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Teng Zaijin,1,* Yu Miao,1,* Ding Yang,1,* Zhang Huaqing,1 Yan Shen,1 Jiang Menglao,1 Liu Peixin,1 Yaw Opoku-Damoah,2 Thomas J Webster,3 Zhou Jianping1 1 Pharmacy Department, State Key Laboratory of Natural Medicine, China Pharmaceutical University, Nanjing 210009; 2 Institute of Bioengineering and Nanotechnology, University of Queensland, St Lucia, Brisbane, QLD 4072, Australia; 3 Department of Chemical Engineering, Northeastern University, Boston , MA 02115, USA* These authors contributed to this work for the same purpose: Nimodipine (NMP) is a clinical dihydropyridine calcium antagonist.However, the clinical application of NMPs is limited by poor water solubility and low oral bioavailability.To overcome these shortcomings, optimal NMP-incorporated nanostructured lipid carriers (NLCs) were designed in this study.Methods: NMP-NLC was successfully prepared by high pressure homogenization, and the morphology of nanoparticles was observed by transmission electron microscopy.The existing forms of NMP in NMP-NLC were investigated by powder X-ray diffraction, differential scanning calorimetry and Fourier transform infrared spectroscopy, respectively.In vitro release studies were performed by dialysis, and in situ intestinal perfusion and pharmacokinetics in rats were measured by high performance liquid chromatography.RESULTS: The obtained NMP-NLCs were spherical in shape of ~70 nm, with a smooth surface, and the encapsulation efficiency was as high as 86.8%±2.1%.The spectrum showed that the drug was in an amorphous state.NMP-NLC exhibited sustained release and diverse release profiles under different release media, simulating the physiological environment.Furthermore, in situ intestinal perfusion experiments showed that NMP-NLC was mainly absorbed by the small intestine.Significant improvements in Cmax and AUC0-∞ of NMP-NLC were obtained by pharmacokinetic experiments, and the relative bioavailability of the NMP-loaded nanostructured lipid system relative to the NMP suspension was 160.96%.Conclusions: Overall, NLC significantly enhanced the oral bioavailability of NMPs and may provide a promising nanoplatform for hydrophobic drug delivery.Keywords: nimodipine, nanostructured lipid carrier, increase drug solubility, improve bioavailability
Nimodipine (NMP), a new-generation dihydropyridine calcium channel blocker, was originally developed by Bayer for the treatment of brain dysfunction due to its high selectivity for brain tissue receptors and high blood-brain barrier permeability .It is considered the drug of choice for the prevention of subarachnoid hemorrhage and a good opponent for Alzheimer’s disease, stroke and hypertension.1 Has been one of the top ten products sold by Bayer over the past 30 years, generating an economic value of $120 million.At present, with the development of pharmacy, NMP has been widely used in various marketed products such as injections, tablets, capsules, and oral liquids, which has promoted its clinical application.However, in terms of pharmacological properties, NMP belongs to class II of the Biopharmaceutical Classification System with a half-life in plasma of 7-8 hours.2 In clinical applications, NMP is limited due to its low solvency and first excess, which affects low bioavailability.3 Thus, even for the best oral formulation of NMP tablets, Nimotop® marketed by Bayer, patients had to take 60 mg (about two tablets) every 4 hours for 21 consecutive days.Such high doses have triggered a series of non-compliance and inconvenience, and even limited its market prospects.4 Although intravenous injection can make up for this deficiency, various safety issues cannot usually be ignored.5 Therefore, there is an urgent need to design suitable formulations to enhance the biological activity of NMPs.
Since the 1990s, nanotechnology has been widely used at the intersection of biology and medicine.Notably, nanoparticle drug delivery systems including liposomes, nanoemulsions, and polymer nanoparticles have gradually become an emerging hotspot.Lipid nanoparticles, including solid lipid carriers and mixed lipid carriers in nanostructures,6 have been identified as suitable carriers for the delivery of drugs or other active ingredients.The first generation lipid carrier solid lipid nanoparticles (SLNs) contain biocompatible solid lipid matrices.Likewise, nanostructured lipid carriers (NLCs) include both liquid and solid lipids, inspired by SLNs.7 More importantly, the potential of mixed lipid systems to guarantee excellent bioavailability, controlled drug release, and large-scale industrial-scale production has been explored.compared to SLN.8 In addition, the heterolipidoid components in NLC can form internal defect structures and amorphous states to encapsulate more drugs and avoid drug leakage during storage.9,10 In addition to high drug-loading efficiency, NLC has many advantages for various delivery methods via oral, pulmonary, dermal, and ocular, which expand the application of nanoparticle delivery systems.For the oral route, NLC showed superior drug-controlled release capabilities over other lipid carriers in the gastrointestinal tract, and different release mechanisms were achieved by adjusting the liquid lipid content of the solid form.In promoting the absorption of drugs with low water solubility, due to its small particle size and lipid solubility, it can maintain sufficient solubility at the intestinal absorption site.11 When interacting with bile salts to form micelles, lipid components in NLC are degraded into mono- and diglycerides triggered by digestive enzymes to improve drug solubility in the static aqueous layer on the surface of intestinal villi.8 In addition, NLC can enhance internal drug absorption via lymphatic circulation or Peyer’s patches for selective uptake, thereby avoiding elimination from the liver.12 These clues suggest that NLC is a promising oral vehicle.
In general, it is necessary to achieve new purposes of traditional medicine by developing more suitable formulations of medicines to enhance their therapeutic capabilities.13 Therefore, in this study, an NMP-loaded NLC (NMP-NLC) was designed to evaluate NLC for enhancing oral bioavailability.Here, particle size, zeta potential, encapsulation efficiency (EE%), morphology, physicochemical properties, and in vitro properties were evaluated to characterize the developed NMP-NLC.In addition, the biopharmaceutical properties of the optimized NMP-NLC were evaluated by in situ intestinal perfusion studies and rat pharmacokinetic studies, which verified the feasibility of NMP-NLC for NMP absorption and bioavailability, respectively.
NMP was purchased from Xinhua Pharmaceutical Co., Ltd. (Shandong, China).Precirol ATO 5 and glycerol monostearate (GMS) were purchased from Gattefosse (Saint-Priest, France), medium chain triglycerides (MCT), isopropyl myristate (IPM), ethyl oleate (EO) , oleic acid (OA), and propylene glycol diddecanoate (Labrafac PG) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).Poloxamer 188 and Tween 80 were purchased from Thai Chemical Co., Ltd. (Shanghai, China).Vitamin E polyethylene glycol succinate (TPGS), polyoxyethylene 12-hydroxystearate (Solutol HS-15), polyoxyethylene castor oil (ELP), and hydrogenated polyoxyethylene castor oil (RH 40) by Courtesy of BASF GmbH (Ludwigshafen, Germany).All reagent chemicals meet analytical grade standards.
The method for formulation screening was introduced on the basis of previous studies that explored the partitioning behavior between different solid lipids and NMP through drug solubility studies.13 Among the various solid lipids, ATO5 and GMS were selected due to the higher solubility of NMP.14 Then, liquid lipid screening was performed by exploring the saturation solubility of NMP in various types of lipids.Briefly, the drugs were dissolved in liquid lipid (1 mL) and incubated in a shaking bath (HZC-250; Bing Lab Equipment Co. Ltd, Beijing, China) at 200 rpm at 37 °C ± 0.5 °C Incubate for 48 hours. 15 The mixture (KL05R; Aedas Scientific Instruments Co., Ltd., Changsha, China) was centrifuged at 3,000 rpm for 30 min, and the supernatant was filtered using a 0.22-micron membrane filter.The concentration of NMP in the supernatant was determined by high performance liquid chromatography (HPLC) (Agilent 1260; Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (250 mm × 4.6 mm, 5 μm). 16 The mobile phase was water and methanol (30:70, v/v) at a flow rate of 1 mL/min to detect NMP at 356 nm.In addition, different batches of NMP-NLC were prepared with mass ratios of solid lipid and liquid lipid of 2:1, 7:3, 3:1, 4:1 and 5:1.17, respectively.Particle size and EE%.
As shown in Figure 1, NMP-NLC was prepared by high pressure homogenization.18 Briefly, Labrafac PG (0.9 g) and Precirol ATO 5 (2.1 g) were mixed and melted at 80°C to form an oil phase. Then, NMP coarse powder (100 mg) was dissolved in ethanol (5 mL), Add to the above oil phase and heat to evaporate the ethanol.The resulting mixture was added dropwise to hot water (30 mL, 80 °C) containing Tween 80 (1.4 g), and then stirred at 15,000 rpm for 5 minutes by high-speed shearing (FS-400D; sold by Zhibolian Testing Instrument Co., Ltd. , Tianjin, China).19 The obtained pre-emulsion was transferred to a high pressure homogenizer (AH-2010; ATS Engineering Inc, Suzhou Branch, Jiangsu, China) and homogenized at ​​100 MPa for six cycles.20 The dispersion was then coagulated at room temperature to form nanoparticles, filtered through a 0.22 micron membrane, collected and stored at 4°C for further use.
Fig. 1 The preparation flow chart of NMP-NLC.Abbreviation: NMP-NLC, nimodipine-loaded nanostructured lipid carrier.
Particle size and zeta potential of NMP-NLC were detected by dynamic light scattering (DLS) at 25°C using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK).Three measurements were performed after five-fold dilution with ultrapure water.
EE% was detected by ultracentrifugation.21 Briefly, 1 mL of the NMP-NLC suspension was diluted in methanol and demulsified by an ultrasonic water bath.The dispersion was added to an ultrafiltration centrifuge tube and centrifuged at 4,000 rpm for 20 minutes.The ultrafiltrate containing free drug was filtered through a 0.22 micron membrane filter and detected by HPLC as described above.Likewise, LE% is the ratio of incorporated drug to lipid (w/w).EE% and LE% are calculated by the following equations.(1) and (2), respectively.
In the formula, WTotal is the total drug content in the NLC, WFree is the unencapsulated drug content in the ultrafiltrate, and WLipid is the lipid content in the system.
TEM analysis was used to observe the morphology of the nanoparticles.Briefly, diluted NMP-NLC solutions were deposited on formvar-coated copper grids and then negatively stained with 1% (w/v) phosphotungstic acid for 10 min.The obtained sample was observed by TEM (H-7650; Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 200 kV.
The in vitro release study of NMP in NMP-NLC was detected according to the dialysis method.22 dialysis bags (MWCO 12-14 kDa; Ruida Henghui Technology Development Co., Ltd., Beijing, China) according to the manufacturer’s initial activation protocol.To study the NMP release profiles of NMP-NLC in response to different physiological pH values, HCl (0.1 M, pH 1.2, 0.5% Tween 80) and PBS (0.01 M, pH 6.8, 0.5% Tween 80) were introduced as release media to stimulate gastric juice, respectively and intestinal fluid.The resulting NMP-NLC (1 mL, 2.5 mg NMP) and an equal amount of NMP suspension were added to the dialysis bag, then immersed in the released medium (100 mL, 37 °C) in a shaking bath at 100 °C Incubate turn.At predetermined time intervals, including 0.5, 1, 2, 4, 8, 12, 24, and 48 hours, 1 mL of release samples were withdrawn for HPLC analysis as described above.Refresh the entire medium with warm release medium.Release profiles were obtained by calculating the percentage of cumulative drug release versus time and estimated from the equation.(3):
A preliminary study on the physical stability of NMP-NLC was carried out.Freshly prepared NMP-NLC were stored in closed glass vials at 4°C and 25°C, respectively.During the 2-week monitoring period, particle size and polydispersity index were detected at 0, 1, and 2 weeks, respectively.
DSC analysis Analysis was performed using a differential scanning calorimeter (DSC 8000/8500; PerkinElmer Inc., Waltham, MA, USA).23 The samples consisted of NMP meal, a physical mixture of NMP and lipids in mass ratios with NMP-NLC and lyophilized NMP-NLC powders were accurately weighed to 3 mg and sealed in aluminum pans, and then incubated at 40°C to 200°C Heating at a rate of 10°C/min at the specified temperature of C.At the same time, the whole process is protected under nitrogen protection.An empty aluminum pan was used as a reference.Carry indium and zinc standards to calibrate the enthalpy scale and temperature, respectively.
PXRD analysis is used to identify composition and structure based on the diffraction signatures produced when X-rays pass through the crystal lattice of a substance.PXRD patterns were obtained using a powder X-ray diffractometer (XRD-6000; Shimadzu Group Co., Ltd., Kyoto, Japan).24 different samples, including NMP meal, physical mixture, and lyophilized NMP-NLC powder were lightly held in aluminum holders.The detection conditions are as follows: the radiation source is a Cu-Kα tube with a wavelength of 1.5406 Å.The tube voltage was 40 kV and the current was 40 mA.The sample was scanned from 3° to 40° (2θ) in 0.02° steps at a scan rate of 1.2°/min.X-ray diffraction patterns were evaluated by DIFFRAC plus EVA (ver.9.0) diffraction software.
The infrared absorption properties of NMP drug substance, physical mixture, and NMP-NLC lyophilized powder were compared by FT-IR (IRTracer-100; Shimadzu, Kyoto, Japan).The crystalline form of the drug is detected by the change of the characteristic absorption peak or the formation of hydrogen bonds between the components.The samples were prepared by the KBr tablet method, and the FT-IR spectra were recorded by an FT-IR analyzer.25 Briefly, the sample was extruded into a fine powder, and anhydrous KBr (sample: KBr) was added in a mass ratio of 1:5 to compact into fine particles.The pressure was 5 tons, the pressure was 3 minutes, the scanning range was 500-4,000 cm-1, and the resolution was 1.0 cm-1.
The SPIP study was a slight modification of the previous study.26 The sample preparation procedure was as follows: NMP-NLC and NMP solutions were diluted to 30 μg/mL in Krebs Ringer buffer.27 All animal experiments were conducted in accordance with the protocols approved by the Ethics Committee of China Pharmaceutical University and in accordance with the Guidelines for the Care and Use of Laboratory Animals.Sprague-Dawley rats (200-250 g; Qinglongshan Animal Center, Nanjing, China) were injected with 10% chloral hydrate (3 mL/kg) by intraperitoneal injection.They were then immobilized on rat plates with infrared light to maintain body temperature.Open an abdominal cavity (3-4 cm in length) at the midline of the rat’s abdomen.Separation of intestinal segments (small intestine and colon).Then normal saline at 37°C was pushed into the intestinal segment to wash the intestinal contents.Insert a silicone tube into the end of the intestinal segment.Place pre-weighed donor and recipient vials on either side of the intestinal segment.Before perfusion, the intestine was perfused at 3 mL/min for 15 minutes to reach a steady state.Then, perfusate (NMP-NLC and NMP solution, 30 μg/mL) was injected at 1 mL/min.Donor and recipient vials were rapidly changed every 15 minutes, and perfusate was collected at 60, 75, 90, 105, and 120 minutes.Samples were stored at –20°C prior to analysis.After the rats were sacrificed, each intestinal segment was excised and the length (l) and inner diameter (r) were recorded.Finally, the lysed samples were centrifuged at 12,000 rpm for 10 minutes.After diluting the supernatant with methanol, samples (20 μL) were analyzed by HPLC.
Absorption (Ka) and apparent permeability (Papp) are calculated based on the following equations:
where Cout and Cin represent the drug concentration at the outlet and inlet, respectively; Vout and Vin are the volumes of perfusate in the recipient vial and donor vial, respectively; Q is the flow rate (mL/min), l is the intestinal length (cm), and r is the Intestinal radius (cm).
All animal experiments in this study were approved by the Ethics Committee of China Pharmaceutical University and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Male Sprague-Dawley rats of 200–250 g were housed at a temperature of 25°C ± 2°C and a relative humidity of 45%–50%.All rats were randomly divided into two groups (n=7) and fasted for 12 hours (water ad libitum) before the experiment.The NMP suspension was prepared by dissolving NMP powder in 0.5% CMC-Na solution.28 Then, NMP-NLC and NMP suspension were orally administered to rats at a dose of 40 mg/kg, respectively.Blood samples (0.5 mL) were collected via the infraorbital vein at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 8, 12, and 24 hours post-dose.All blood samples were immediately centrifuged at 5,000 rpm for 5 minutes to obtain clear plasma and then stored at -20°C prior to HPLC analysis.29
In this study, diazepam solution (50 μL) was used as internal standard (50 μg/mL), and 100 μL plasma samples were mixed.After vortexing for 1 minute, 500 μL of tert-butyl methyl ether was added and the drug was extracted by vortexing for 5 minutes at room temperature.Then, the mixture was centrifuged at 3,000 rpm for 10 minutes.The organic layer was transferred to a new container and evaporated in a vacuum desiccator.The residue was dissolved with 200 μL of mobile phase.The final mixture was centrifuged at 3,000 rpm for 5 minutes to obtain the supernatant for HPLC analysis.30
The main pharmacokinetic parameters were calculated by the pharmacokinetic program PK Solver 2.0.31 to obtain the maximum concentration (Cmax) and the time to Cmax (Tmax) obtained from the experimental data.The area under the concentration-time curve (AUC) was calculated by the linear trapezoidal method.The relative bioavailability of NMP-NLC was calculated using the following formula:
where Fr represents relative bioavailability, AUC is the area under the plasma concentration curve, D is the dose administered, T is the determinant formulation (NMP-NLC), and R is the reference formulation (NMP suspension).
All results are expressed as mean ± standard deviation, and t-test was performed using SPSS 22.0 software.Numerical data were used to assess the significance of differences, and *P<0.05, **P<0.01, and ***P<0.001 were defined as statistically significant.
Given that lipid properties are critical to the drug loading capacity, stability and even drug release ability of NLCs, lipids were screened for optimal nanostructured lipid systems.Therefore, we first screened out solid lipids according to different characteristic parameters.Relevant data, including particle size, polydispersity index (PDI), zeta potential and EE%, are shown in Table 1.It can be found that the particle size of NLCs prepared with ATO5 is the smallest, which is 76.1±0.2 nm, and the highest EE% is 87.5%±2.4%.Furthermore, a suitable liquid lipid plays a crucial role in NLC as it can form an incomplete lattice to store more drugs.Therefore, drug solubility in appropriate liquid lipids is a key factor for formulation screening.Therefore, a solubility study of NMP in liquid lipids was performed.As shown in Table 2, the best solubility in Labrafac PG was 24.88±2.1 mg/g, which was significantly higher than 4.65±0.8 mg/g for OA, 12.34±1.4 mg/g for IPM, and 13.58±1.2 mg/g for EO. g, MCT was 18.74±1.6 mg/g.Therefore, Labrafac PG was selected as the most suitable liquid lipid.To investigate the optimal ratio of ATO5 and Labrafac PG, the ratios of various mixtures were investigated (Fig. 2A).When the ratio (g/g) of solid lipid and liquid lipid was 7:3, no separation between lipids was observed.The smallest particle size of NLCs prepared with this ratio was 86.1±0.2 nm, and the highest EE% was 86.8%±2.8%.In addition, the effects of different drug contents were evaluated by changes in drug and lipid mass ratios (Fig. 2B).The results show that when the mass ratio (drug:lipid) exceeds 0.03:1, the EE% starts to decrease.It is believed that when the dose is higher, the amount of drug exceeds the steric capacity of the lipid mixture, leading to drug precipitation resulting in lower EE%.Therefore, a mass ratio of 0.03:1 (drug:lipid) was considered the most appropriate.
Table 1 Effects of different solid lipids on particle size, PDI, zeta potential and EE% Note: All results are expressed as mean ± standard deviation (n=3).Abbreviations: ATO5, Precirol ATO 5; EE%, encapsulation efficiency; GMS, glycerol monostearate; PDI, polydispersity index.
Table 2 Apparent solubility of NMP in various liquid lipids Note: All results are presented as mean ± standard deviation (n=3).Abbreviations: EO, ethyl oleate; IPM, isopropyl myristate; MCT, medium chain triglycerides; NMP, nimodipine; OA, oleic acid; PG, propylene glycol dicaprate.
Figure 2. Formulation screening of nanostructured lipid carriers.NOTE: (A) Effects of different ratios of solid lipid and liquid lipid (g/g) on ​​particle size and EE%.(B) Effects of various ratios (g/g) of drug and lipid on particle size and EE%.(C) Effects of different surfactants on particle size and EE%.(D) The effect of different Tween 80 content (g) on ​​particle size and EE%.Results are expressed as mean ± SD (n=3).Abbreviations: EE%, retention efficiency; HS 15, polyoxyethylene ester of 12-hydroxystearic acid; RH 40, hydrogenated polyoxyethylene castor oil; TPGS, vitamin E polyethylene glycol succinate.
Emulsifiers can effectively reduce the particle size of lipid nanoparticles and increase their physical stability because they can coat the surface of hydrophobic particles and reduce the surface tension between the particles and the aqueous medium, thereby preventing particle aggregation or growth.32 Therefore, six nonionic surfactants, including Tween 80, Poloxamer 188, TPGS, Solutol HS-15, ELP, and RH 40, were first screened based on particle size and EE%, as shown in Figure 2C.Among all the surfactants, Tween 80 has the smallest particle size of 78.6±1.3 nm and the highest EE% of 87.7%±3.8%.On the other hand, surfactant content also had a significant effect on particle size and EE% (Fig. 2D).When the Tween 80 content increased above 1.4 g, the particle size and EE% increased.Higher levels of Tween 80 lead to drug deposition and micellar aggregation, disrupting the stability of the system.Therefore, the Tween 80 content of 1.4 g was chosen as the optimal formula.
It has been reported that NLCs can be prepared by high-pressure homogenization or ultrasonic dispersion.33 High-pressure homogenization has been considered as a method for large-scale production of NLCs, which can reduce the use of organic solvents.Produces and produces a more uniform particle size distribution.Therefore, NMP-NLC was prepared by using high pressure homogenization.Notably, prior to homogenization, high-speed shearing of the coarse dispersion was used to pulverize large particles and avoid clogging of the high-pressure homogenizer.The effect of different shear rates on particle size distribution is shown in Fig. 3A.When the shear rate was 15,000 rpm, no oil slick adhered to the surface of the dispersion.NLC at this rate showed a minimum particle size of (71.9±0.9) nm and a uniform distribution.In addition, pressure and number of homogenization cycles also affect particle size and PDI.As shown in Figures 3B, C, and 6, a cycle of 100 MPa was confirmed as the final homogenization condition for optimal formulation.
Figure 3. Technical screening of nanostructured lipid carriers.Note: (A) Effects of different shear rates (×103 rpm) on particle size and PDI.(B) Effects of different homogenization pressures (MPa) on particle size and PDI.(C) Homogenization cycles of different particle sizes.Results are expressed as mean ± SD (n = 3).Abbreviation: PDI, Polydispersity Index.
The resulting NMP-NLC was a light blue clear solution with no insoluble components, clumps or drug precipitation.In addition, it achieves satisfactory fluidity.The average particle size of NMP-NLC was 71.0±1.3 nm, measured by DLS (Fig. 4A), and the PDI was 0.057±0.027.This narrow and uniform particle size distribution can increase the surface area and wettability of the drug, which, according to the Noyes-Whitney equation, will lead to increased drug solubility in the gastrointestinal tract and facilitate drug absorption.The negative zeta potential of NMP-NLC at -24.8 ± 2.0 mV can guarantee the physical stability of the nanosystem, because it is generally believed that when the zeta potential of the nanoparticles is ~ ± 30 mV, unwanted aggregation and agglomeration can be avoided by the following means.Static Barrier.34 In addition, the mean LE% of NMP-NLC was 3.2% ± 1.5% and the EE% was 86.8% ± 2.1%.
Figure 4 In vitro pharmacological properties of NMP-NLC.NOTE: (A) Particle size and PDI of NMP-NLC analyzed by DLS.(B) TEM morphology of NMP-NLC.bar = 50 nm.(C) In vitro drug release profiles of NMP-NLC and NMP suspensions in simulated gastric fluid at pH 1.2.(D) In ​​vitro drug release profiles of NMP-NLC and NMP suspensions in simulated intestinal fluid at pH 6.8.(E) Particle size and PDI of NMP-NLC stored at 4°C for 2 weeks, and (F) Particle size and PDI of NMP-NLC stored at 25°C for 2 weeks.Results are expressed as mean ± SD (n = 3).Abbreviations: NMP-NLC, nimodipine-loaded nanostructured lipid carrier; PDI, polydispersity index.
As shown in Figure 4B, the TEM image shows that the particles are spherical and monodisperse in diameter.In addition, the average size of NMP-NLC was 57.755 ± 9.508 nm, which was smaller than that detected by DLS.It can be explained that the particle size measured by DLS is the hydrodynamic diameter, whereas the particle size measured by TEM is the true size of the dried particles.35
In this study, tank conditions were critical to simulate a physiological environment.36 Therefore, the saturated solubility of NMP in different media was measured.The saturated solubility in distilled water, 0.3% Tween 80 solution, 0.5% Tween 80 solution, and 0.7% Tween 80 solution were 13.98, 74.45, 96.33, and 132.56 μg/mL, respectively.When the Tween 80 content is 0.5%, 0.7% and 0.9%, the sinking conditions are satisfied.However, since the surfactant concentration cannot be too high, a 0.5% Tween 80 solution was chosen as the optimal surfactant concentration.A release study was conducted in various media at different pH values ​​designed to mimic physiological pH gradients (pH 1.2 as gastric fluid and pH 6.8 as intestinal fluid).Comparing the release profiles of NMP-NLC at pH 1.2 and pH 6.8, only 10% of NMP was released in the first 4 h, indicating that free drug was attached to the particle surface.As shown in Figure 4C and D, 29.77% of NMP was released from NMP-NLC at pH 6.8 for 48 hours, which was higher than that at pH 1.2 (20%).However, NMP suspensions at pH 1.2 tended to release 90% of NMP at 24 hours.The results showed that the lipid carrier can encapsulate and protect NMP from gastric acid damage, and then deliver NMP to the small intestine.It can be absorbed by intestinal cells and transported to the body’s circulation.On the other hand, the amorphous lattice of NMP in NMP-NLC leads to a higher dissolution rate than the lattice under certain conditions.37
Physical stability is critical during the exploration of lipid carriers, as it is necessary to ensure that the nanoparticles are sterile and have a stable particle size.Here, short-term stability tests were performed to evaluate the physical stability of NMP-NLC at 4°C and 25°C.As shown in Figure 4E, within 2 weeks, the particle size of NMP-NLC at 4°C increased slightly from 75.6 ± 1.2 nm to 88.1 ± 2.4 nm, and the PDI increased from 0.104 ± 0.018 to 0.168 ± 0.015.Likewise, the particle size of NMP-NLC was observed to increase from 75.6±1.2 nm to 107.3±1.4 nm and the PDI from 0.104±0.018 to 0.213±0.023 at 25°C (Fig. 4F).NMP-NLC continued to increase in particle size after storage at 25°C for 1 week, while no significant change was observed at 4°C.Therefore, NMP-NLC is stable for 2 weeks at 4°C and 25°C, and is more suitable for storage at 4°C.
DSC is a fundamental method to study phase transition temperature and energy changes to explore drug lattice morphology in hybrid systems.38 Therefore, DSC was performed to detect drug-excipient interactions.The DSC thermograms of NMP, physical mixture and lyophilized NMP-NLC powder are shown in Figure 5A.The melting peak of NMP in the thermogram is about 124.9 °C, which belongs to the crystalline nature of NMP.The DSC curve of the physical mixture has peaks at 50.9°C and 123.8°C.For lyophilized NMP-NLC, there were two melting peaks at 43.0 °C and 54.7 °C, which belonged to the lipid mixture.This pattern can be explained by melt recrystallization during heating.Furthermore, in the spectrum of NMP-NLC, the peak of NMP around 124.9°C disappeared, which indicated that NMP was embedded in NLC and appeared amorphous rather than crystalline.The results are consistent with those of in vitro release experiments where the drug achieves a reasonable release rate due to the amorphous state.
Figure 5 The crystal form of NMP-NLC.Notes: (A) DSC thermograms of NMP coarse powder, lipid physical mixture, NMP and lyophilized NMP-NLC powder.(B) X-ray diffraction patterns of NMP coarse powder, lipid physical mixture, NMP, and lyophilized NMP-NLC powder.Abbreviations: DSC, dynamic light scattering; NMP-NLC, nimodipine-loaded nanostructured lipid carrier.
The individual components and physical mixtures in the PXRD pattern show different peaks, which indicate different characteristic lattice forms.Here, the diffraction patterns of free and encapsulated NMP were detected by PXRD analysis.Performed using NMP meal, physical mixture and lyophilized NMP-NLC powder.The PXRD pattern is shown in Fig. 5B.In the PXRD pattern of NMP coarse powder, significant strong and clear diffraction peaks of NMP can be observed at two scattering angles of 6.556°, 12.368°, 12.912°, 17.387° and 20.335°.The pattern of the physical mixture shows that the peak intensity of pure NMP coarse powder is reduced, which indicates that the NMP in the physical mixture is still in a crystalline state, apparently the presence of lipids affects the peak intensity.However, compared with pure NMP, there are two different diffraction peaks (5.524° and 22.951°) in the pattern of NMP-NLC, which are considered to be characteristic peaks of Precirol ATO 5 .Furthermore, no spikes were found in the NMP lattice in the NMP-NLC powder, indicating that the NMP in the NMP-NLC is in an unformed state rather than a crystalline structure.These results are consistent with DSC analysis.
FT-IR has been used to assess the molecular interactions between NMPs and lipid matrices.In the FT-IR spectrum (Fig. 6), the characteristic bands of NMP include 3,296.9 cm-1 and 1,309.5 cm-1, which belong to the stretching vibration peaks of NH.The wavenumbers at 3,097.6 cm-1, 1,621.6 cm-1, 1,495.9 cm-1 and 809.4 cm-1 represent the benzene ring.Ester bands are 1,695.3 cm-1, 1,270.7 cm-1 and 1,022.6 cm-1, -NO2 are 1,523.3 cm-1 and 1,346.2 cm-1, COC is 1,098.9 cm-1, CH is 2,982.3 cm-1, 2,933.9 cm-1 -1, 1,454.9 cm-1 and 1,381.7 cm-1.These results are in good agreement with the 2015 edition of the Chinese Pharmacopoeia and previous studies.39 For the FT-IR data of the physical mixture and NMP-NLC, the new characteristic absorption bands are considered a lipid matrix.However, in the physical mixing spectrum, the intensity of the NMP characteristic band is significantly reduced, and for NMP-NLC, the peak even disappears, indicating that there is no chemical interaction between NMP and lipids.The entrapment of NMP in NLC depends only on the solubilization between drug and lipid.All the results of this experiment are consistent with previous studies.40
Fig. 6 FT-IR spectral analysis of NMP coarse powder, lipid and NMP physical mixture and lyophilized NMP-NLC powder.Abbreviations: FT-IR, Fourier transform infrared spectroscopy; NMP-NLC, nimodipine-loaded nanostructured lipid carrier.
Previous studies have shown that NMP is stable in the perfusate prior to perfusion.45 Typically, gravimetric and phenol red markers are used to indicate volume changes.In this study, the gravimetric method was used to calibrate the volume change of the perfusate.Because phenol red is partially absorbed in the gut, this interferes with the original extent of absorption or transport of poorly soluble drugs in rat intestinal perfusion.41 The obtained absorption rate constant (Ka) and apparent effective permeability coefficient (Papp) of the SPIP study are shown in Fig. 7.The results showed that the Ka order of NMP-NLC was small intestine>colon (P<0.05), and the Papp value of NMP-NLC in the small intestine was significantly higher than that in the small intestine.Colon (P<0.05).Furthermore, the Ka of NMP-NLC in the small intestine increased from (2.99±0.08) × 10-3 min-1 to (5.98±0.20) × 10-3 min-1 (twofold higher) compared with the NMP solution. The Papp of NMP-NLC in the small intestine also increased twofold compared to the solution (Table 3).This intuitively suggests that NMP-NLC can be absorbed mainly in the small intestine.The increased absorption of NMP-NLC in rats can be explained by some possible reasons.First, the nanoscale particle size dramatically increases the drug surface area, which significantly improves the apparent solubility and dissolution of NMP in NMP-NLC.Second, lipid carriers are mainly digested by lipase to form micelles or vesicles after oral administration.These newly formed drug carriers are readily absorbed by the small intestine through the static aqueous layer.42 The lipid composition in the carrier has a certain similarity with the composition of the intestinal epithelial cell membrane, which can increase the affinity with the cell membrane and promote the absorption or transport of drugs.Furthermore, p-glycoprotein (p-gp) has been reported to be widely expressed in cells such as intestinal epithelial cells, hepatocytes, and renal tubular epithelial cells.However, Tween 80 in NMP-NLC formulations is a p-glycoprotein inhibitor that reduces p-gp activity and reduces drug efflux, acting as an absorption enhancer.Finally, it may also be related to the physiology of the small intestine.The shrinkage and villi of the small intestine greatly increase the surface area of ​​the intestinal lumen, which provides the possibility for the effective absorption of drugs and is conducive to the absorption of drugs by the intestinal mucosa.
Fig. 7 Comparison of in situ absorption of NMP-NLC in rat intestinal segment with NMP solution.Notes: (A) Absorption rate (Ka) and (B) Effective permeability coefficient (Papp).Results are expressed as mean ± SD (n = 3).*P<0.05, compared to corresponding parameters of NMP solution.Abbreviation: NMP-NLC, nimodipine-loaded nanostructured lipid carrier.
Table 3 Absorption parameters of NMP solution and NMP-NLC in different intestinal segments Note: The concentration of NMP in each preparation is 30 μg/mL.Results are expressed as mean ± SD (n = 3).*P<0.05, compared to corresponding parameters of NMP solution.Abbreviations: Ka, absorption rate constant; NMP-NLC, nimodipine-loaded nanostructured lipid carrier; Papp, effective permeability constant.
Figure 8 shows the plasma concentration-time curves after a single gavage of NMP-NLC and NMP suspensions.After 24 hours of oral administration, the NMP plasma concentration was still 0.89 μg/mL in the NMP-NLC group, whereas the drug was not even detected in the NMP suspension group.Table 4 shows the mean parameters calculated by non-compartmental analysis.It was found that NMP-NLC exhibited higher Cmax (32.516±3.151 μg/mL vs 11.887±3.464 μg/mL, P<0.05) and faster Tmax (0.250±0.500 h vs 3.000±0.540 h, P<0.01).The data indicate higher uptake of NMP when loaded into NLC.The AUC0-∞ after oral administration of NMP-NLC was 73.546±8.933 μg/mL*h, which was about 1.6 times higher than that of NMP suspension 45.691±7.667 μg/mL*h.Therefore, the relative bioavailability of NMP-NLC was 160.96% compared to the NMP suspension.The results showed that NMP-NLC could significantly enhance the systemic absorption of NMP.NLCs show great potential to improve the oral bioavailability of poorly soluble drugs.This can be explained by various factors enhancing the oral bioavailability of NMP-NLC.First, according to the Noyes-Whitney equation, drug dissolution rate is positively related to surface area.The particle size of NMP-NLC is less than 200 nm, which can maintain a huge specific surface area, thereby enabling faster drug dissolution.31 In addition, smaller particles also facilitate the adhesion of mucosa to gastrointestinal surfaces, thereby ensuring sufficient retention time to improve oral bioavailability.Second, lipids in NLCs can be enzymatically degraded in the digestive tract to form emulsifiers such as monoacylglycerols and fatty acids, which enter the duodenum.At the same time, bile salts, cholesterol and pancreatic juice are stimulated to be excreted from the gallbladder into the intestinal lumen.NMP-NLC mixes with bile salts to form micelles that facilitate transport to the lymphatic circulation, thereby protecting it from the first-pass effect of the liver.43,44 Alternatively, orally administered nanoparticles are absorbed into Peyer patches by granulosa cells.After the nanoparticles are phagocytosed, they are transported to the basal cavity of M cells by vesicular transport.Nanoparticles are either unbound or phagocytosed by macrophages and transported from the lymphatic circulation into the blood circulation.This process also avoids the first-pass effect of the liver.These results are consistent with previous findings that adequate absorption and lymphatic transport pathways for nanoparticles contributed to enhanced oral bioavailability.32 In addition, results from intestinal perfusion studies suggest that Tween 80 can inhibit p-pumped drugs.gp efflux acts as a p-gp inhibitor, which may lead to improvements in Cmax and AUC0-∞.In addition, it has also been reported that NLCs can enhance cell membrane fluidity to increase intracellular transport or open tight junctions on intestinal mucosal epithelial cells, thereby facilitating the intercellular transfer of ionized drugs and hydrophilic macromolecules.
Figure 8. Plasma concentration-time curves of pharmacokinetic experiments.Note: Rats were given a single oral dose of 40000 μg/kg of NMP-NLC and NMP suspension.Results are expressed as mean ± SD (n=7).Abbreviation: NMP-NLC, nimodipine-loaded nanostructured lipid carrier.
Table 4 Pharmacokinetic parameters Note: Rats were given a single oral dose of 40 mg/kg of NMP-NLC and NMP suspension.Results are expressed as mean ± SD (n = 7).*P<0.05, **P<0.01, ***P<0.001, compared to corresponding parameters for NMP suspensions.Abbreviations: AUC0-∞, area under the concentration-time curve; AUMC, area under the bending moment curve; Cl/F, clearance in plasma; Cmax, maximum concentration; MRT, mean residence time; NMP-NLC, loaded nimodipine of nanostructured lipid carriers; Tmax, time to maximum concentration; t1/2, half-life in plasma.


Post time: Jun-10-2022