peaks with a strong intensity of C–H stretching at 2916 cm⁻¹ and 2849 cm⁻¹, C=O stretching at 1736 cm⁻¹ from the carboxyl group, C–O stretching at 1179 cm⁻¹, CH₂ scissoring at 1471 cm⁻¹, and CH₂ rocking at 717 cm⁻¹, respectively, which were the typical bands of the lipid in the formulation.^29,30 The pure drug presented characteristic peaks of the carbonyl functional group at a wavenumber of 1651 cm⁻¹.³¹ But the appearance of characteristic peaks of ATC loaded in the SLNs and CSK-SLNs indicated that the matrix lipid successfully encapsulated the drug. No unique and distinct peak was presented in the SLNs and there was no shifting of the peaks compared to the pure lipid. This clearly confirmed that there was no chemical interaction between the drug and the excipients. Moreover, from the spectra of the ATC CSK-SLNs, three characteristic peaks at 1649, 1562 and 798 cm⁻¹ were observed which may be due to SA-CSK as aforementioned.

Fig. 5 FT-IR spectra of ATC (a), GTP (b), blank SLNs (c), ATC SLNs (d), and ATC CSK-SLNs (e).

3.3. Drug loading and release

The entrapment efficiency (EE, %) and drug loading (DL, %) are also presented in Table 1. It shows that higher drug encapsulation efficiencies were obtained. The EE% of the ATC SLNs and ATC CSK-SLNs was around 80%. The incorporation of the CSK peptide had no significant effect on the EE%.

In this study, dynamic dialysis was chosen for separation of free ATC from the SLNs. The solubility of ATC in phosphate buffer (pH 6.8) at 25 °C is only 0.1 mg mL⁻¹. Thus, 0.8% SLS in which the solubility of ATC is 2.1 mg mL⁻¹ was chosen as a receptor medium. The drug release curves of ATC, the ATC SLNs and ATC CSK-SLNs are shown in Fig. 6. It was obvious that the release of free ATC through the dialysis membrane was much faster, with approximately 98% of the drug being released within 8 h. In contrast, only 40% of ATC was released from the SLNs within 8 h, which indicated that a certain amount of ATC was incorporated at the surface of the SLNs. Subsequently, an ATC SLN and ATC CSK-SLN suspension exhibited a sustained release property in the latter stage and the accumulated drug release percentages at 72 h were about 80.9% and 77.9%, respectively. These results indicated that the SLNs could be effectively loaded with ATC and the drug behavior of the CSK peptide-modified SLNs was similar to that of the SLNs. Hence, CSK would be considered to negligibly interfere with the drug release.

Fig. 6 In vitro release profiles of ATC, ATC SLNs and ATC CSK-SLNs in pH 6.8 phosphate buffer.

3.3.1. In vitro cytotoxicity study. Currently, a Caco-2 cell monolayer was used as an intestinal model to evaluate the effect of delivery systems on drug permeation via transcellular, and paracellular transports, a carrier mediated route and by transcytosis.^32–34 Meanwhile, HT-29 subclones have been used in co-cultures with Caco-2 cells to mimic the small intestinal epithelial layer by containing both mucous and columnar absorptive cells.³⁵ To assess the safety of the prepared SLNs and CSK-SLNs, the viability of Caco-2 cells and HT29 cells in the presence of this compound was evaluated separately in the MTT assays. The cell viability was studied in different concentrations, and Fig. 7A and B show the cytotoxicity effect of the blank SLNs, blank CSK-SLNs, ATC SLNs and ATC CSK-SLNs. The results after incubation for 4 h at the tested concentration showed cell viabilities higher than 80% of that of the negative control, which indicated that all of the formulations had no obvious cytotoxicity for the Caco-2 cells and HT29 cells.

Fig. 7 Cell viability of the intestinal cells after exposure to SLNs and CSK-SLNs assessed by the MTT assay. The viability of Caco-2 (A) and HT29 (B) cells after 4 h incubation with different nanoparticle concentrations at 37 °C.

3.3.2. Cellular uptake study. The targeting effect of CSK peptide modification was evaluated by confocal laser microscopy. Therefore, a co-cultured cell model consisting of both absorptive enterocyte-like Caco-2 cells and the mucus-producing HT29 cells could provide a better simulation of natural conditions.⁹ This co-culture model simulates the intestinal epithelium due to the existence of a mucus layer that is near the true condition of the TEER of the intestinal epithelium.²² It was selected to study the transport efficiency of the SLNs and CSK-SLNs.

To clarify the internalization of the SLNs, the cell nuclei were stained with Hoechst 33342 (blue) while FITC (green fluorescence) was used to label the SLNs. As shown in Fig. 8, the stained nucleus surrounded by FITC was observed, suggesting the successful internalization of SLNs and CSK-SLNs into the cytoplasm by the co-culture cell monolayer. The images indicate that the cellular uptake of the SLNs and CSK-SLNs is time dependent, and the maximal signals were presented at 4 h. According to the images, the significantly strong green fluorescence in the cytoplasm of the co-cultured cell monolayer treated with CSK-SLNs revealed the potent targeting binding of CSK to intestinal epithelium cells.

Fig. 8 Confocal microscopy images show the cellular uptake of SLNs and CSK-SLNs (200 μg mL⁻¹) by Caco-2/HT29 co-cultured cells for different incubation times.

From the results, CSK peptide modification could greatly promote the internalization of SLNs on Caco-2/HT29 co-cultured cells which was consistent with the results in previous studies.¹⁰

3.4. In situ single-pass intestinal perfusion study

In order to account for the shortcomings of the cell monolayer model such as the lack of a three-dimensional macrostructure and cells of varying degrees of differentiation, the absorptive behaviours of ATC, the ATC SLNs, and ATC CSK-SLNs in three different intestinal segments were investigated using the in situ single-pass perfusion method in rats. Comparison of three different intestinal segments of the absorption parameter (K_a) and the apparent permeability (P_app) are presented in Table 2. It was interesting to notice that not only for ATC but also for the ATC SLNs and ATC CSK-SLNs do the values of both K_a and P_app appear to be significantly higher in the duodenum than those in the jejunum and ileum. For the ATC-loaded CSK-SLNs, the absorption in the duodenum segment (K_a, 0.076 ± 0.23 min⁻¹) was 1.71- and 2.97-fold higher, and the apparent permeability (P_app, 0.011 ± 0.63 cm min⁻¹) was 1.31- and 2.99-fold higher than for the ATC SLNs and ATC solution, respectively. There was also a significant increase in permeation produced by the ATC CSK-SLNs in the jejunum and ileum segments. As a result, SLNs and CSK-SLNs could effectively improve the absorption of insoluble ATC in the whole intestinal segment, especially in the duodenum. Moreover, the values of both K_a and P_app of ATC were significantly improved as formulated into the CSK-SLNs in various intestinal segments.

Table 2 Absorption rate constant (K_a) and permeability coefficients (P_app) of ATC, the ATC SLNs and ATC CSK-SLNs measured by in situ single-pass intestinal perfusion^a

Intestinal segment	Ka × 10^−2 min^−1			Papp × 10^−3 cm min^−1
	ATC	ATC SLNs	ATC CSK-SLNs	ATC	ATC SLNs	ATC CSK-SLNs
a *p < 0.05, **p < 0.01, compared with ATC.
Duodenum	2.56 ± 0.91	4.45 ± 0.43*	7.62 ± 0.23**	3.62 ± 0.68	8.29 ± 0.79**	10.82 ± 0.63**
Jejunum	2.08 ± 0.89	2.94 ± 0.15*	4.89 ± 0.12**	2.97 ± 0.48	6.93 ± 0.61**	8.68 ± 0.33**
Ileum	1.48 ± 0.23	2.77 ± 0.12*	3.45 ± 0.21*	2.47 ± 0.42	4.16 ± 0.29*	6.45 ± 0.28**

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K_a and P_app were markedly higher than those of the ATC solution. It could be interpreted that the efficient modification of CSK to the particle surface allowed nanoparticles to rapidly penetrate through the viscoelastic small intestinal mucus, and transported nanoparticles from intestinal lumen to systemic via specific intestinal mucosal binding. On the other hand, numerous studies highlighted the potential of nanoparticles in improving the gastrointestinal absorption and oral bioavailability in vivo.³⁶ Overall, the in vivo ligated intestinal loop showed good correlation with the improved absorption and transport of SLNs in the cell study in vitro.

4. Conclusions

ATCSLNs and ATC CSK-SLNs were successfully prepared and characterized. Both of them have a spherical morphology, uniform size, positive zeta potential, and high drug entrapment efficiency with a sustained release profile in vitro. The in vitro transport study suggested that the CSK peptide-modified SLNs showed an excellent mucus penetrating ability across the intestinal cell monolayer, although mucus was an impediment to the transport of SLNs. The intestinal absorption (K_a, 0.076 ± 0.23 min⁻¹, P_app, 0.011 ± 0.63 cm min⁻¹) of the ATC CSK-SLNs was 2.97-fold (p < 0.01) and 2.99-fold (p < 0.01) higher compared with that of the ATC solution. The overall investigations present an alternative drug delivery system for increasing the atorvastatin calcium bioavailability through CSK peptide ligand-modified solid lipid nanoparticles. Moreover, the targeting peptide-modified solid lipid nanoparticles are promising vehicles for oral delivery of hydrophobic drugs.

Acknowledgements

The authors acknowledge the National Science Foundation of China (NSFC) (no. 21172148, 21472126) and Shanghai Excellent Young Program (no. 4521ZK11YQ02) funding support.

Notes and references

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