Nanostructured lipid carriers of ivabradine hydrochloride: Optimization, characterization and in-vivo estimation for management of stable angina

3.2.1 Particle size and zeta potential

A Delsa-Nano C particle size analyser was used to measure the particle size and PDI of prepared IBH-PNPs (Beckman Coulter, UK), which uses photon correlation spectroscopy as its basis. The observations were carried out utilising scattering light generated by a typical He-Ne laser at a fixed angle of 165⁰ at a temperature of 25 ⁰C. In order to calculate, the electrophoretic mobility of charged particles under the influence of an applied electric field was measured from the zeta potential of IBH-PNPs. Before all measurements, IBH-NLCs were diluted and sonicated.

3.2.2 Estimation of entrapment efficiency (EE)

Entrapment efficiency of IVB-NLCs are controlled via estimating free IVB in the water stage, detached utilizing centrifugation (Sun et al. 2014). 500 μL IVB-NLCs was added to the centrifugal filter Unit 100 kDa (Millipore Co, USA) and uses a UniCen MR (Herolab, Germany) to radiate for 20 min at 40°Celsius at 3000 rpm (Lakshmi et al. 2018). In order to analyse the filtrate, HPLC was used with a Dionex UltiMate 3000 (Sunnyvale, USA) by way of the C8 segment. The filtrate was degraded in methanol. A portable stage or mobile phase made of distilled water and acetonitrile (55:45 v/v) was used for the chromatographic analysis, which was carried out at 300C and 284 nm. The infusion volume was 20 L, and the stream speed was 1.0 mL/min. The 0.22 m membrane is used to describe the ultrapure water at the front. Using a set of criteria, the EE channel was identified (Mishra et al. 2023a):

Where w_total is quantity of initial IVB used and w_free is quantity of free IVB in the aqueous phase.

3.2.3 Morphology by TEM and SEM

In Transmission Electron Microscopy (TEM, JEM-2100F electron microscope, JEOL Ltd., Tokyo, Japan) at 200 kV, the morphological IVB-NLC traits were observed. The samples were dropped onto a copper grid made of Formvar/Carbon with a 230 mesh size, air-dried for 24 h at normal temperature, and then negatively saturated with phosphotungstic acid (1% w/v) about 25 min before examine.

For Scanning Electron Microscopy (SEM: Zeiss, Evo Research Ltd., Tokyo, Japan), a small drop of the nanoformulation was applied to the glass stub and allowed to air dry to found the thin film. After the dried coated with gold using a glass sputter coater in a high vacuum evaporator and then observed in SEM microscope.

3.2.4 In-vitro IVB release estimation

Employment of dialysis membrane technique, for activation of membrane it was placed over night in the phosphate buffer solution. IVB-NLCs (5 mL) filled in the dialysis membrane and it was kept in the phosphate buffer (150 mL, pH 7.4), stirred at (100 rpm at 37 ± 5 ⁰C). At a time up to 72 hrs, interval sampling was done and maintain the sink condition. HPLC, at 284 nm, was used to analyse samples. All experiment was performed in the triplicate manner.

3.2.5 Interaction study by DSC and FTIR

The IVB-NLCs and IVB were analysed by Differential Scanning Calorimetry (DSC Q20, thermograms (TA Instruments, New Castle, USA). The temperature raised from 25 to 240 °C during the temperature or cooling scan of 10 °C min⁻¹ when 3 mg of samples were placed in normalised aluminium pans. Dry nitrogen was employed as a liquid gas during the hot scales, and a reference for analysis was an empty pan (Dewangan et al. 2021; Ahmadian-Fard-Fini et al., 2021).

The Fourier transform infrared spectroscopy (FTIR: SHIMADZU 8400, Japan) was also used to determine compatibility. The characteristic bands and peaks of poloxamer, lecithin, IBH, physical mixture, and IBH-NLCs were examined in order to comprehend the interaction (Ahmadian-Fard-Fini et al., 2019; Ahmadian-Fard-Fini et al., 2020). The samples were thinly compressed with potassium bromide (KBr) using a pressed pellet method. The pellets were next exposed to the IR path length for scanning against a KBr background that was left empty. The scanning area was between 400 and 4,000 cm⁻¹.

3.2.6 X-ray severity scales

It was decided to gather and compare XRD data using lyophilized IVB-NLCs, lyophilized blank NLCs, and IVB using the X-ray diffractometer X'SPert3Power (PANalytical B.V., Almelo, Netherlands). Under CuK radiation, 40 kV of voltage, and 40 mA of current, an XRD examination was conducted (step size: 0.013; scan step time: 8.67 s) over an angle size of variation of 0 to 2 to 90 (Deepika et al. 2019).

3.4.1 Evaluation of hemolysis

Evaluation of hemolysis is required to demonstrate heme compatibility when NLCs are reached in circulation. Placebo-NLCs, IVB and IVB loaded NLCs have been performed using reported methods (Dewangan et al. 2018; Hung et al. 2004). In short, human blood was obtained from an authorized blood bank. The blood plasma membrane is carefully decomposed, mixed in an equal volume and gently mixed with normal saline solution. Small volumes of cell suspension (5 mL) were centrifuged at 1344 × g for 10 min in a clean graduate centrifuge tube at room temperature. Erythrocyte suspension was then centrifuged. The same process for washing was thrice. Finally, the solution is dilute to 50 mL and resuspended in regular saline solution (Dewangan et al. 2022).

Samples such as placebo IVB, and IVB loaded NLCs (equivalent to 10 and 20 ng/mL of IVB), placebo-nanoparticles (equal to volume of IVB loaded nanoparticles) were mixed with erythrocyte suspension (2 mL) in sterile tubes. As positive controls, 100% of the erythrocytes were lysed; as spontaneous negative controls, samples of normal saline and erythrocyte suspension were diluted with 1% Triton X-100, respectively. Every 15 min, the samples were gently mixed while being incubated at 37oC. At 0.5, 1, 2, 4, and 8 h, aliquots (200 L) were collected and centrifuged at 1344 g for 10 min. Thereafter, 100 L of the supernatant was oxidised to haemoglobin for 30 min at room temperature. to oxymoglobin after oxidation. The absorbance was measured spectrophotometrically at 284 nm (Synergy H1 Hybrid, Biotech, USA). The following formula is used to determine the proportion of hemolysis: $$\%Haemolysis=\frac{A_{\mathit{Sample}}-A_{\mathit{SpontaneuousControl}}}{A_{\mathit{PositiveControl}}}\times 100$$

Where A_sample is the absorbance of the erythrocyte supernatant after being incubated with IVB loaded NLCs formulations, A_{Spontaneous Control} is the absorbance of the erythrocyte supernatant after being incubated with normal saline equivalent to the volume of the samples, and A_{Positive Control} is the absorbance of the erythrocyte supernatant after being incubated with 1% triton X-100 solution. The data is shown as mean ± SD (n = 3) and experiments were carried out in triplicate.

3.4.2 Qualitative platelet aggregation

Peripheral blood smears were microscopically evaluated after whole blood is incubated with test specimens (10 and 20 ng/mL of plain IVB, IVB loaded NLCs and placebo-NLCs). After the peripheral blood smears were incubated, the samples were prepared, spread on a clean glass slide, and then air dried after 5 min of Leishman stain (Span Diagnostics, India). After washing the slides, the cover glass was placed on it, analysed with an optical microscope for immersion, and images were taken using a digital camera.

3.4.3 In-vivo study

In propylene cages male wister rates (200–250 g) were placed and kept in 12 h light/dark cycle run for the stabilization of standard laboratory atmosphere. All experiment was under the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals). The animals were sacrificed by Euthanasia; disposal by incineration.

3.4.4 Ex-vivo intestinal permeation study

Intestinal permeation potential of IVB-NLCs across GIT was evaluated by non-everted gut sac technique (Singh et al. 2010). The experimental rat was divided into two groups (=3) and following the ether anaesthesia that the cervical dislocation sacrificed. A cold saline solution is placed immediately after the small intestine is split in the stomach. A body saline solution was then administered using a syringe to remove any intestinal contents and cut components from a 20–30 cm section of small intestine that had been separated from the pyloric sphincter. Using a laboratory aerator and a solution of IVB and IVB-NLCs, constant aeration (carbogen, 95:5 O₂/CO₂) was applied to the intestines at a temperature of 5 °C. Samples were taken periodically from the receptor chamber and repeated with an identical volume of warm, fresh water in front of the solution. The withdrawal sample was sonicated for 15 min before being filtered using 0.2-m membrane filters. After the suitable dilution analysed in UV at 284 nm (Lakshmi et al. 2022; Gandhi et al. 2014). Using the formula below, IBH solution and IBH-PNPs' apparent permeability coefficients (Papp) were calculated and expressed in cm/s. The formula was used to get the Permeability Enhancement Ratio for IVB. $$P_{\mathit{app}}=\partial \mathrm{Q}/\partial \mathrm{t}\ast 1/{\mathit{AC}}_0$$

Where A is the exposed intestinal tissue surface area (cm²), ∂Q/∂t is the steady-state appearance rate of IVB-NLC in the receiver compartment, and Co is the initial concentration of the IVB in the donor compartment at zero time.

3.4.5 In-vivo anti-anginal single-dose study

The experimental rat was divided into 4 groups; normal control, disease control, test and marketed formulation. In each groups contain 6 rats. Calculated equivalent dose 1.54 mg/kg for test and marketed formulation as per dose conversion formula. After the 1 h induce the angina and the dose was 2 mL orally administered by gavage tube to all groups.

3.4.6 Induction of myocardial ischemia measurement of ST-segment depression

The experimental male rats Inhalation anaesthesia (Isoflurane) used to anesthetize the experimental male rats and placed on a heating pad to maintain a temperature 37 °C. During these study for maintenance of aesthesia given 0.2 L/min was used, which is the mixture of 2% of isoflurane in 100% oxygen. Vasopressin was given IV after the stabilization of aesthesia with the dose 2 IU/kg (Hirata et al. 2005; Ikeda et al. 2006). 2 mL IVB-NLC or dispersed IVB tablet in purified water was given after the 1 h. The cited ischemia generation was used for three consecutive days. Vasopressin was administered intravenously, and Lab Scribe software used the ST-segment depression as a measure of myocardial ischemia.

4.1.1 Effect on mean particle size

As indicated in Table 2, the mean particle size (Y1) ranged from 95.21 nm (Formulation 7) to 189.05 nm (Formulation 9). The quadratic equation has been used to illustrate how independent variables affect mean particle size as follows:

The negative incentive preceding the variable in the quadratic condition showed that condition (3) exposed negative impact on factors X1, X2, and X3 on Y1. A larger emulsifier-to-lipid percentage may encourage the formation of smaller particles and enable higher adjustability of nano framework due to the reduction in interfacial strain between the lipid grid and the hydrophilic stage. Fig. 1(a)–1 show the 3D surface plots showing the effect of association between X1 and X2, X1–X3, and X2-X3 on molecule size (c). The fluid lipid-to-strong lipid proportion (X3 and X32), in particular, has a notable impact on the mean molecule size of IVB-NLC, as shown in Table 3. The low value demonstrated that the observed characteristics matched those that were predicted, and connection coefficient (R2) was 0.6822 with a 19.27% coefficient of variation. “Adeq Precision” (Raghuvanshi et al. 2022) estimated that the model's sign-to-commotion ratio was 4.673 (>4), indicating that it was adequate for the typical molecule size (Ye et al. 2016).

4.1.2 Impact on entrapment efficiency

The value for EE with the selected levels of variables ranged from 59.95% (Formulation 14) to 88.25% (Formulation 3), with an average value of 76.30%. The quadratic formula below demonstrated how different independent factors affected EE:

Moreover, the terms X1-X2 and X2-X3 had negative effects on particle size, whereas X1-X3 had beneficial effects on the EE of IVB-NLC (Pradhan et al. 2015). Fig. 2(a)-2 show the 3D response surface graphs that depict how X1-X2, X1-X3, and X2-X3 interact to affect EE (c). The model's importance for EE was suggested by the F-value of 0.48. There was a strong correlation between predicted and experimental values, as indicated by the low values of the coefficient of variation (13.27%) and the coefficient of correlation (R2 = 0.3798).

4.1.3 Optimization and validation

Based on requirements of satisfying minimum mean particle size and maximising value of EE produced via Design Expert software, optimization of IVB-NLCs preparation was selected. Lecithin to Poloxamer 188 ratio and, liquid lipid to solid lipid ratio were selected as 1.35 and 0.95, and emulsifier to lipid ratio of 1.76 were chosen as the optimised compositions, which were advised to achieve fundamentals of optimised IVB-NLCs formulation. The mean particle size and EE for this chosen batch were determined to be 114. 45 ± 5.14 nm and 83.45 ± 3.23 nm, respectively, while the anticipated values of 112.15 nm and 86.04%, produced by Design Expert programme, were establish to be in good arrangement (Yadav et al. 2022). The optimized batch zeta potential was found to be −21.3 mV.

4.7.1 Evaluation of hemolysis

Hemolytic evaluation is needed to prove safety. Comparative hemolysis results of placebo-NLCs, plane IVB and IVB nanoparticles, at concentrations of 10 and 20 ng/mL, respectively. IVB nanoparticles showed an equivalent hemolysis value of 1% over the study period (8 h). The hemolysis of 10 ng/L equivalent placebo-nanoparticles was found to be statistically similar to that of IVB nanoparticles. Similarly, at an equivalent volume of 20 ng / mL, placebo-nanoparticles showed significantly higher hemolysis values ​​at 8 h compared with IVB nanoparticles. The hemolysis values ​​of placebo-nanoparticles were found to be significantly higher at 4 and 8 h compared with IVB nanoparticles at an equivalent volume of 30 ng/mL. Although IVB nanoparticles contain all components of placebo-nanoparticles, IVB-loaded nanoparticles show significantly less hemolysis at some points in the same concentrations of 10 and 20 ng / mL over time. Furthermore, IVB nanoparticles at multiple time points of three equivalent versions showed lower hemolysis values ​​than nanoparticles and placebo-nanoparticles.

4.7.2 Qualitative platelet aggregation

Total IVB, IVB nanoparticles (equivalent to 10 and 20 ng/mL of IVB), placebo-nanoparticles (equal to volume of IVB nanoparticles) were analyzed by total microscope by plane microscope. Erythrocytes, white blood cells and the platelets were seen under a microscope. and imaging cap. Aggregation of platelet aggregation was not observed at three different concentrations of the test samples (Fig. 5). Platelets were distributed on whole blood smears on microscopic examination of all test specimens. Observations indicate that IVB nanoparticles are nontoxic and hem compatible in nature.

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Fig. 5

Light microscopy images (100X resolution) of Leishman’s stained whole blood samples after treating with PBS, pure IVB, placebo and IVB-NLCs, in concentration of 10 and 20 ng/mL.

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4.8.1 Ex-vivo intestinal permeation study

Higher permeation of IVB-NLCs as compared to the IVB solution due to the large surface area (Fig. 6: A and B). 0.2826 ± 0.023 × 10⁻⁵ and 0.5253 ± 0.036 × 10⁻⁵ cm/s, the permeability coefficients for IVB solution and IVB-NLCs respectively.

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Fig. 6

(A) Ex-vivo permeation study of IVB solution and IVB-NLCs across rat intestinal membrane. (B) Apparent permeability coefficients (Papp) for IVB solution and IVB-NLCs.

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4.8.2 In-vivo anti-anginal activity

Impact of Ivabradine Hydrochloride on ST-Segment depression time, duration, and onset: As compared to disease Control group, IVB-NLCs (1.54 mg/kg) dose considerably delays the start of ST-segment depression (P 0.05) and significantly shortens its duration (P 0.05). IVB marketed tablets (1.54 mg/kg) initially produced significant effects on growth and decrease ST-segment depression period but only after the first day and statistically non-significant in disease control group.

Effect of ivabradine hydrochloride on the severity of ST-segment depression brought on by vasopressin: After the injection of vasopressin, IVB-NLCs (1.54 mg/kg) the ST-segment produced a substantial drop in height at 0, 1, 3, and 6 min, respectively. IBH pills that have been marketed have also resulted in notable shortages. When compared to the disease control group, the ST-segment only peaks for one day before declining again. Due to the ongoing release of the IBH-NLCs Property covered in the in-vitro part, there were three days of difficult activity.