Surface modified targeted nanostructured lipid carrier system for Axitinib: development and characterization

Currently, a variety of techniques are used to treat cancer, including cancer surgery, chemotherapy, irradiation, targeted therapy, and immunotherapy. Early-stage and less malignant tumours are frequently operated on, but later metastatic tumours do not benefit much from surgery. One of the most widely utilized techniques for eliminating cancer cells and treating advanced tumours is chemotherapy. Since chemotherapy is non-invasive or minimally invasive, it is preferred over surgery ^[1]. Consequently, a broad range of chemotherapeutic drugs been created for the chemotherapy of cancer, including cisplatin, paclitaxel, docetaxel, and doxorubicin (DOX).Despite having long- recognized applications, antineoplastics are generally extremely hazardous substances. In order to decrease toxicity and/or boost the efficacy of the current chemotherapeutic drugs, research remains essential to find more specific substances or different strategies ^[2]. Some of the disadvantages of antineoplastics include low bioavailability, distribution, non-specific body distribution, ineffective tumour delivery, nephrotoxicity and hepatotoxicity ^[3]. Numerous colloidal delivery systems for pharmaceuticals on a nanoscale that are Lipid-based nanoparticles (LBNs), such as Solid Lipid Nanoparticles (SLNs) and NLCs, have been thoroughly investigated, along with polymeric nanoparticles, niosomes, liposomes, exosomes and LBNs.Axitinib is an inhibitor of tyrosine kinase used in the course of treatment cancer which is classified under BCS II, which shows low oral bioavailability (58%) in commercial products due to its low solubility as well as cytochrome P450-mediated hepatic metabolism. Axitinib also shows shorter halflife of 2.5 to 6.1 hrs. It shows various side effects such as diarrhea, hypertension, fatigue, dysphonia, nausea, decreased appetite, hypothyroidism, rash, stomatitis, dyspnea, hand-foot syndrome and many more in cells other than cancerous cells.Due to their special qualities and benefits, nanostructured oils (NLC), which were first used in the late 1990s, are widely used in the delivery of chemotherapy drugs. Solid lipid frameworks and spatially inconsistent liquid lipids make up NLC, which is derived from SLNs. This leads to crystal structural flaws and enhances drug loading ^[4]. NLC offers a number of advantages, including high drug loading, controlled drug release, passive and active targeting, biocompatibility and biodegradability, and the possibility of large quantity production ^[5]. Additionally, because of the impact of EPR in cancers, NLC can increase amount of medication that accumulates in tumors and prolong the duration that PTX is in circulation.
Consequently, NLC might be the best lipid-based carrier of a system that delivers PTX.Low antitumor effect and serious side effects are typically the result of non-specific targeting to the tumor. Thus, there is an urgent need for An active method for delivering chemotherapy medicines that targets tumors. Numerous targeting chemicals, including Arg-Gly-Asp (arginylglycylaspartic acid) tripeptide, have been used to direct anticancer drugs to tumor tissues ^[6–10]. Because of its special qualities, hyaluronic acid continues to be the subject of extensive research. N-acetyl-D-glucosamine (GlcNAc) and glucuronic (GlcUA) repeating units are joined by alternating b-1,3 and b-1,4 glycosidic linkages to form Hyaluronic acid (HA), a negatively charged linear polymer. The primary extracellular matrix component, HA, binds selectively to the extracellular protein CD44 on cell membranes and controls a variety of cellular reactions. A cell outer marker called CD44 is underexpressed in healthy tissues and overexpressed in malignancies. The macromolecules' EPR impact in malignancies and the particular binding of HA to CD44 offer new possibilities for active tumor targeting.Drug conjugates containing hyaluronic acid, hyaluronic acid nanogels, and hyaluronic acid-covered nanoparticles are among the other formulations that have been created ^[11]. The following benefits have led to a great deal of research on hyaluronic acid conjugated nanoparticles: (a) HA may be used to modify the majority of NPs; (b) HA is present on the particle's outer shell and can bind to CD44 specifically; (c) HA can shield NPs and control their circulation time and biodistribution; and (d) additional HA or NP modifications can result in multifunctional NPs. Covalent bond modification can be used to create hyaluronic acid-conjugated nanoparticles. or the force of electrostatics. Chemical reactions are necessary for the alteration of covalent bonds, and these reactions are intricate and difficult to replicate. However, without the use of chemical reagents, electrostatic attraction is a straightforward and regulated process. Consequently, an alternative to covalent bond alteration could be electrostatic attraction. changes made to HA or NPs.In order to target AXT to tumors, we created a novel, active system for delivering tumor targets in this work: hyaluronic acid coated nano lipid carriers (HA-NLC), which are acquired through the process of electrostatic attraction. A schematic representation of the HA-NLC's structure is shown in Fig. 1. The following are some of the system's benefits: (a) HA-NLC was made of materials that are biocompatible and biodegradable; (b) it had a elevated medication loading levels; (c) it is an active cancer target delivery system; and (d) it is created by electrostatic attraction, meaning it was easy to manage and didn't require complicated chemical reactions. To the best of our understanding, this work is the initial effort to create HA-NLC for tumor targeting via electrostatic attraction. Prior to studying the characterisation, the cationic AXT-NLC was produced. After that, HA to a molecular weight (MW) of 300 kDa was chosen to make HA-NLC. We looked into the HA-NLC's cytotoxicity in cell lines that overexpress CD44 in vitro.

3. MATERIAL AND METHOD

3.1 Material

Gattefosse offered Compritol ATO 888 as a free sample. Pluronic F-68 and Capmule MCM C8 were gifts from Subhash Chemical in Pune, India, and Mohini Organics in Mumbai, respectively. Giiava India Pvt. Ltd. Wai and Research Lab Fine Chem Industries, both in Mumbai, provided the hyaluronic acid and soy lecithin. From Sisco research laboratories Pvt. Ltd. (India), cetyl trimethyl ammonium bromide were kindly acquired.

Screening of solid lipid

To help assess AXT solubility in solid-lipids, 10 mg drug were precisely weighed and placed to the test tube. After that, the solid-lipid mixture was added in 0.5 g increments. Until the clear melt formed, the test tube's temperature was hold at 80°C in water bath. The experiment's endpoint determined by visually inspecting the vials to check for any undissolved drug. This method yielded the lipid needed to dissolve 10 mg of AXT.

Screening of liquid lipid

The choice of liquid lipids was based on AXT's maximal saturation solubility. Each glass vial holding one milliliter of lipids received an excess of axitinib. After being plugged, the vials were left in isothermal shaker for 72 hours at 25 ±0.5°C to reach equilibrium. For 30 minutes, the resultant mixtures were centrifuged around 5000 rpm. Following this, the supernatant was gathered and then further diluted with methanol.  Each glass vial holding 1 ml with lipids received an excess of axitinib. Each sample's AXT concentration was evaluated using a UV spectrometer at 330 nm ^[12].

3.2 Preparation of cationic nanostructured lipid carrier (AXT-CNLC):

AXT-NLC was formulated by emulsion evaporation and solidification at low temperatures with some modifications [13]. Compritol ATO 888, Capmul MCM, and AXT were made soluble  in 4 ml of ethanol at an elevation that is 5 °C higher than a lipid's Melting point. The desired amount of CTAB (0.2% w/v) was dissolved in 20 ml of pure water containing the surfactants Tween 80 and soy lecithin, and this aqueous solution was also being heated with similar degree. After complete melting, the organic phase was slowly introduced in aqueous solution on a magnetic stirrer at 600 rpm to about 1 hour. The resulting warmed nanoemulsion was then promptly added to 20 milliliters of distilled water in an ice bath at 0 °C. After two more hours of stirring in a cold bath, ATX-NLCs were collected. Every type of NLC that was collected was kept between 2 and 8 °C.

3.3 Preparation of HA coated NLC (HA-AXT-CNLC):

Hyaluronic acid is a naturally occurring ligand that binds to CD-44 receptor. Hyaluronic acid was applied to the lipid nanocarrier by ionic conjugation. For effective surface coating, The CNLC solution (10 mL) was added dropwise in 5 mL of the 0.05% hyaluronic acid solution were combined. Stirring was carried out continuously for 6 hours [14].

3.4 Systematic optimization using 3² factorial design.

3² factorail design was select for optimization of AXT-NLC. Nine runs are produced by taking into account three levels—low, a medium, and high (-1, 0, +1) —at two separate variables. The selected independent were total volume of lipid (X1) and volume of surfactant (X2). And dependent variables were particle size zeta potential.

Table 1. Factors investigated using 32 full factorial experimental design.

Table 2. Based on the 32 FD, schematic representation of an experimental design.

To find the best NLC formulation with the smallest particle size and highest percent entrapment efficiency, a numerical optimization method was used.

3.5 In vitro NLCs characterization

3.5.1. Measurement of particle size (PS) and Polydispersibilty Index (PDI)

The produced formulations' PS and PDI were measured using a Zetasizer (Horiba SZ100). The apparatus operates according to the principles of dynamic light scattering, which evaluates variations luminous intensity of light scattered by NLC’s particles moving with Brownian motion. With the use of double distilled water, the samples were diluted (1:10). Following that, these samples were assessed at a 25°C temp and a 90° angle [15].

3.5.2 Zeta potential (ZP)

Using the Horiba Zetasizer device, the zeta potential and particle size examination of NLC were performed through the dynamic dispersion of light approach. The stability of NLC produced is mostly determined by ZP measurement. Due to particle-particle repulsion, the ZP test uses colloidal stability to determine the particle's surface charge. The colloidal dispersion's stability is also assessed using zeta potential (ZP). ZP was determined by placing the sample in a special type of electrode (a Zeta cell), diluting it with water, and recording the results [16].

3.5.3 Drug Entrapment efficiency (EE)

The literature was used to determine the EE. To dissolve the free AXT, the required quantity of AXT-NLC was dissolved in phosphate buffer solutions (PBS of pH 6.8) and vortexed over five minutes. To separate the free AXT from AXT-NLC, The mixture was subsequently centrifuged for45 minutes at 35,000 rpm (REMI R-8C). Following centrifugation, UV spectrophotometry was used to measure the quantity of AXT in the supernatant. 1ml of surpernatant was dissolved in ethanol upto 10ml and absorbance was determined at 330nm.

          % Entrapment efficiency=  Total amount of drug-Amount of free drugTotal amount of drug

3.5.4 Drug release study

To estimate the release behaviour AXT from HA-AXT NLC, the dialysis technique was used. In brief, a dialysis bag was filled with a suspension of 2 mL HA-AXT-NLC. Then, at 37 ± 0.5 °C and 100 rpm, it was submerged in 50ml of Phosphate buffer (pH 6.8). 1 mL of the dissolution medium was pipetted for the quantitative analysis and the same amount of release media was reintroduced to keep the sink condition in place. The obtained 1ml of sample was examined using UV spectroscopy at 330 nm after being diluted using ethanol upto 10 ml.

3.5.5 Differential scanning calorimeter:

DSC was employed to examine the crystallinity and melting behaviour of the optimized NLC. An aluminium pan containing 1-2 mg of lyophilized AXT-CNLC and AXT-HANLC was sealed, and thermograms were taken using thermogravimetric analyzers (Model-SDT Q600, USA). The nitrogen purging rate was set to 20 mL/min, and the instrument was running between 40 and 400°C at a heating rate for 10°C/min. An unfilled sealed pan served as the experiment's reference [17].

3.5.6 Structural integrity by FTIR:

FTIR spectroscopy was used to examine the structural integrity. The ALPHA-II bruker performed FTIR on the pure drug, the physical mixture of the drug and the excipients employed, and the powdered HA-AXT-NLC. To assess the coating of HA to AXT-CNLC, FTIR was also used. To verify the HA coating, FTIR analysis was performed on formulations of AXT-NLC and HA-AXT-NLC.

3.5.7 Electron microscopic analysis:

By using Transmission electron microscopy, the actual size, shape, and surface coating of the prepared NLCs were further investigated. For staining, small carbon-coated grid was used to dry the particle suspension.The material was examined under a microscope (JEOL JEM 2100 plus, Japan) after drying.

3.5.8 Cell line study:

To confirm that produced coated NLC is targeted to cancer cells while avoiding adverse impacts on noncancerous cell, in-vitro cytotoxicity and brine shrimp lethality assays were carried out.

In-vitro Cytotoxicity:

 Cell toxicity of the HA-AXT formulation was assessed with the MTT test. The culture media was used to cultivate the MC7 cells for 24 hours at 37°C with 5% CO2. A density of 70 µl or 104 cells/well was used to seed the cells in 100 µl of culture fluid and 100 µl of sample, respectively, in 96-well tissue culture grade microplates. The cell line was incubated with DMSO (0.2% in PBS) in control wells. Three copies of each sample were grown. The viability of the control cells and the proportion of viable cells after culture were assessed by maintaining controls. In a Thermo Scientific BB150 CO2 incubator, cell cultures were kept at 37°C and 5% CO2 for 24 hours. After incubation, the medium was completely removed and 20 µl of MTT reagent (5 mg/min PBS) was added. After adding MTT, cells were cultivated for four hours at 37 ^oC in a CO2 incubator. examined the wells under a microscope to check for the formation of formazan crystals. After the media was completely removed, only the yellowish MTT was transformed into a darker-colored formazan by live cells. After adding 200 µl DMSO, the mixture was incubated for 10 minutes at 370°C covered with aluminum foil. In order to analyze the triplicate samples, the absorbance of all sample was evaluated at 570 nm utilizing Elisa microplate analyzer (Benesphera E21) [18].

Brine Shrimp Lethality Assay:

Following the procedures described by Meyer et al. in 1982, the Bioassay experiment carried out. Ten of these shrimps were transferred to each sample vial containing 4.5 ml of a brine mixture (specific volume brine and yeast suspension) after their counting in the stem of the glass capillary against a light background. Water and Nauplli were drawn into a glass capillary. In each experiment, 0.5 ml of Samples 01 and 02's solutions were combined with 4.5 ml of brine solution containing the appropriate amounts. The control container was filled with synthetic seawater (4.5 ml) as well as synthetic seawater (0.5 ml) that contained 0.2% DMSO. Following a 24-hour period, the percentage of deaths was calculated by counting the survivors under 3X magnification or standing in front of a light source.

 4. RESULT AND DISCUSSION

Researchers are interested in lipid containing nanocarriers such SLNs or NLCs because of their tiny size, ease of scaling up, and utilization of physiological lipids in their creation. Because of these benefits, these nanotechnology systems have been documented in the literature to increase the oral bioavailability, solubility, and light stability of RSV. To the knowledge, however, this is the first publication to systematically optimize AXT-loaded NLCs using the QbD technique for site specific targeting of the created nanocarrier to tumor cells that are CD44 receptor positive.

Selection of solid lipid (SL)

Based on AXT's highest solubility in several lipids, the lipid was chosen. Glyceryl monostreate, Compritol 888 ATO, Precirol ATO, and Steric acid were the lipids employed for screening. AXT demonstrated the highest degree of solubility in Compritol 888 ATO among the several lipids used.The results showed the drug has exhibited solubility in the following order: Stearic acid< Precirol ATO< Glyceryl monostreate< Compritol 888 ATO. Based on its best solubility profile for AXT, Compritol 888 ATO was chosen as the SL for the NLC preparation.                                   

Figure 1. Solubility of AXT in different solid lipid.

Selection of liquid lipid:

Similar to solid lipid, maximal solubility was taken into consideration when selecting liquid lipid. Capmul MCM, Capmul MCM PG-8, Isopropyl myristate, Captex 355 and Isopropyl palmitate were the lipids employed for screening. The results showed that drug has exhibited solubility in the following order: Isopropyl palmitate< Isopropyl myristate< Captex 355< Capmul MCM PG-8< Capmul MCM. Based on its best solubility profile for AXT, Compritol 888 ATO was chosen as the SL for the NLC preparation.

Figure 2. Solubility of AXT in different Liquid lipid.

Optimization of NLCs

In order to determine potential factors that could significantly affect the size of particles and entrapment efficiency of produced nanocarriers, first batches of AXT-NLCs were prepared using the emulsification solvent

evaporation approach.

Table 3. Layout for 3² Full Factorial Experimentation Design.

The factor combination produced varying size and percentage of values of entrapment efficiency, which range from 102.4 to 356.7 nm and 87.5% to 93.5%, respectively, according to the experimental runs. Design Expert software 9.0 was used to evaluate the data, and the following equations may be used to estimate the quantitative impacts of the two influencing elements at various levels on both answers, namely particle size and percent entrapped efficiency:

Particle size = +180.00 +89.95 *A +32.73 *B +18.47 *A *B +22.05 *A² +7.90 *B²The above quadratic equation can be utilized to derive conclusion after taking into account amount of coefficient and mathematical sign it conveys, (i.e., positive otherwise negative). Considering equation when the amount of total lipid grows, the particle size also increases, as indicated by the positive sign for the coefficient of A. The positive sign for coefficient of B indicates increase in the particle size with increase in level of surfactant. The combinations also show similar results.

Entrapment efficiency = +90.56 +2.17 *A +0.75 *B

The above linear equation can be utilized to derive conclusion after taking into account amount of coefficient and mathematical sign it conveys, (i.e., positive otherwise negative). The positive sign for coefficient A indicates increase in entrapment efficiency with increase in the concentration of total lipid. The positive sign for coefficient of B indicates increase in entrapment efficiency with increase in level of surfactant.

Table 4. Quadratic model of particle size and entrapment efficiency.

F value (192.45 and 524.08): A very high F-value indicates that the model is statistically significant, i.e., the variation described by model is much greater than the unexplained variation.

Prob > F (0.0500): This is the p-value. A value at or below 0.05 suggests the model is significant at the 95% confidence level.

Adj R-square (0.9917 and 0.9924): The total amount of predictors is taken into consideration by adjusted R². A value close to 1 indicates an excellent model fit, meaning the model explains almost all variability in the data.

Pred R-square (0.9659 and 0.9875): Predictive R² shows how well the model predicts new data. The high value confirms the model has strong predictive capability.

Adeq. Precision (38.896 and 58.244): This measures the signal-to-noise ratio. A ratio above 4 is desirable; here, the very high value shows the model has an excellent signal and can be used for reliable optimization or prediction.

Figure 3. Contour plot illustrating the impact of  factors on the Particle size and Entrapment efficiency

Figure 4. Plotting 3D responses to selected variables' effects on Particle size

4.1 Particle size:

Particle size of optimized batch (F5) of AXT-CNLC AND HA-AXT-NLCs was measured successfully using zetasizer. Formulations of AXT-CNLC was prepared in nanoscale successfully with a particle size of 136.9± 0.7 nm as shown in figure 7. However, the size increased to 327.8± 1.3 nm after coating with hylauronic acid as shown in figure 8. As a result, it was possible to develop nanoscale particles with effective ligand coating and an affinity for the CD-44 biomarker.

      Figure 5. Particle size of AXT-CNLC              Figure. 6 Particle size of HA-AXT-NLC

PDI was found to be less than 0.5 in both cases which indicated a narrow size distribution.

4.2 Zeta potential:

Cationic NLC (AXT-CNLC) is formed with the cationic surfactant CTAB. Zeta potential for AXT-CNLC was thus positive, measuring +27.90 mV. After the surface had been modified with HA, there was a considerable charge reversal. The zeta potential for HA-AXT-NLC was negative, measuring -30.2 mV. This demonstrated that surface coating had been done successfully.

Figure 7. Zetapotential of AXT-CNLC                       Figure 8. Zetapoteintial of HA-AXT-NLC

4.3 Entrapment efficiency:

Entrapment efficiency of HA-AXT-NLC was found to 89.42 ± 0.23 % which was carried out using centrifugation.

4.4 Drug release study:

Using dialysis bag method the drug release of HA-AXT-NLC pattern was found out. The HA-AXT-NLC release profiles displayed a biphasic release pattern, includes a two-hour initial burst release, followed by ongoing sustained release behavior throughout the remaining hours.

Table 5. Invitro % drug release study

Figure 9. % Drug release graph

4.5 Differential scanning calorimetry:

AXT and compritol 888 ATO showed their endothermic peaks at 215.09? and 77.05±?? respectively, which specified the drug to be crystalline as as shown in figure 12. In the NLC thermogram, the peak of AXT diminished, which was caused by the partial formation of less energy lipid forms leading to a decrease of crystallinity. It also suggested the solubilization of drug and molecularly dispersed state of AXT in the lipid matrix. The thermogram of NLC showed peaks at 248.86°C which resembled with the melting point of hyaluronic acid (241-247?) the polymorphic transition might be responsible for the peak shift as shown in figure 13.

Figure 10. DSC thermogram of A. Mixture of AXT and Compritol 888 ATO

B. HA-AXT-NLC

4.6 Structural integrity by FTIR study:

FTIR was used to evaluate chemical composition and compatibility the optimised formulation. The overlay spectrum of HA-AXT-NLC, physical mixture, and AXT is shown in the above figure. The distinctive absorption bands for the pure drug were found in the FTIR spectra at 1612.55 (C=O), 1146.92 (amine C-N), 1584.58 (aromatic C=C), 3202.03 (secondary amine N-H), 3043.29 (aromatic C-H). The NLC FTIR spectra confirmed the presence of some of the AXT-specific peaks that indicated the drug's presence but no evidence of any chemical interactions. The shifting of the peaks was attributed to a change in the molecular environment brought on by the molecular dispersion of medications inside the lipid matrix [19].

Figure 11. FTIR peak of a) pure drug b) physical mixture c) final HA-AXT-NLC.

4.7 Transmission electron microscopy:

TEM images (Figures 15 and 16) clearly demonstrate the morphological difference between AXT-CNLC and HA-AXT-NLC. The observed increase in particle size after HA coating indicates successful surface modification of AXT-CNLC with hyaluronic acid, confirming the formation of HA-AXT-NLC.

12. TEM image of AXT-CNLC                  Figure 13. TEM image of HA-AXT-NLC

4.8 CELL LINE STUDY:   

Cell cytotocicity study:

Table 6. Anticancer activity of 5-FU and HA-AXT-NLC against MCF-7 cells.

The standard drug 5-Fluorouracil (5-FU) showed a dose-dependent increase in % inhibition of cell growth (77.84% at 10 µl → 91.60% at 100 µl), confirming its strong anticancer potential. The prepared HA-AXT-NLC also exhibited dose-dependent cytotoxicity, with % inhibition increasing from 30.93% (10 µl) → 50.33% (100 µl). Compared to the standard, the sample showed moderate anticancer activity, but the trend confirms that encapsulated AXT in NLCs is biologically active and effective against MCF-7 cells.

Brine Shrimp Lethality Assay:

Table 7. Brine Shrimp Lethality Assay of HA-AXT-NLC.