Hispolon-Loaded Liquid Crystalline Nanoparticles: Development, Stability, In Vitro Delivery Profile, and Assessment of Hepatoprotective Activity in Hepatocellular Carcinoma

Abstract

The present work describes the development and characterization of liquid crystalline nanoparticles of hispolon (HP-LCNPs) for treating hepatocellular carcinoma. HP-LCNPs were prepared by a top-down method utilizing GMO as the lipid and Pluronic F-127 as the polymeric stabilizer. The prepared formulations (HP1–HP8) were tested for long-term stability, where HP5 showed good stability with a particle size of 172.5 ± 0.3 nm, a polydispersity index (PDI) of 0.38 ± 0.31 nm, a zeta potential of −10.12 mV ± 0.05, an entrapment efficiency of 86.81 ± 2.5%, and a drug loading capacity of 12.51 ± 1.12%. Optical photomicrography and transmission electron microscopy images demonstrated a consistent, low degree of aggregation and a spherical shape of LCNPs. The effect of temperature and pH on the optimized formulation (HP5) indicated good stability at 45 °C and at pH between 2 and 5. In vitro gastrointestinal stability indicated no significant change in the particle size, PDI, and entrapment efficiency of the drug. The drug release study exhibited a biphasic pattern in simulated gastric fluid (pH 1.2) for 2 h and simulated intestinal fluid (pH 7.4) for up to 24 h, while the best fitting of the profile was observed with the Higuchi model, indicating the Fickian diffusion mechanism. The in vivo pharmacokinetic study demonstrated nearly 4.8-fold higher bioavailability from HP5 (AUC: 1774.3 ± 0.41 μg* h/mL) than from the HP suspension (AUC: 369.11 ± 0.11 μg* h/mL). The anticancer activity evaluation revealed a significant improvement in antioxidant parameters and serum hepatic biomarkers (SGOT, SGPT, ALP, total bilirubin, and GGT) in the diethyl nitrosamine-treated group of rats with the optimized LCNP formulation (HP5) vis-à-vis HP suspension.

1. Introduction

Hepatocellular carcinoma (HCC) or liver cancer is a dreadful cancer worldwide. It is the world’s fifth most frequent malignancy and the third leading cause of cancer-related deaths.¹ There are various etiological parameters including aflatoxin B1 exposure and hepatitis B and C virus infections that have been identified as major risk factors for HCC. Its therapy options are traditionally divided into curative and palliative categories. Surgical excision can be a key curative approach in the early stages of HCC, but it is relatively limited in patients with numerous or metastatic tumors.¹ As a result, an effective anticancer drug must be sought or developed to improve the survival rate of patients with advanced or recurring HCC following surgical treatment. Surgical resection, liver transplantation, and non-surgical local ablation procedures such as percutaneous ethanol injection and radiofrequency ablation (RFA) are the only treatments available for HCC in its early stages.² In a small proportion of patients with confined solitary tumors and no cirrhosis, surgical excision is the preferred treatment option.² Liver transplantation is seen to be preferable due to the simultaneous eradication of HCC and other liver diseases, but it does have drawbacks such as a scarcity of donor organs.

Anticancer drugs have good therapeutic action but are limited in use due to serious adverse effects including hepatic dysfunction, neurotoxicity, cardiotoxicity, and hypersensitive reactions, which may exacerbate the condition.¹⁻⁴ Therefore, because of the above-mentioned demerits of chemotherapeutic agents, researchers emphasize the use of phytoconstituents, and they have gained wider acceptance in the management of liver cancer therapy.³

Phellinus linteus (PL) is a Phellinus genus mushroom that is well known in Taiwan as “Sangwhang”. It has a long history of use in both food and medicinal applications in the oriental countries.⁵ PL consists of various bioactive constituents that have beneficial effects on human health and prevents various ailments including gastroenteritis, lymphatic disease, and cancers.⁶ Hispolon (HP) is a phenol molecule derived from PL that has anti-inflammatory, antiproliferative, and antimetastatic properties. However, this molecule suffers from biopharmaceutical challenges such as low solubility, poor bioavailability, instability, and rapid metabolism leading to its fast elimination from the body.^6,7

Nanostructured carriers are colloidal particles with diameters ranging from 10 to 200 nm and have been extensively explored in drug delivery.^2,3 The designed nanocarriers must be biodegradable and have the ability to entrap drug molecules and circulate in the bloodstream, delivering the appropriate amount to the intended spot.^2,3 Nanocarriers smaller than 200 nm promote passive targeting to the liver through greater sinusoidal fenestration (Hodoshima et al. 1997).⁸ Anticancer agents are entrapped in the network, adsorbed on the surface, or both for drug targeting applications.^5,8,9 The potential of nanoparticle (NP) use for anticancer drug delivery is enormous, and they may be used for a wide range of therapeutic applications. Small particles having a larger surface area and a faster dissolution rate help in achieving better anticancer activity.^5,8 Literature search on nanostructured systems of HP shows only one report on codelivery with docetaxel in liposomes for improving the efficacy against melanoma cells.¹⁰

In the recent decade, liquid crystalline nanoparticles (LCNPs) have been found as a promising tool for oral drug delivery applications.¹¹⁻¹³ LCNPs are self-assembled architectures formed when polar lipids are dispersed in an aqueous surfactant solution.¹² Furthermore, they changed into different secondary structures after being diluted with gastrointestinal fluid.^12,14 The specific positioning of lipids imparts stiff and fluid distinctiveness to them, assisting secondary transformations such as mixed micelles and hexagonal and cubic nanoparticles in the gastrointestinal environment.¹² These secondary structures have characteristics such as enhanced oral absorption, good stability, sustained drug release, drug protection, penetration enhancement, and so forth.¹⁵

The current research work, therefore, focuses on a systemic development of HP-loaded LCNPs, followed by extensive characterization of formulation quality parameters, gastrointestinal stability, in vitro drug release, bioavailability, and biodistribution, and hepatoprotective activity evaluation in a rat model induced with experimental HCC.

2. Materials and Methods

Glyceryl monooleate [GMO, Monomuls 90-O18] lipid was purchased from BASF (Ludwigshafen, Germany). Sigma-Aldrich Chemical Co (St. Louis, MO, USA) provided HP. Accurex Biomedical Pvt. Ltd., Mumbai, India provided measurement kits for alanine aminotransferase (ALT), malondialdehyde (MDA), and aspartate amino transferase (AST). Merck, India provided high-performance liquid chromatography (HPLC)-grade solvents such as acetone, acetonitrile, and isopropyl alcohol. Apart from these solvents, all other reagents of analytical grade were obtained from local sources. Gibco, Massachusetts, USA provided fetal bovine serum (FBS). Sigma–Aldrich Chemical Co., Mumbai, India provided sodium chloride (NaCl) salt and phosphate buffered saline (PBS) tablets, which were dissolved in sufficient quantities in HPLC-grade water.

2.1. Analytical Method Development

HPLC was used for the chromatographic separation of HP. An Agilent 1260 Infinity HPLC system equipped with a Zorbax C18 column and isocratic elution of the mobile phase containing a 1:2 (v/v) mixture of deionized water and acetonitrile was used. A 0.4 mL/min flow rate was used during the separation process, which was carried out at 35 °C. HP was detected at 302 nm and appeared in the chromatogram at 5 min. Validation of the method’s linearity range was performed to determine its precision and accuracy, robustness, lower limit of quantification (LLOQ), and limit of detection (LOD).

2.2. Formulation Development of HP-LCNPs

The drug-loaded LCNPs were prepared using a top-down method as per the methods described in literature reports.^16,17 An accurate amount of GMO was weighed into glass vials and heated to 45 °C until it is free flowing. Furthermore, the required dose of HP as the drug and melted GMO as the lipid base were dissolved in isopropyl alcohol (IPA) in a glass vial, and rotary evaporation was performed at a temperature of 45 °C for evaporating IPA to form a dry thin film. The HPLC-grade water (pH 6.0) containing Poloxamer P407as the polymeric stabilizer was added to the vial containing lipid (GMO/polymeric stabilizer in different ratios) to achieve a concentration of 100 mg. This mixture was sonicated for 3 min at 45 °C using a probe sonicator (UP 200 S, Dr Hielscher GmbH, Berlin, Germany) at an amplitude of 40% and a pulse of 60 s to produce a milky dispersion. Furthermore, the dispersion of HP-LCNPs was freeze-dried (Vir Tis, Wizard 2.0, New York, USA, freeze dryer) using 6% w/v sucrose as a cryoprotectant. The total concentration of HP in all the formulations was fixed at 20 mg/mL.

2.3. Long-Term Stability and Physicochemical Characterization of HP-LCNPs

The long-term stability evaluation of the LCNPs was performed at room temperature (25 °C) for up to 90 days in tightly closed containers protected from light (Chong et al. 2012). After 30 days, the aforementioned formulation was evaluated for the particle size, PDI, zeta potential, entrapment efficiency, loading capacity, and pH. Furthermore, the measurements were carried out in triplicate after each time interval, and the results were presented as the mean ± standard deviation.

2.3.1. Particle Size

The size (hydrodynamic radius Rh, nm) and size distribution (PDI) of the developed HP-LCNPs were measured by dynamic light scattering (DLS) using a Zetasizer instrument (Malvern, Worcestershire, UK).¹⁸ Before measurement, the aforementioned formulation was diluted 1:9 (v/v) in water and filtered through a membrane filter (0.45 μm). The measurements were taken in triplicate for each sample, and the results were reported as mean ± standard deviation.

2.3.2. Zeta Potential

The zeta potential of HP-LCNPs was measured by a Zetasizer using disposable plastic cells.¹⁷ The samples were diluted (1:9) with 10 mmol NaCl solution and filtered through a membrane filter (0.45 μm). The measurements were presented as a three-replicate mean ± standard deviation.

2.3.3. pH Evaluation

The pH of HP-LCNPs was measured using a pH meter (Mettler Toledo AG, Giessen, Germany) with an In Lab 410 NTC electrode 9823 (Switzerland). Furthermore, the buffer solutions were used to calibrate the equipment (pH 7.0 and 4.01). The pH measurements of the formulations were performed three times, and the results were expressed as mean ± standard deviation.

2.3.4. Encapsulation Efficiency and Drug Loading Capacity

The entrapment efficiency of HP-LCNPs was measured by analyzing the drug content in LCNPs after removal of the unentrapped drug and total drug content in the formulation.^2,3 The loading capacity (LC) is the quantity of the drug loaded per unit weight of the LCNPs, indicating what percentage of the LCNPs’ weight is attributable to HP loaded. For entrapment efficiency and loading capacity, 2 mL of the formulation was centrifuged at 5000 rpm for 25 min, and the resultant supernatant was collected. From this, 100 μL of the dispersed suspension of HP-LCNPs was extracted with a 1:2 (v/v) mixture of deionized water and acetonitrile, and the drug amount was estimated using HPLC in triplicate to report the values in mean ± standard deviation. Equations 1 and 2 were used to calculate the encapsulation efficiency and drug loading capacity, respectively.

1

2

2.4. Particle Morphological Characterization

2.4.1. Photomicroscopy

An optical microscope (Medilux, Kyowa Optical Co. Ltd, Hashimoto, Japan) was used to identify the optimized HP-LCNPs for morphological surface characterization.^2,3

2.4.2. Transmission Electron Microscopy

The prepared HP-LCNPs were microscopically tested using a M-10 (Philips, The Netherlands) transmission electron microscope.^2,3 With a phosphotungstic acid solution, one drop of HP5 was put on a carbon-coated grid, treated, and dried for 30 s. The dry exposed grid was transferred to the slide, covered with a cover slip, and examined using the transmission electron microscope at 60–80 KV.

2.5. Stability Evaluation of HP-LCNPs

2.5.1. Effect of Temperature on Particle Size and PDI

The HP5 was placed in a screw-capped transparent glass tube and heated in a water bath. The temperature was elevated from 25 to 75 °C at a constant rate (3 °C/min) with 10 °C intervals between measurements. Furthermore, HP5 was evaluated to detect instability signs such as turbidity or precipitate and phase separation, followed by measurement of the particle size and PDI. All the measurements were carried out in triplicate, and the results were expressed as mean ± standard deviation.¹⁹

2.5.2. Effect of pH on Particle Size and PDI

Titration was carried out using a Malvern MPT-2 titrator (Malvern, Worcestershire, UK), which was connected to a dynamic light scattering system with a Malvern Nano ZS Zetasizer (Malvern Instruments, UK).^18,19 Using an experimental setup, the effect of pH on the particle size and PDI was investigated. The buffer solutions at pH 3, 5, and 7 were employed to calibrate the Mettler Toledo Seven Multi pH meter (Mettler Toledo, AG, Giessen, Germany) with InLab 410 NTC electrode 9823. To avoid abrupt changes in the solution, the titrator was calibrated for the addition of 1–3 mL of titrating solutions. Furthermore, the experiment was repeated thrice at a temperature of 25 °C and a voltage of 150 V.

2.6. In Vitro Studies

2.6.1. Gastrointestinal Stability

In vitro gastrointestinal stability was evaluated using biorelevant gastric fluids that mimicked the human body’s environment.²⁰ The stability of HP-LCNP was tested in various simulated GIT fluids [simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and simulated colon fluid (SCF; pH 7.4)]. Furthermore, freeze-dried HP-LCNP was reconstituted with 1 mL of SGF and SIF and suspended in a dialysis bag in 20 mL of SGF/SIF medium for 2 h in the case of SGF and 4 h in the case of SIF and SCF for 42 h to stimulate the stomach state. In addition, the stability of HP-LCNP was assessed in terms of the particle size, PDI, and entrapment efficiency.

2.6.2. In Vitro Drug Release Study

The in vitro drug release for HP was determined by using a dialysis method as per the procedure described in the literature.²¹⁻²³ The dialysis tube (MWCOs 14 kD, Sigma, USA) was pretreated in distilled water at least overnight prior to performing the drug release study. The optimized LCNP formulation (HP5) equivalent to 20 mg of HP and pure drug (20 mg) taken in the dialysis tube were suspended in the SGF (pH 1.2) and SIF (pH 6.8) containing 2% IPA and kept under stirring at 100 rpm on a water shaker bath maintained at 37.0 ± 1 °C. The release study was performed for 2 h in SGF, while the experiment was continued up to 90 h in SIF. Aliquots (2 mL) of samples were withdrawn at intermittent time intervals, followed by replacement with an equal volume of the fresh release media. The drug content in the samples was quantified by HPLC and graphically demonstrated as percentage cumulative release versus time. The drug release data were fitted using different release mathematical models to recognize the best drug release kinetic model.

2.7. Cell Culture and Cell Viability Study

The human hepatocellular cell lines (HepG2) were grown using supplement medium containing Dulbecco modified eagle medium (DMEM), 10% (v/v) fetal bovine serum (FBS), 1 mL l-glutamine, 100 mg/mL of penicillin, and streptomycin. To test the cytotoxicity of HP on the HepG2 cell line, the cells must be maintained at 37 °C in a 95% CO₂/5% O₂ environment.³ After confluency, the cells were taken in 96-well plates, treated with HP (10–40 μg/mL), and incubated for 24 and 48 h at 37 °C. The cells were then washed with PBS solution and incubated at 37 °C for 2 h with MTT solution. The cells were used for optical density measurement using an ELISA microplate reader (BioTek, United States), and cell vitality was determined vis-à-vis the control cells.

2.8. In Vivo Preclinical Study

For preclinical experiments, Albino Wistar male rats were employed (175–200 g body weight). The rats are collected from the central animal house and maintained in polypropylene cages in accordance with standard experimental protocols.^2,3 These were housed at a constant temperature of 25 °C with a 12 h light/dark cycle and fed rat chow and water ad libitum. Animal studies were conducted according to the regulations of the Institutional Animal Ethics Committee (IAEC) guidelines, by Patliputra University, Patna, Bihar, India (1840/PO/ReBi/S/15/CPCSEA Reg. no). Furthermore, both animal-related experimental studies were conducted in accordance with IAEC standards and regulations.

2.8.1. Pharmacokinetic Studies

A randomized study design was employed, and a single-dose pharmacokinetic study was performed. The rats were divided into two groups, each group containing six rats. The animals in group I received a single oral dose of HP5 (20 mg/kg), and animals in group II were administered with a single oral dose of HP-suspension (20 mg/kg) (HP dispersed in 3% sodium carboxymethylcellulose, CMC-Na). At different time intervals (0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h), 0.5 mL blood was taken from the retro-orbital plexus under light ether anesthesia and kept in heparinized Eppendorf tubes, which were then centrifuged at 5000 rpm for 15 min. Following centrifugation, the plasma was kept at −20 °C until analysis. As per the procedure described by us in the literature, the drug was extracted from rat plasma.^2,3 Plasma proteins were precipitated by adding 500 μL of acetonitrile and thereafter 1 mL of ethyl acetate was added to 100 μL of plasma. The plasma samples were vortexed for 5 min and centrifuged at 10,000 rpm for 15 min. The supernatant organic fraction was collected and dried under vacuum at 40 °C. Furthermore, the residue was reconstituted with 100 μL of ethanol, and 10 μL was taken for HPLC analysis. The pharmacokinetic data analysis was performed by non-compartmental analysis using Kinetica version 5.0 software (Thermo Fisher Scientific, Massachusetts, USA) to compute various absorption and elimination parameters.

2.8.2. Biodistribution Studies

Male Wistar rats were divided into two groups with six animals each for the biodistribution studies. The animals were administered with a single dose of the HP5 and HP suspensions, each containing 20 mg of HP. Organs such as heart, liver, spleen, kidney, lung, and liver tumor were removed from each group (n = 3) at regular time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h), blotted dry with tissue paper, accurately weighed, and homogenized by adding 1 mL ice cold KCl solution per 0.5 g tissue. Furthermore, the obtained supernatant was kept at −20 °C until analysis. The drug content was determined by HPLC analysis, as given in Section 2.1.

2.8.3. Pharmacodynamic Studies

For HCC development, the rats were administered with a single intraperitoneal injection of diethyl nitrosamine (DEN, 200 mg/kg) in phosphate buffer solution. The rats were divided into four groups at random with six animals in each group (n = 6). The control group (group I) of animals was given a single daily dose of 0.9% w/v normal saline 5 mL/kg/day p.o. (orally). DEN at a dose of 200 mg/kg was given to group II. For 14 weeks, group III rats that received DEN (200 mg/kg) were also administered with HP suspension (20 mg/5 mL) at a single dose of suspension (5 mL/kg/day p.o). For 14 weeks, the group IV DEN group of rats was administered with HP-LCNPs (HP5) (20 mg/5 mL) at a single dose of formulation (5 mL/kg/day p.o).

2.8.3.1. Antioxidant Parameters

Blood samples were collected to measure antioxidant parameters as per procedures reported in previous literature reports.^2,3 A somewhat modified approach was used for assessing phase I enzymes such as NADH cytochrome b5, cytochrome reductase P450, and cytochrome reductase P420. Moreover, the various antioxidant parameters [viz., lipoxygenase (LPO), catalase (CAT), dismutase superoxide (SOD), peroxidase glutathione (GPX), and transferase (GST) glutathione] were determined as per the procedures reported in our previous reports.^2,3

2.8.3.2. Evaluation of Hepatic Enzymes

The rats were randomly split into four groups (n = 6): normal control (NC) (group I), DEN (group II), DEN-treated HP suspension (group III), and DEN treated with HP-LCNPs (HP5) (Group IV). The rats in Group II received a 200 mg/kg intraperitoneal injection of DEN, which induced HCC. Groups III and IV were orally administered with HP suspension and HP5 at a dose of 20 mg/kg/day for 7 days, respectively. Normal saline was administered to the rats in the normal control group. At the end of 14 weeks, 1 mL blood was collected from the retro-orbital plexus, while the rats were under ether anesthesia. The blood was collected in non-heparinized tubes, centrifuged at 1500 rpm for 15 min, and the serum was separated and stored at −80 °C until testing. According to the instructions on the assay kits (COGENT, India), serum was tested for several enzymes such as SGOT, SGPT, ALP, total bilirubin, AFP, and GGT.^2,3

2.8.3.3. Statistical Data Analysis

The one-way ANOVA test was used to assess possible statistical differences between three or more groups of data. Statistical differences of p < 0.05 were considered significant. Origin 7.0 software was used to create some graphs (Origin Lab Corporation, USA). The Zetasizer software was used to generate a zeta potential graph and analyze the particle size distribution (Malvern, USA). All quantitative data were provided as an average ±SE for the various animals in each group (with a 95% confidence range). For comparison, two-tailed Student’s t-test was utilized, where P-values less than 0.05 were considered statistically significant.

3. Results

3.1. Development of HP-LCNPs

Using a top-down method, 20 mg of HP was added to IPA containing GMO, and the mixture was subjected to rotary evaporation process at 45 °C to form a dry thin film. Then, Pluronic F-127 (Poloxamer P407) and the lipid mixture were added in varying ratios as shown in Table 1 to prepare the LCNPs. Furthermore, the dispersion was sonicated for a fixed time (3 min) at a fixed temperature (45 °C) using a probe sonicator, producing HP-LCNPs (HP1-HP8). The prepared formulations were subjected to various characterization studies as described in the below sections.

Table 1. Composition of HP-Loaded LCNPs with Different Amounts of Lipids and Polymeric Stabilizers^a.

formulation code	lipid quantity (GMO; mg)	polymeric stabilizers (poloxamer P407; mg)
HP1 (hispolon 20 mg/mL)	50	50
HP2 (hispolon 20 mg/mL)	60	40
HP3 (hispolon 20 mg/mL)	40	60
HP4 (hispolon 20 mg/mL)	70	30
HP5 (hispolon 20 mg/mL)	75	25
HP6 (hispolon 20 mg/mL)	80	20
HP7 (hispolon 20 mg/mL)	45	55
HP8 (hispolon 20 mg/mL)	55	45

^aTotal quantity of GMO/poloxamer P407 is 100 mg. HP; hispolon, LCNPs; liquid crystalline nanoparticles.

3.2. Long-Term Stability and Physicochemical Characterization of HP-LCNPs

Table 2 shows the values of the particle size distribution, PDI, zeta potential, EE, loading capacity, and pH of all the formulations (HP1 to HP8), which were maintained at room temperature (25 ± 2 °C). All the selected formulations were kept in screw-capped glass tubes, which were protected from light for 30, 60, and 90 days. The particle size distribution showed almost non-significant variation for HP5 (172.5–172.73 nm) with time (shown in Table 2), whereas the polydispersity index (PDI) was also found to be constant (PDI = 0.380–0.391) with long-term stability. The value of zeta potential, on the other hand, was steadily decreasing over time (shown in Table 1). The values of particle size, PDI, zeta potential, EE, LC, and pH of all eight selected LCNPs with various quantities of polymers and lipids are shown in Table 2. All the prepared formulations showed good stability for a 24 h storage duration after the formulation preparation. After regular intervals (30 to 90 days), HP1–HP4 and HP6 to HP8 showed a significant variation in the particle size (continued increasing), PDI, and zeta potential (decrease in the value, as shown in Table 2). However, significant changes in the value lead to instability in the aforementioned formulation. On the other hand, the particle size of HP5 remained unaltered with a miniscule reduction in the PDI (a narrower size distribution). Furthermore, DLS analysis of HP5 displayed a monomodal distribution (particle size and particle size distribution shown in Figure 1A,B) and revealed stable nature of the formulation.

Table 2. Properties of Different HP-Loaded LCNPs (HP1–HP8) at a Temperature of 25 °C ± 2 °C and under 60% RH ± 5% RH after Stability Testing at 30, 60, and 90 Days.

	particle size ± SE (n = 3)a			PDI ± SE (n = 3)a			zeta potential ± SE (n = 3)a			EE % ± SD (n = 3)			LC ± SD (n = 3)			pH ± SD (n = 3)
formulation code	30 days	60 days	90 days	30 days	60 days	90 days	30 days	60 days	90 days	30 days	60 days	90 days	30 days	60 days	90 days	30 days	60 days	90 days
HP1	173.10 ± 2.01	173.23 ± 2.21	176.67 ± 1.02	0.421 ± 0.01	0.51 ± 0.04	0.52 ± 0.03	–7.68 ± 0.03	–9.23 ± 0.07	–6.22 ± 0.08	71.2 ± 4.42	70.1 ± 3.21	68.34 ± 2.21	9.12 ± 2.21	9.01 ± 4.12	8.5 ± 2.31	5.9 ± 0.04	6.2 ± 0.05	6.4 ± 0.03
HP2	175.73 ± 1.85	175.92 ± 1.78	179.21 ± 0.89	0.32 ± 0.06	0.34 ± 0.02	0.38 ± 0.03	–0.421 ± 0.08	–0.231 ± 0.06	–0.170 ± 0.04	74.71 ± 2.20	72.21 ± 3.11	71.21 ± 2.51	10.34 ± 2.21	10.1 ± 3.20	9.7 ± 2.21	5.21 ± 0.01	5.7 ± 0.02	6.2 ± 0.04
HP3	178.01 ± 1.70	179.21 ± 1.52	184.01 ± 1.71	0.53 ± 0.04	0.56 ± 0.11	0.58 ± 0.22	–0.441 ± 0.04	–0.323 ± 0.02	–0.231 ± 0.03	77.76 ± 1.12	75.34 ± 2.45	74.12 ± 2.20	11.23 ± 2.21	11.0 ± 3.33	10.72 ± 1.81	5.88 ± 0.02	5.93 ± 0.03	6.12 ± 0.04
HP4	182.20 ± 1.80	184.11 ± 3.10	186.21 ± 2.22	0.27 ± 0.23	0.29 ± 0.21	3.1 ± 0.02	–4.12 ± 0.05	–6.21 ± 0.05	–9.45 ± 0.05	80.22 ± 3.72	78.12 ± 2.81	75.21 ± 2.71	9.45 ± 3.12	9.21 ± 4.12	8.89 ± 3.21	5.2 ± 0.01	5.4 ± 0.03	6.8 ± 0.02
HP5	172.5 ± 0.3 nm	172.61 ± 0.21 nm	172.73 ± 0.41 nm	0.380 ± 0.31 nm	0.383 ± 0.03	0.391 ± 0.02	–10.12 ± 0.05	–10.24 ± 0.06	–11.12 ± 0.03	86.81 ± 2.5	86.21 ± 2.8	86.01 ± 2.71	12.51 ± 1.12	12.43 ± 1.34.	12.31 ± 1.16.	5.50 ± 0.02	5.54 ± 0.04	5.57 ± 0.06
HP6	190.35 ± 1.78	192.12 ± 1.82	196.89 ± 2.12	0.51 ± 0.20	0.53 ± 0.15	0.54 ± 0.23	–2.20 ± 0.04	–1.06 ± 0.06	–0.21 ± 0.04	82.34 ± 1.31	80.45 ± 1.41	78.21 ± 1.45	10.11 ± 1.50	9.89 ± 3.21	9.76 ± 2.21	5.12 ± 0.01	5.43 ± 0.02	5.91 ± 0.03
HP7	188.11 ± 1.10	189.11 ± 1.21	193.21 ± 1.31	0.43 ± 0.21	0.47 ± 0.01	0.48 ± 0.02	–0.412 ± 0.01	–0.521 ± 0.031	–0.220 ± 0.041	83.01 ± 1.21	80.12 ± 1.86	76.45 ± 2.10	9.75 ± 1.31	9.32 ± 1.42	8.88 ± 2.61	5.71 ± 0.01	5.78 ± 0.03	6.59 ± 0.02
HP8	184.31 ± 1.20	185.21 ± 2.21	188.23 ± 1.11	0.62 ± 0.31	0.67 ± 0.02	0.72 ± 0.03	–3.21 ± 0.02	–6.12 ± 0.033	–8.67 ± 0.012	79.66 ± 1.51	78.12 ± 1.52	75.23 ± 2.10	10.61 ± 2.12	10.01 ± 2.01	9.41 ± 1.05	6.01 ± 0.01	6.21 ± 0.20	6.91

^aStandard error at 95% confidence interval (n = 3).

Figure 1.

Particle size distribution of optimized HP5 (A,B), optical photomicrograph of the said formulation (C), and TEM image of the said formulation (D). HP5; hispolon-loaded LCNPs.

3.2.1. Encapsulation Efficiency, Drug Loading Capacity, and pH Measurement

Encapsulation efficiency of the formulation HP1 to HP4 and HP6 to HP8 decreased after a time interval (30 to 90 days). In the case of HP5, EE (86.81 to 86.01%) remained unaffected and superior to the abovementioned formulation. On the contrary, the loading capacity for HP1 to HP4 and HP6 to HP8 decreases over the passing of days, and for HP5, the drug loading capacity (12.51 to 12.31%) remained unaffected throughout the study.

Furthermore, Table 2 displays the pH of the aforementioned composition. Moreover, HP1 to HP4 and HP6 to HP8 were unstable due to the significant variations in the pH. On the contrary, for HP5 with pH in the range of 5.50 to 5.57, no significant variation observed over 90 days (shown in Table 2). Overall, the HP5 formulation was chosen for further investigation.

3.3. Particle Morphological Characterization

Optical photomicrographs revealed that the morphology of HP5 was uniform and had a low degree of aggregation (as shown in Figure 1C). The TEM image (Figure 1D) represented the spherical shape and uniformly distributed particles, which also confirmed their nano-sized structure.

3.4. Characterization of the Prepared HP-LCNPs (HP5)

3.4.1. Effect of Temperature on the Particle Size and PDI

After 15 days of preparation, the effect of temperature (25 to 75 °C) on the particle size and PDI of HP5 was evaluated. The particle size remained constant throughout the temperature from 25 to 45 °C, as shown in Figure 2A. After that, up to 65 °C, the particle size rapidly decreased, then up to 75 °C, the particle size increased rapidly. In the temperature range of 25–45 °C, PDI of HP5 remained almost constant, as shown in Figure 2B. For 45 to 55 °C, PDI decreases; upon increasing the temperature again up to 75 °C, the PDI value increases ultimately reaching a value of 0.43.

Figure 2.

(A,B) Effect of temperature on the particle size and PDI of HP5.

3.4.2. Effect of pH on the Particle Size and PDI

The effect of pH on HP5 formulation is shown in Figure 3A. It revealed that change in pH from 2 to 5 did not show any significant variation in the particle size. For pH 5 to 9, a rapid enhancement occurred in the particle size reaching up to 289.12, as shown in Figure 3B. In the pH range of 2 to 5, PDI was found to be 0.38 and 0.38, respectively. From pH 5 to 9, PDI increased rapidly to reach 0.71 at pH 9.

Figure 3.

(A,B) Effect of pH on the particle size and PDI of HP5.

3.5. In Vitro Studies

3.5.1. Gastrointestinal Stability

The stability of HP5 (HP-LCNPs) in gastric fluid is an utmost important quality attribute, and the particle size, PDI, and encapsulation efficiency were measured under different GI fluid conditions.²²Table 3 shows the non-significant differences in the particle size, PDI, and entrapment efficiency of HP5 when incubated with different GI fluids (p < 0.05).

Table 3. In Vitro Gastrointestinal Stability of HP5 (HP-LCNPs) in Different Dissolution Media.

	SGF (pH 1.2)		SIF (pH 6.8)		SCF (pH 7.4)
stability parameters	before	after	before	after	before	after
particle size (nm)	172.5 ± 0.3	172.7 ± 1.10	172.5 ± 0.3	172.3 ± 1.35	172.5 ± 0.3	172.4 ± 0.1
PDI	0.380 ± 0.21	0.382 ± 0.32	0.380 ± 0.12	0.385 ± 0.21	0.380 ± 0.21	0.383 ± 0.11
entrapment efficiency (%)	86.81 ± 2.5	86.34 ± 0.22	86.81 ± 1.31	86.45 ± 1.01	86.81 ± 2.5	86.33 ± 1.21

3.5.2. In Vitro Drug Release

Figure 4 depicts the in vitro drug release profile of HP-loaded LCNPs (HP5) and HP suspension in SGF medium (pH 1.2) for 2 h and SIF (pH 7.4) for up to 90 h. In the case of HP-loaded suspension, more than 80% drug release was observed within first 7 h. In contrast, HP5 has a persistent biphasic release pattern, with substantially quicker release (40.23%) in the first 10 h and then steady release (89.25%) until 90 h (shown in Figure 4A). Furthermore, the drug release data curve fitting analysis revealed Higuchi type (shown in Supporting Information Figure S1, Table 4) release kinetics, implying HP release from the matrix of LCNPs. Furthermore, HP release from LCNPs is proportional to LCNPs, and HP release from LCNPs diminishes over time.

Figure 4.

In vitro release profiles of HP suspension and HP5.

Table 4. Drug Release Correlation Coefficients of HP5 after Fitting in Various Release Models.

formulation	zero-order model	first-order model	Higuchi model	Hixon–Crowell root model	Korsmeyer–Peppas model
HP5	0.8382	0.9669	0.9912	0.978	0.9892

3.6. In Vitro Cytotoxicity

At 24 and 48 h, MTT results demonstrated that HP-loaded LCNP, plain LCNP, and HP were all cytotoxic to HepG2 cells treated with them (Figure 5A,B). A decrease in cell viability over time versus concentration was seen in the cytotoxicity experiments of formulations at various concentrations. As a result, it was discovered that HP5 dramatically reduces the HepG2 cell line’s viability within 24 h when compared to the HP suspension alone. However, in 48 h, the HP5 cells with least viable population were detected at a lower IC50 (as shown in Figure 5B, Table 5).

Figure 5.

Evaluation of cell viability of HP suspension, HP5, and blank LCNP formulation at various concentrations following incubation with HepG2 cells: (A) for 24 h and (B) for 48 h.

Table 5. MTT Viability Assay of HP Suspension and Optimized HP5^a.

	IC50 (μg/mL)
formulations	24 h	48 h
hispolon suspension	44.13 ± 1.21	33.04 ± 0.89
HP5	24.01 ± 2.22	18.12 ± 0.75

^aHP5 shows significant (p < 0.05) cytotoxicity compared to HP suspension after 24 and 48 h. Data are expressed as mean ± SD (n = 3); IC50 = half-maximal inhibitory concentration; HP5: optimized HP-loaded crystalline nanoparticles; and ± SD: standard deviation.

3.7. In Vivo Studies

3.7.1. Pharmacokinetic Studies in Rats

The objectives of this research were to determine the amount of drug in the systemic circulation following oral delivery of HP5 at various time intervals. Figure 6 shows a comparison graph of the pharmacokinetic profiles of HP5 and HP suspensions. Table 6 lists important pharmacokinetic characteristics such as C_max, T_max, and AUC_0→∞, which indicated a significant improvement in the drug absorption parameters. The HP5 formulation (AUC: 1774.3 ± 0.41 μg h/mL, C_max: 230 ± 0.02 μg/mL) exhibited 4.8-fold improvement in the oral bioavailability than HP suspension (AUC: 369.11 ± 0.11 μg h/mL, C_max: 55 ± 0.04 μg/mL).

Figure 6.

Comparative curves showing pharmacokinetic profile of HP suspension and HP5; data expressed as mean ± S.D. (n = 6).

Table 6. Pharmacokinetic Parameters of HP Suspension and HP-LCNPs (HP5).

formulations	Cmax (μg/mL)a	Tmax (h)	AUC0–∞ (μg* h/mL)a	t1/2 (h)	MRT0–t
HP suspension	55 ± 0.04	4	369.11 ± 0.11	2.84 ± 0.13	6.07 ± 2.21
HP-LCNPs (HP5)	230 ± 0.02	4	1774.3 ± 0.41	5.58 ± 0.11	8.93 ± 1.32

^aStandard error at 95% confidence interval (n = 3). HP: hispolon; LCNPs: liquid crystalline nanoparticles.

3.7.2. Biodistribution Studies

In biodistribution studies, two groups of animals were given a single oral dose of HP5 (HP-LCNP) and HP-suspension, and biodistribution of HP5 to numerous important organs was studied. Explicit results were acquired from the supernatant collected after the homogenization of the organs. As compared to the HP suspension, a significantly higher amount of drug reached the liver (p < 0.05). Besides, some quantities of HP were also found in other organs due to the partitioning of nano-sized LCNPs through endothelial fenestrations. Figure 7 depicts a graphical representation of biodistribution of HP5 and HP suspension in various organs. Overall, a higher drug concentration was achieved in the liver tumor and liver compared to other vital organs. In all organs, however, HP suspension had lower AUCs (p < 0.05).

Figure 7.

Biodistribution profiles of HP suspension and HP5 at various time intervals showing relatively enhanced uptake of HP5 by liver and the liver tumor. Statistical significance compared with HP suspension and HP5 (HP-LCNPs): p < 0.01 and p < 0.01. HP; hispolon.

3.7.3. Pharmacodynamic Studies in Rats

3.7.3.1. Effect of Treatment Formulations on Macroscopic Existence of HCC

No hepatic nodules were found macroscopically in the control group. Table 7 reveals that rats in the DEN and DEN-treated groups had nodules. In the DEN group of rats that received HP suspension and HP5 at a dose of 20 mg/kg, a total of five out of six and two out of six rats were found with hepatic nodules, respectively. However, in the rest of the treated rats, no hepatic nodules were observed. Tables 7 and 8 show the total number of hepatic nodules and percentage of tumor incidence. In rats administered with DEN only, 100% expansion of nodules was observed and a total of 272 hepatic nodules were found in the dimensions of ≤1 mm (140), <3 mm >1 mm (80), and ≥3 mm (52), respectively. On the contrary, the rats administered with DEN, followed by HP suspension showed 83.3% tumor incidence and a total 130 hepatic nodules in the dimensions of ≤1 mm (55), <3 mm >1 mm (40), and ≥ 3 mm (35) with a dose of 20 mg/kg. Furthermore, animals treated with DEN, followed by HP5 showed a total of 55 nodules with 33.3% tumor incidence, and their dimensions were measured to be ≤1 mm (26), <3 mm >1 mm (21), and ≥3 mm (8), respectively (shown in Tables 7 and 8). In a nutshell, the study indicated that rats administered with HP5 were found to be superior in reducing the tumor incidence as compared to the HP suspension.

Table 7. Effect of HP Suspension and HP5 on the Number of Rats, Number of Nodules, and Average Number of Nodule-Bearing Rats^a.

				relative size (% of number size)
s. no	groups	number of rats with nodules/number of rats	total number of nodules	≤1 mm	<3 mm >1 mm	≥3 mm
1	normal control (saline)	0/6	0	0	0	0
2	DENA control	6/6	272	140	80	52
3	DEN + HP suspension (20 mg/kg)	5/6	130	55	40	35
4	DEN + HP5(20 mg/kg)	2/6	55	26	21	08

^aDEN: diethyl nitrosamine; HP5:optimized hispolon-loaded crystalline nanoparticles; group I did not show any sign of hepatic nodules; and group IV showed a few signs of hepatic nodules.

Table 8. Effects of HP and HP5 Suspensions on the Number of Rats with Tumor Incidence^a.

s. no	groups	number of rats/number of rats with tumor	tumor incidence (%)
1	normal control (saline)	0/6	0
2	DEN control	6/6	100
3	DEN + hispolon suspension (25 mg/kg)	5/6	83.3
4	DEN + HP5 (25 mg/kg)	2/6	33.3

^aDEN: diethyl nitrosamine; HP5:optimized hispolon-loaded crystalline nanoparticles; group I did not show any sign of hepatic nodules; and group IV showed a few signs of hepatic nodules.

3.7.3.2. Effect of Treatment on Antioxidant Enzymes

A significant increase in lipid peroxidation (LPO) was reported in the DEN control group (16.12 ± 0.49). The LPO value reached 12.12 ± 1.04 after administration of HP suspension (20 mg/kg). At 20 mg/kg dose, HP5 showed a reduction in the LPO value (10.81 ± 0.74), which reached a near normal value, as indicated in Table 9. The DEN control group, on the other hand, had significantly lower enzyme levels of CAT (1.12 ± 0.21), SOD (1.41 ± 0.51), GPX (5.12 ± 0.41), and GST (0.61 ± 0.31). In the case of HP suspension group, an enhancement in the levels of CAT (2.412 ± 0.06**), SOD (3.10 ± 0.31**), GPX (7.32 ± 0.21**), and GST (0.81 ± 0.31**) was observed. However, HP5 (20 mg/kg) administration resulted in a significant increase in the values of CAT (3.65 ± 0.12**), SOD (4.71 ± 0.42**), GPX (8.63 ± 0.21**), and GST (1.02 ± 0.31**), which were observed to be close to those of the control group (as shown in Table 9).

Table 9. Effect of HP Suspension and HP5 Treatment on the Antioxidant Parameters of DEN-Induced HCC Rats^a.

group	LPO (μM/mg protein)	CAT (nmol/min/mL)	SOD (U/mL)	GPx (μmol)	GST (U/min/mg protein)
normal control(saline)	10.12 ± 0.31	4.12 ± 0.23	5.10 ± 0.21	9.12 ± 0.73	1.12 ± 0.04
DENA control	16.12 ± 0.49a	1.12 ± 0.21a	1.41 ± 0.51a	5.12 ± 0.41a	0.61 ± 0.31a
DENA + hispolon suspension (20 mg/kg)	12.12 ± 1.04**	2.412 ± 0.06**	3.10 ± 0.31**	7.32 ± 0.21**	0.81 ± 0.31**
DENA + HP5 (20 mg/kg)	10.81 ± 0.74**	3.65 ± 0.12**	4.71 ± 0.42**	8.63 ± 0.21**	1.02 ± 0.31**

^aHP5: optimized hispolon-loaded crystalline nanoparticles, values are expressed as mean ± SEM (n = 3). ^aP < 0.001 as compared to the normal group. *P < 0.05 as compared to the DENA control group. ***P < 0.001 as compared to the DENA control group.

3.7.3.3. Assessment of Hepatic Enzymes via Serum Biochemical Estimation

The rats administrated with DEN showed hepatotoxicity, which were thus estimated for assessment of the effect of treatment formulations on liver biomarkers. The observed results indicated an increase in the serum biochemical markers such as SGOT, SGPT, ALP, total bilirubin, AFP, and GGT of (78.63 ± 1.664) (U/L), 75.72 ± 3.05 (U/L), 13.12 ± 0.12 (U/L), 2.51 ± 0.36 (mg/dL), 295.10 ± 20.21 (mg/dL), 4.23 ± 0.41 (mg/dL), respectively, in the DEN group of rats as compared to the normal control group of rats (shown in Table 10). HP5 and HP suspensions showed varied levels of hepatoprotective efficacy. HP suspension produced a considerable reduction in the enzyme levels, while HP5 produced a significant reduction as compared to the HP suspension (p < 0.05). The reduction in the levels of biomarkers was observed as 64.25 ± 1.36 (U/L), 69.32 ± 1.21(U/L), 7.12 ± 0.6 (U/L), 0.39 ± 0.06 (mg/dL), 50.31 ± 1.46 (mg/dL), and 2.81 ± 0.46 (mg/dL) for SGOT, SGPT, ALP, total bilirubin, AFP, and GGT, respectively. In the case of HP5-treated group of rats, a significant reduction in the levels of parameters SGOT, SGPT, ALP, total bilirubin, AFP, and GGT of 58.18 ± 1.36 (U/L), 62.19 ± 1.28 (U/L), 7.90 ± 0.13 (U/L), 0.71 ± 0.09 (mg/dL), 34.16 ± 1.26 (mg/dL), and 2.34 ± 0.16 (mg/dL), respectively, was observed (shown in Table 10).

Table 10. Effect of Hispolon Suspension and HP-LCNPs (HP5) on Serum SGOT, SGPT, ALP, Total Bilirubin, AFP, and GGTP in Various Groups against HCC^a.

group	SGOT(U/L)	SGPT (U/L)	ALP (U/L)	total bilirubin (mg/dL)	AFP (mg/dL)	GGT(mg/dL)
NC (saline)	55.01 ± 1.044	60.12 ± 0.33	8.12 ± 0.41	1.01 ± 0.01	28.12 ± 1.28	2.12 ± 0.07
DENA control	78.63 ± 1.664a/***	75.72 ± 3.05a/***	13.12 ± 0.12a/***	2.51 ± 0.36ns	295.10 ± 20.21b/***	4.23 ± 0.41ns
DENA + HP suspension	64.25 ± 1.36a/***	69.32 ± 1.21b/***	7.12 ± 0.6a/***	0.39 ± 0.06ns	50.31 ± 1.46b/***	2.81 ± 0.46b/*
DENA + HP-LCNPs (HP5)	58.18 ± 1.36b/***	62.19 ± 1.28b/***	7.90 ± 0.13b/***	0.71 ± 0.09ns	34.16 ± 1.26b/***	2.34 ± 0.16b/*

^aHP; hispolon; LCNPs: liquid crystalline nanoparticles; HCC: hepatocellular carcinoma; and GGTP; gamma glutamyl transpeptidase. Values are expressed as mean ± SEM, (n = 6). P < 0.001 as compared to normal group. b ***P < 0.01 as compared to disease control.

4. Discussion

To improve the oral drug absorption of phytopharmaceuticals across the gastrointestinal tract, several oral lipid-based formulations have been developed and proven to be very effective. HP was effective in preventing HCC, but it has a number of disadvantages that could be addressed by lowering the dose and improving the distribution at the sites by using a novel formulation. LCNPs are effective drug delivery carriers for the treatment of HCC due to their small size, prolonged drug release properties, biocompatibility, and biodegradability.

In the present study, HP-LCNPs were developed using a top-down method to yield a uniform size and stable nanoparticles. In this formulation development, GMO/Poloxamer P407 aids in efficient solubilization of the drug within the lipid matrix, thus aiding in increasing the drug loading. After the formulation development, HP-LCNPs (HP1 to HP8) were stored for up to 90 days at room temperature (25 °C). At the end of 90 days, formulations HP1 to HP4 and HP6 to HP8 were found to be unstable showing significant variations in the particle size, PDI, zeta potential, entrapment efficiency, loading capacity, and pH. Among eight formulations prepared, the formulation HP5 was found to be stable when evaluated for various formulation attributes (as shown in Table 2). The PDI value remained constant (0.380), which revealed the homogeneous nature. The zeta potential for HP5 was observed to be −10.12 to −11.12 mV, which was responsible for thermodynamic stability of the nanoparticles. Higher anionic and cationic potentials signify a larger repulsion between the particles, thus enhancing the physical stability of the dispersion. Furthermore, it showed a narrow size distribution and a monomodal distribution profile of the formulation during the stability study, and pH was also found to be between 5.5 and 5.57 throughout the stability testing period. Therefore, HP5 has been selected for further study.

The effect of temperature (25 to 75 °C) (stressed temperature) on the characteristics of HP5 was studied after 15 days of formulation development, and the particle size was found to remain constant up to 45 °C. Above this temperature up to 65 °C, the particle size rapidly decreased, and beyond 65 up to 75 °C, the particle size increased rapidly (Figure 2A). Similarly, PDI was found to remain constant in the temperature range of 25–45 °C (shown in Figure 2B). From 45 to 55 °C, PDI decreased, which increased again up to a temperature of 75 °C (Figure 2B). The study on the effect of pH on HP5 showed no significant variation in the particle size and PDI in the pH range of 2–5 (Figure 3A,B). From pH 5 up to 9, an increase in the particle size as well as in PDI was observed, as depicted in Figure 3A,B. In vitro gastric stability testing in GI fluids revealed stability of the HP5 in the GIT. An in vitro drug release study of HP5 showed sustained drug release with a relatively faster release profile (40.23%) compared to the HP suspension during the initial 10 h, followed by a sustained release profile (89.25%) up to 90 h. In addition, Higuchi drug kinetics were revealed using a curve fitting study of HP release (Table 4).^17,22 The longer duration of drug release may be attributed to the irregular channels in the lipidic matrix, resulting in diffusion and delay of the drug release from HP5. The in vivo pharmacokinetic studies revealed a significant enhancement (p < 0.005) in the AUC value of HP5 over the oral HP suspension, which is ultimately 4.8-fold higher bioavailability over the same. This may be attributed to the preventive action of enzymes in the release medium and LCNPs cannot not be degraded further, which result in a lag phase of release. The second factor is the amorphous or molecular state of HP in LCNPs, which significantly enhanced its solubility over the free HP. Moreover, this was confirmed by the comparative biodistribution study.

According to the literature, solid tumors have high vascular permeability.²⁴ As a result, passive targeting allows the nanoparticles (200 nm) to concentrate extensively in tumors.^25,26 The particle size of the optimized formulation (HP5) in our experiment was roughly 172.5 nm. Furthermore, the biodistribution plot revealed that the liver tumor had the largest trapping and retention of HP5, followed by the spleen and other organs. This concentration effect was not observed in any other organs. Furthermore, this effect could be due to increased HP5 trafficking into the liver via the hepatic portal system, resulting in cumulative entrapment and retention within the liver and liver tumor. This event could be interpreted as evidence of size-based passive targeting, as nanoparticles smaller than 200 nm easily pass through the liver endothelial lining.²⁴

Free radicals, one of the primary sources of tumor-induced damage, were slowed down by antioxidants.^2,3 ROS was mainly inhibited by antioxidants, which controlled the production of ROS and generated oxidative stress. The overproduction of free radicals by lipid peroxidation occurred in HCC and was exacerbated by the administration of DEN.^2,3 The level of LPO, which may be used to determine the precise redox state of cells and to trigger cancer development by ROS production, was found to be linked to HCC.^2,3 LPO levels increased in DEN-induced HCC rats, which indicates abnormalities in the redox cell state and oxidative stress.³ HP5 (P < 0.001) significantly lowered and inhibited the LPO content over HP suspension. The first-generation antioxidants, CAT and SOD, are made up of natural protein components. The great antioxidant effect of HP prevented oxidation. SOD’s principal role is to reduce the generation of superoxide during the oxidation of hydrogen peroxide and water.³ The conversion of H₂O₂ to H₂O is facilitated by CAT and GPx. DEN-mediated HCC showed a marked decrease in enzymes and an improvement in free radical formation and degradation of the endogenous redox system. DEN, an electrophilic carcinogen that communicates with nuclear GSH sites, is linked to macromolecules. Defending the cell state by eliminating free radicals is an important duty of GSH. HP5 elevated GSH levels more quickly in DEN-induced HCC-treated rats over the DEN group of rats. DEN-induced HCC rats were shown to have decreased levels of SOD, CAT, GSH, and GPx, all of which were connected to increased tumor formation. These endogenous antioxidants and scavengers of damaging free radicals are strengthened and best protected by HP5.

In cancer, increased enzyme activation is a sign of the relationship between multiple altered cells.^2,3 The transport function of hepatocyte cell organelles is disrupted during cancer, allowing these enzymes to escape via plasma membrane permeability regulation, resulting in enzyme alteration in the cell and blood serum.²⁷ Furthermore, hepatic cell damage causes cell breakdown, which releases biochemical enzymes (SGOT, SGPT, and ALP) into the serum, which is a quantitative metric that can be used to quantify liver damage. When HP5 was compared to HP suspension, there was a considerably greater reduction in increased blood enzyme levels caused by DEN intoxication in HCC. The following is the sequence in which these formulations reduced high enzyme levels: HP-LCNPs (HP5) > HP-suspension. The explanation for this is that HP5 appears to flow via endothelium fenestrations and into the space of capillaries, where HSCs are located. Hepatic impairment can be caused by DEN, as evidenced by the increased plasma generation of AFP. HP5, on the other hand, dramatically lowered AFP, indicating a chemoprotective effect on the liver and its functional performance.²⁸ Each group of rats had significantly different liver parameters. In fact, the DEN rats had a greater alpha-fetoprotein (AFP) serum content, which remained high throughout the experiment. The HP5-treated rats had the greatest reduction in AFP compared to the DEN and other treatment groups. In the DEN-induced group of rats, total bilirubin (2.51 mg/dL) was higher than that in the control group. In comparison to the DEN group of rats, HP5 showed the greatest reduction in total bilirubin in DEN-induced HCC-treated rats. Gamma glutamyl transferase (GGT) is a serum marker, whose higher value is linked with liver cancer, and it increased similar to the DEN group of rats.^28,29 When compared to other treatment groups of rats, HP5 demonstrated the most significant reduction in GGT over the DEN group of rats, approaching the closest value of the normal control group. The presence of a lipid carrier also facilitates its uptake by the liver and other RES organs. The antioxidant and biochemical levels in the DEN-treated group of rats fed with HP5 are proof of the positive protective effects of HP.

5. Conclusions

HP-LCNPs (HP1 to HP8) were developed using a top-down method. HP1 to HP4 and HP6 to HP8 were found to be unstable at the end of 3 months, showing significant variations in the particle size, PDI, EE, and LC. HP5 remained stable throughout the 3 months and showed no variations of the abovementioned characteristics at temperatures from 25 to 45 °C and pHs from 2 to 5. Moreover, DLS representation analysis of HP5 showed a homogeneous distribution and a low degree of aggregation. The HP5 formulation was found to be gastric stable and showed the Higuchi drug release kinetics with a sustained biphasic pattern. The cellular cytotoxicity studies on HP5 cells found a least viable population at a lower IC50 at the end of 48 h. The in vivo experiment in rat plasma revealed a 4.8-fold increase in bioavailability of HP5 and a greater accumulation of HP in the liver tumor and liver compared to other vital organs over the HP suspension. Pharmacodynamic studies in DEN-intoxicated rats treated with HP5 revealed most significant considerable restoration of hepatoprotective action via antioxidant enzymes and hepatic enzyme markers. Finally, it can be concluded that HP5 may be an effective treatment option for HCC. In the future, more in vivo data on safety will be required to demonstrate definite outcomes.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c06796.

• Drug release kinetic modeling of data with various mathematical models (PDF)

The authors declare no competing financial interest.