Physicochemical characterization and biological evaluation of amorphous solid dispersions of an anticancerous drug: Erlotinib HCl

Abstract

Erlotinib hydrochloride (ERL), a tyrosine kinase inhibitor, is effective in treating various cancers. However, low aqueous solubility limits its bioavailability and therapeutic efficacy. We developed an amorphous solid dispersion (ASD) of ERL with biocompatible polymers, polyvinylpyrrolidone (PVP-K30), and polyethylene glycol (PEG-4000) for enhanced amorphization, miscibility, and molecular interactions. The present study focuses on the physicochemical characterization of formulated ASD of ERL using Fourier Transform Infrared Spectroscopy (FTIR), X-ray Powder Diffraction (PXRD), UV–Visible Spectroscopy, and High-Performance Liquid Chromatography (HPLC), along with biological evaluation including antioxidant, cytotoxicity, and antitumor studies in mouse tumor models. FTIR analysis confirmed the retention of ERL’s characteristic peaks in ASDs with PVP, PEG, and PVP/PEG, with shifts to lower frequencies for C=O bending, CH₂ deformation and CH symmetric deformation, indicating reduced molecular vibration energy, increased molecular flexibility, and strong drug–polymer interactions. PXRD analysis confirmed the transformation of crystalline ERL into an amorphous state in ASDs, as evidenced by the diminished ERL peaks at 11.7°, 16.2°, 21.7°, 24.75°, 25.56°, and 29.37°. UV spectroscopy revealed shifts in absorption peaks (256 nm), suggesting favorable drug–polymer interactions. HPLC demonstrated enhanced release rates at 4.72 retention time. In dissolution studies, the ERL + PEG formulation attained the greatest dissolution rate (80%). ERL + PVP showed superior DPPH radical scavenging activity with an IC₅₀ value of 100 µg/mL, while ERL + PEG demonstrated stronger hydroxyl radical scavenging activity with an IC₅₀ of 200 µg/mL. In the MTT assay, ERL + PEG exhibited the most potent cytotoxicity against MCF-7 cells, with an IC₅₀ of 19 μM, whereas the ERL + PEG + PVP combination was most effective against HCT-116 cells, with IC₅₀ of 19.5 μM. In vivo, ERL + PEG significantly reduced tumor volumes to 0.167 ± 0.002 g and 0.063 ± 0.004 g, corresponding to a tumor reduction of 98.78%. This study highlights the successful development of erlotinib ASD, particularly with PEG, which significantly improved ERL’s solubility, dissolution rate, antioxidant activity, cytotoxicity, and antitumor efficacy. These enhancements are attributed to physical modifications such as enhanced amorphization and strong drug–polymer interactions, without any chemical alteration of ERL, underscoring the potential of this formulation as an effective and promising drug delivery strategy for cancer therapy.

Introduction

Globally, cancer remains a leading cause of morbidity and mortality. Despite advances in understanding cancer biology and the identification of numerous potential targets, has a greater failure in transforming these breakthroughs into effective therapies. Though oral delivery is the most favored route for cancer due to practicality, affordability, and convenience, 90% of pharmaceuticals on the market and more than 40% of newly developed chemical entities have low water solubility, which causes problems with bioavailability^1,2. After surveying oral anticancer medications, Sawicki et al. classified 72 of them in the Biopharmaceutical Classification System (BCS) class as class II drugs. Many attempts were made to address the solubility issues of anticancer drugs, including physical mixtures, co-solvents, lipid-based systems, prodrugs, and amorphous solid dispersions (ASD). Among them, ASD is an emerging technology that is increasingly being utilized to enhance the bioavailability of poorly water-soluble drugs. Amorphosisation of the drug can be achieved by quench cooling, solvent evaporation, vapor condensation, and disruption of the crystalline lattice. The biggest drawback of this method is the recrystallization tendency of the drug from its formulation, resulting in decreased shelf life and physical stability. To reduce the recrystallization tendency and to improve the physical stability and shelf life, ASD can be prepared by entrapping active pharmaceutical ingredients (API) in water-soluble, bio-compatible polymers or oligomers.

This study focuses on formulating ASD, containing the potent anticancer agent, erlotinib HCl (ERL)³. ERL has shown single-agent activity in non-small cell lung (NSCLC), head and neck cancers^4,5 and ovarian carcinomas⁶. ERL targets one of the most important pathways in lung carcinogenesis and can exploit the phenomenon of ‘oncogene addiction’, with different efficacy according to EGFR gene mutational status in tumors (Fig. 1). ERL is very slightly soluble in water (hydrochloride salt—maximal solubility of approximately 0.4 mg/mL occurs at a pH of approximately 2), slightly soluble in methanol, and practically insoluble in acetonitrile, acetone, ethyl acetate, and hexane. Hence, ERL is classified as a class II drug in the BCS, which is characterized by low solubility and high permeability. Many studies were already been done to increase the rate of dissolution using traditional formulations, such as the binary solid ERL dispersions made by one-step electrospray method⁶, liquisolid compact technique⁷, solid self-nano emulsifying drug delivery systems⁸, nano sponge complex with cyclodextrin⁹, etc., However, these studies resulted in limited drug absorption and compromised physical stability.

Fig. 1.

(a) The chemical structure of ERL, (b) targeted pathways of ERL predicted from STITCH network pharmacology. (Stronger associations are represented by thicker lines. Protein–protein interactions are shown in grey, chemical-protein interactions in green, and interactions between chemicals are shown in red.)

Many strategies were explored yet to enhance the solubility and bioavailability of ERL. Recent studies highlight combinational therapies to overcome drug resistance. Parimala et al. reported co-encapsulation of erlotinib and quercetin in solid lipid nanoparticles (EQNPs), significantly inhibiting n-EGFR/PI3K/AKT expression in resistant A549/ER cells and reducing lung tumor progression in mice¹⁰. Chauhan et al. developed optimized nanoemulsions for deep lung delivery, achieving a 2.8-fold IC₅₀ reduction in A549 cells and enhanced efficacy in 3D spheroid models¹¹. The liquisolid compact technique using PEG 400 and Avicel PH200 improved erlotinib dissolution, with optimized formulations showing stable release profiles⁷. Jahangiri et al. enhanced erlotinib’s dissolution using PVP-based binary solid dispersions via electrospray, confirming successful amorphization without chemical interaction⁶. Mugnier et al. formulated solid amorphous dispersions of ERL with Soluplus®, HPMC-AS-L, and HPMC-AS-H, concluding that cryo-milled HPMC-AS-L extrudates offered superior solubility and crystallization resistance during gastric-intestinal transfer, supporting oral administration¹².

In response to these constraints, this study aims to develop polymer-based ERL-ASDs to enhance solubility and dissolution rates, with a key innovation of the physical transformation of ERL into an amorphous state without any chemical modification. This work reports the physicochemical characterization and biological evaluation of an ASD for ERL by entrapping it in polymer matrices, specifically utilizing polyethylene glycol (PEG)¹³, polyvinyl pyrrolidone (PVP)¹⁴, and their blend. With the use of sophisticated analytical methods like Fourier Transform Infrared Spectroscopy (FTIR), X-ray Powder Diffraction (PXRD), UV–Visible Spectroscopy, and High-Performance Liquid Chromatography (HPLC), this study delves into the structural, functional and crystalline properties of the ASD. The biological evaluation encompasses antioxidant activity through DPPH and hydroxyl radical scavenging assays, cytotoxicity studies using MTT assays, antiproliferative activity via clonogenicity assays, and in vivo assessments of anticancer efficacy in mouse tumor models, aiming to provide a comprehensive understanding of the ASD’s efficacy and potential therapeutic impact. This work improves solubility and dissolution rates and significantly enhances chemotherapeutic efficacy. The physical transformation of ERL into an amorphous state without altering its chemical structure is a key innovation in drug delivery systems, presenting a promising strategy for enhancing the therapeutic potential of anticancer drugs.

Materials and methods

Materials

Erlotinib HCl (ERL, CAS No: 183319-69-9, Mw ≈ 429.90), polyvinylpyrrolidone- K30 (PVP), CAS No: 9003-39-8, and polyethylene glycol-4000 (PEG), CAS No: 25322-68-3, with purity > 98% were bought from Sigma-USA Aldrich. All chemicals used for the in vitro studies including DPPH, deoxyribose, ferric chloride (FeCl₃), ethylenediamine tetraacetic acid (EDTA), hydrogen peroxide (H₂O₂), ascorbic acid, potassium dihydrogen phosphate (KH₂PO₄)-potassium hydroxide (KOH) are of analytical grade and are products of Sigma-Aldrich (St. Louis, MO) and Merck (Boston, MA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS), penicillin,streptomycin, and other cell culture reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified. Tissues were fixed in 10% neutral-buffered formalin (Sigma-Aldrich, St. Louis, MO, USA), processed, and stained with hematoxylin and eosin (Merck, Darmstadt, Germany). Every reagent and solvent, such as acetonitrile, sodium dihydrogen phosphate, and ortho-phosphoric acid, was of analytical and HPLC quality.

Synthesis of ASDs of ERL

ASDs of ERL were prepared using PVP, PEG, and a PVP + PEG blend as polymeric carriers to enhance solubility and dissolution. The formulations were designed at a drug-to-polymer ratio of 10:90 (w/w). ERL and the respective polymers (PVP, PEG, or PVP + PEG blend) were individually dissolved in methanol to ensure homogeneous mixing. The solutions were stirred continuously for 2 h at 333.5 K on a hotplate magnetic stirrer at 800 rpm. To further enhance molecular mixing and facilitate the interaction between ERL and the polymer matrix, the solutions were subjected to an additional 2 h of agitation at 333.5 K at an increased stirring speed of 1000 rpm. Following thorough mixing, the solvent was removed using a rotary evaporator under reduced pressure to obtain the solid residue. The resulting product was further dried in a vacuum oven at 333.5 K for 12 h to eliminate residual solvents and ensure complete amorphization. No further purification was required, as the process ensured complete removal of residual solvents and homogeneous dispersion of ERL in the polymer matrix. The final dried ASD formulations were stored in desiccators until further characterization.

Physicochemical characterization

The ERL quantification from its ASD was adapted from a previously published and verified HPLC (Shimadzu SPD-10A VP system, Merck, Germany) approach at 300 K reported by Latha et al.¹⁵. The mobile phase comprised acetonitrile and potassium dihydrogen phosphate buffer (0.02 M, with 0.1% orthophosphoric acid added to set pH 5) at a ratio of 30:70% v/v. By comparing the chromatograms from a phosphate buffer that had been tipped with samples, the specificity of the new approach was determined^15,16. The spectroscopic characterization using FTIR was done using the JASCO-4100 spectrometer for powder samples (KBr pellets) in the ATR mode with the diamond crystal from the range 4000–400 cm⁻¹ at 1 cm⁻¹ spectral resolution. The electronic spectrum of the samples has been recorded using a JascoV-550 UV–Vis spectrometer in the regime of 200–800 nm. The crystallinity of the neat and ASD of ERL were assessed using an X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) operating in the angular range from 10^° to 80^° with Nickel-filtered CuKa radiation (k = 1.5418 Ǻ)¹⁷. The dissolution study was conducted using UV-Spectrophotometers. The calibration curve ERL (1 mM) in deionized water were measured at 242 nm. Then saturation solubility of ERL and prepared ASDs were quantified via kinetics method.

Biological evaluation

Antioxidant activity

The evaluation of free radical scavenging activity of ERL and its formulations was evaluated by DPPH and HRS scavenging photometric assays. In the DPPH assay, 50, 100, 200, and 300 μM dosages of the drug were prepared by dissolving the drug (1 μM) in 90 mM methanolic DPPH. The absorbance was observed at a wavelength of 517 nm using a microplate reader (Varioskan Flash, Thermofisher Scientific, Waltham, MA) after incubation for 30 min at 298 K¹⁸. Additionally, the hydrogen radical scavenging (HRS) assay was employed to assess the scavenging activity of ERL and its formulations on OH radicals. For this assay, different concentrations of ERL were added to a reaction mixture containing FeCl₃ (0.1 mM), deoxyribose (2.8 mM), EDTA (0.1 mM), ascorbic acid (0.1 mM), H₂O₂ (1 mM), and KH₂PO₄-KOH buffer (20 mM, pH = 7.4). The reaction mixture was incubated for 1 h at 37 °C, and deoxyribose degradation was measured using thiobarbituric acid (TBA).

In both case, the percentage of inhibition was calculated as follows.

This comprehensive assessment provides insights into the antioxidant properties of ERL and its formulations against DPPH and OH radicals.

Sterility and pyrogen testing of formulations

To ensure the safety and suitability of the formulated ASDs of ERL for further biological evaluation, sterility and pyrogen testing were conducted according to pharmacopeial guidelines. Sterility testing was performed using the direct inoculation method in compliance with USP < 71 > guidelines¹⁹. The formulations were aseptically transferred into fluid thioglycollate medium (FTM) and soybean-casein digest medium (SCDM) and incubated at 30–35 °C and 20–25 °C, respectively, for 14 days. Any microbial growth was monitored daily. The absence of turbidity confirmed the sterility of the formulations.

The presence of pyrogens was assessed using the Limulus Amebocyte Lysate (LAL) assay, following USP < 85 > guidelines²⁰. The test was conducted using an endpoint chromogenic method, where formulations were incubated with LAL reagent at 37 °C for 60 min, and absorbance was measured at 405 nm. The test was performed in triplicate, and formulations with endotoxin levels below the specified threshold (< 0.25 EU/mL) were considered pyrogen-free.

The experimental protocol for this study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Amala Cancer Research Centre under project proposal number ACRC/IAEC/22(1)-P11. The approval was granted in a meeting held on 06.07.2022. All procedures were conducted in accordance with institutional guidelines and regulatory requirements.

Anticancer studies

1. Cell lines

The cell lines used for this study, MCF-7 and HCT-116 were purchased from National Centre for Cell Science, S.P, Pune University Campus, Pune, India, 411,007. Research Resource Identifier RRID for HCT-116:CVCL_0291 and for MCF-7:CVCL_0031. Methodology: Sixteen short tandem repeat (STR) Ioci were amplified using a commercially available AmpFISTRo Identifiler@ Plus PCR Amplification Kit from Applied BioSystems. The cell line sample was processed using the Applied Bio systems@ 3500 Genetic Analyser. Data was analyzed using Gene Mapper ID-X v1.5 software (Applied Biosystems). Appropriate positive and negative controls were used and confirmed for each sample. The cell lines used for the study were tested for mycoplasma by Hoechst staining/PCR method and did not detect its presence.

• (b)MTT assay

The MCF-7 and HCT cells (1x10⁵ cells/well) were seeded in 24-well plates and incubated at 310 K with 95% air and 5% CO₂. After the cell reached the confluence, various concentrations of the drugs were added and incubated for 24 h. Then, 50 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was added per well and incubated for 4 h followed by the addition of 1 mL of DMSO. Finally, cell viability was recorded using a UV-spectrophotometer at the absorbance wavelength of 570 nm²¹.

The percentage of inhibition was calculated using the formulae;

• (c)Clonogenicity assay

To assess tumorigenicity (colony-forming ability), cells were seeded in 6 well plates of 5000 cells for MCF 7 and HCT-116 cells and incubated overnight. Cells were then treated with drug ERL and prepared formulations Sat final concentrations of 10 and 20 μM for 72 h. Then, the DMEM culture medium (including drugs) was removed and the cells were washed twice with PBS and allowed to proliferate in fresh medium for 4 days (for MCF 7 cells) and 1 week (HCT-116). When colonies were intensified in control, the number of colonies was determined by staining with 1% crystal violet in 20% ethanol²².

Animal study

1. Animals

Female Swiss albino mice (25–30 g) were purchased from Small Animal Breeding Station (SABS), Kerala Veterinary and Animal Science University, Mannuthy, Thrissur. The animals were kept following standard conditions in the animal house facility of Amala Cancer Research Centre (297–301 K, 60–70% humidity, 12 h dark/light cycle) and fed with standard rat feed bought from Sai Durga Feeds, Bangalore, India, and water ad libitum. All animal experiments were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org) and approved by the Institutional Animal Ethics Committee (IAEC) of Amala Cancer Research Centre (Project proposal no. ACRC/IAEC/22(1)-P11) and were conducted strictly according to the guidelines of the Committee for Control and Supervision of Experiments on Animals (CPCSEA) constituted by Ministry of Environment and Forest, Government India.

• (b)Toxicity testing

An acute oral toxicity study was performed according to the Organization of Economic Co-operation and Development (OECD) guideline 420 for testing the pristine and formulated ASDs. Five female Swiss albino mice were used in each group of Normal, ERL, ERL + PEG, and ERL + PEG + PVP. A single dose of 50 mg/kg, i.e., 1.25 mg in 200 μL in purified water, was orally administered to mice of average weight 25 g. In contrast, the control group only received 2 mL of purified water as a vehicle. All mice were then granted free access to food and water and observed for any signs of acute toxicity for 24 h with special attention to the first 4 h and once daily for 14 days²³.

• (c)DLA-induced solid tumor studies

After 14 days of quarantine, DLA cell lines (1.0 × 10⁶ cells/mice) were injected subcutaneously into the right hind limb of Swiss albino mice and divided into 8 groups containing 5 mice in each group. Group I served as tumor control. Group II was treated with standard cyclophosphamide at a dose of 10 mg/kg body weight dose. Groups III and IV were treated with ERL at 5 and 10 mg/kg body weight doses (i.e., 0.125 mg in 200 μL and 0.25 mg in 200 μL). Groups V and VI were treated with ERL + PEG at 5 and 10 mg of ERL/kg body weight doses (i.e., 1.25 mg in 200 μL and 2.5 mg in 200 μL), and Groups VII and VIII were treated with ERL + PEG + PVP at 5 and 10 mg of ERL/kg body weight doses (i.e., 1.25 mg in 200 μL and 2.5 mg in 200 μL). All the treatments were orally administered on the 6^th day after DLA tumor inoculation and continued once daily for 10 days. From the first day onwards, the diameter of the right hind limb was noted using vernier calipers, measured every third day, and recorded for up to 31 days. The following formula calculated the tumor volume: , where r₁ and r₂ are the radii of tumors in vertical and horizontal directions, respectively²⁴.

• (d)Statistical analysis

The data from in vitro and in vivo studies were presented as mean ± standard deviation (SD), derived from three distinct experiments. Statistical analysis was conducted using one-way ANOVA, followed by Dunnett’s multiple comparison test in GraphPad Prism 8 software. Statistical significance was assigned to p values < 0.05*, < 0.01**, < 0.001***,and < 0.0001****, while p > 0.05 was considered non-significant.

• (e)Histopathological analysis

A small portion of tumor the tissue from the solid tumor was fixed in 5% formalin after washing with 0.5% saline. After several treatments for alcohol dehydration, sections (~ 4 µm thickness) were cut and stained with hematoxylin and eosin and monitored under a light microscope at a magnification of 40ײ⁵.

Results and discussion

Characterization of ASDs

FT-IR, XRD, and UV spectroscopies were utilized to analyze the physical and chemical characteristics of the prepared ASDs of ERL. This study preferred a 10:90 (drug: polymer) weight ratio depending upon the stability and integrity of the ASD (discussed in detail in section "Introduction", “X-ray diffraction”, in the supplementary material).

FTIR spectroscopy

FTIR spectroscopy is used to identify specific functional groups of ERL in formulations and to detect potential interactions between the API and polymer matrices through peak positions and intensities changes. This will give a detailed insight into the chemical compatibility and molecular interactions between the drug and polymers. The spectrum of neat and ASDs of ERL in biocompatible polymers PVP, PEG, and PVP + PEG matrix (10:90) was recorded in the range from 4000 to 400 cm⁻¹ and shown in Fig. 2. The unprocessed ERL showed IR absorption bands at 1666 cm⁻¹ for NH bending, 1603 cm⁻¹ for C=C stretching, 1539 cm⁻¹ for C=N stretching, and 1479 cm⁻¹ for CH₂ sym deformation vibration, 1386 cm⁻¹ for CH sym. deformation vibration, 1301 cm⁻¹ for NCO stretching vibration, 1183 cm⁻¹ for C–N stretching vibrations, 1146 cm⁻¹ for COCN stretching vibration, 1086 cm⁻¹ for –COCN stretching vibration, 1035 cm⁻¹ for C=O stretching, 947 cm⁻¹ for C–H out-of-plane deformation vibration, 893 cm⁻¹ for C–C skeleton vibration, 793 cm⁻¹ for C–O–C stretching vibration, 709 cm⁻¹ for N–H deformation vibrations, 679 cm⁻¹ for N–H out-of-plane bending vibrations and 588 cm⁻¹ for C–C skeleton vibration.

Fig. 2.

FTIR spectra of ERL and its ASDs with polymeric carriers. The spectra represent % transmittance as a function of wavenumber (cm^⁻1) for ERL alone (black), ERL with PEG (red), ERL with PVP (blue), and ERL with blend PVP and PEG (pink).

Interestingly, all the characteristic bands of ERL were retained in its binary dispersion with PVP, PEG, and a blend of PVP + PEG, even at a 10% weight ratio of the drug. In addition, the spectrum is overwhelmed by the IR absorption bands of PVP and PEG. The intensity corresponding to ERL vibrations in ASD was diminished and shifted to the low-frequency side. The C=O– bending vibration at 1666 cm⁻¹ for ERL was shifted to 1642 cm⁻¹ in ERL + PEG, 1648 cm⁻¹ in ERL + PVP and 1658 cm⁻¹ in ERL + PVP + PEG. CH₂ sym. deformation vibration of ERL at 1479 cm⁻¹ was shifted to 1470 cm⁻¹ in ERL + PEG, 1467 cm⁻¹ in ERL + PVP and 1474 cm⁻¹ in ERL + PVP + PEG. C–H sym. deformation vibration of ERL at 1386 cm⁻¹ was shifted to 1346 cm⁻¹ in ERL + PEG and ERL + PVP and 1386 cm⁻¹ in ERL + PVP + PEG. These shifts to the less frequency side imply less energy requirement for the molecular vibration in an amorphous state. The C=C stretching and C=N stretching at 1603 cm⁻¹ and 1539 cm⁻¹, –COCN stretching vibration at 1086 cm⁻¹, C=O stretching at 1035 cm⁻¹, N–H deformation vibrations at 709 cm⁻¹ were diminished, which may be indicative changes in the molecular environment due to interaction between ERL and polymers. The lower intensity is due to lower drug concentration. In contrast, the shift towards the lower frequency side may be attributed to its lesser energy requirement for vibrations because of increased freedom in the amorphous phase. The assignments for vibrational peaks were tabulated in table S1 in the supplementary material.

X-ray diffraction

One of the prominent methods in quantifying crystallinity in ASD formulation is the PXRD technique with a lower quantification limit of crystalline API of 5–10 wt.%. Figure 3 compares the XRD patterns of crystalline ERL with formulated ASD in the polymers of PVP, PEG, and their blend of PVP + PEG. ERL is characterized by the PXRD pattern at about 11.7°, 16.2°, 21.7°, 24.75°, 25.56°, and 29.37°, which coincided with those reported previously^26,27. The excipient PEG showed two sharp peaks for crystallinity at 19.36° and 23.72° (shown in Fig. 2S in supplementary material), whereas another excipient, PVP K-30, showed no peak for crystallinity. Upon preparation of the ASD formulation, most of the crystallinity peak of ERL was demolished except for the peaks of PEG-4000, which signified the conversion of crystalline ERL to an amorphous state. These characteristic peaks of ERL were found to diminish in the formulations, signifying that both polymers PVP and PEG were suitable additives to inhibit the crystallization of ERL²⁸.

Fig. 3.

Powder X-ray diffraction patterns of neat and ASDs of ERL with polymers (at the weight ratio 10:90).The diffractogram of pure ERL (black) exhibits sharp crystalline peaks, indicating its highly crystalline nature. The ERL + PVP dispersion (red) shows a broad halo pattern, confirming amorphization. The ERL + PEG system (green) reduced the crystallinity of ERL but retained the semicrystalline nature of PEG. The ERL + PVP + PEG (blue) also shows reduced crystallinity but retained minor peaks of PEG.

UV–visible spectroscopy

The spectrophotometric method was used to analyze potential interactions between ERL and the polymer blends by detecting shifts in the UV absorption peaks. The pure and its binary absorption spectra were acquired in ethanol at room temperature and are characterized by three noticeable absorption bands, around λ = 252 nm and λ = 336 nm, as shown in Fig. 4. Even though these peaks were retained in the binary mixture, the intensities were found to be reduced. The absorbance at 256 nm shifts towards the higher frequency side (248 for ERL + PVP, 246 for ERL + PEG, and 249 for ERL + PVP + PEG). This may be due to π to π* transition of the C–O group in the PVP matrix since the pure PVP has an absorption at 211 nm (Bahadur et al., 2016), indicating a favorable interaction of ERL with PVP²⁹.

Fig. 4.

UV–Visible absorption spectra of ERL and its ASDs with polymeric carriers. The spectra include neat ERL (green), ERL with PVP (red), ERL with PEG (blue), and ERL with both PVP and PEG (black).

Determination and dissolution testing of ERL in ASDs

HPLC

HPLC is used to quantitatively analyze ERL in the ASD with the polymers PVP K-30, PEG, and their blend. An HPLC technique for the quick (5 min) detection of ERL was developed by optimizing chromatographic parameters in phosphate buffer: acetonitrile (70:30 V/V) mobile phase with phosphate buffer as dissolution medium. The chromatogram of neat, and ASDs of ERL in biocompatible polymers PVP, PEG, and PVP/PEG matrix (10:90) were recorded and shown in Fig. 5. The result obtained from HPLC chromatography studies indicated the presence of ERL in the prepared formulations. Take away the solvent peaks at 2.5 and 18 min, which are well evidently depicted in the figure 5. The peak at 4.72 attributed to the ERL is observed to have grown in height in the formulated ASD, suggesting a more excellent prompt release of ERL that is transmitted to the detector. The maximum absorbance of ERL is found to be 0.0029, while that increases to 0.0119 for ERL + PVP, 0.015 for ERL + PEG, and 0.56 for ERL + PVP + PEG.

Fig. 5.

HPLC chromatograms of ERL and its formulations with polymeric carriers. The chromatograms display retention time (min) on the x-axis and voltage response (volts) on the y-axis. The profiles include solvent (black), ERL alone (purple), ERL with PVP (blue), ERL with PEG (red), and ERL with both PVP + PEG (green).

Dissolution study

The calibration curve of ERL was plotted at different concentrations of the drug in deionized water, was measured at 266 nm and shown in Fig. 6a. All the curves obeyed Beer-Lambert’s law with a linear relationship with an R² value of 0.9995³⁰. Moving on to the in vitro drug release study, Fig. 6 provides illuminating insights into the release profiles of ERL and its ASDs. The inherent challenge of ERL’s low dissolution percentage (< 10%), attributed to its hydrophobic nature and limited aqueous solubility, is effectively addressed through formulation strategies. Remarkably, the ASD formulations exhibit a substantial enhancement in dissolution rates compared to pure ERL. Notably, the formulation incorporating ERL in a PEG matrix achieves the highest release percentage, reaching an impressive 80%. This emphasizes the efficacy of PEG as a matrix in enhancing the solubility and dissolution of ERL. In contrast, ERL in a PVP matrix demonstrates a lower release percentage (20%), suggesting differences in the dissolution behavior influenced by the choice of polymer. The formulation within the blend falls in between, with a release percentage of 50%, indicating a synergistic effect of the combined PVP and PEG matrices. While all release percentages are below 100%, suggesting potential partial precipitation in the dissolution medium, the overall improvement in dissolution rates is evident. Notably, the formulation employing a PEG polymer matrix outperforms that in a PVP matrix, underscoring the impact of the polymer choice on dissolution behavior. This observed difference is attributed to the higher solubility of the formulation entrapped in the PEG matrix, showcasing the critical role of polymer selection in influencing the dissolution kinetics of ERL³¹.

Fig. 6.

(a) Calibration curve of erlotinib showing the linear relationship between absorbance (a.u) and concentration (µg/mL). The linear fit equation, y = mx + c. (b) In vitro drug release profile of erlotinib in water as the dissolution medium. Different formulations—ERL, ERL + PVP, ERL + PEG, and ERL + PVP + PEG—are compared based on drug release concentration (µg/mL) over time (min).

Biological activity

Antioxidant activity

1. DPPH radical scavenging activity

In-vitro scavenging activity of different concentrations of ERL along with its ASDs with PVP, PEG, and PVP/PEG in methanol vehicles with a vehicle control was evaluated and depicted in Fig. 7a. In addition, the DPPH scavenging activity of different concentrations of base polymers PVP K-30 and PEG 4000 was evaluated and depicted in Fig. 3S in the supplementary material. Both polymers have negligible activity, even at higher concentrations. The radical scavenging activities of ERL are found to increase amorphization due to the free availability of the molecule. This may be either due to the presence of polymers or due to the enhanced solubility of the ERL drug in the solvent. The measured IC₅₀ for ERL, ERL + PEG, and ERL + PVP + PEG are > 300 μg/mL while IC₅₀ for ERL + PVP is 100 μg/μL. Notably, the ERL + PVP formulation showed significantly enhanced DPPH scavenging at all tested concentrations, with a maximum effect observed at 200 µg/mL (p < 0.01). However, it is fascinating to note that the IC₅₀ is comparatively less for ERL + PVP ASDs in which PVP has no additional hydrogen donors, in such situation it is compelled to believe that the enhanced antioxidant property is due to the enhanced solubility of the drug in its amorphous form.

• (b)HRS activity

Fig. 7.

(a) DPPH (b) HRS radical scavenging activities of various concentrations of neat and ASDs of ERL with polymers (at the weight ratio 10:90). The results are expressed as mean ± SD, with n = 3. Statistical comparisons were conducted using one-way ANOVA, followed by Dunnett’s multiple comparison test. Statistically significant probabilities are denoted as *p < 0.05 , **p < 0.01 ***p < 0.001 and ****p < 0.0001.

The dose-dependent response of the hydroxyl radical scavenging test for ERL, along with its ASDs with PVP, PEG, and PVP/PEG, was depicted in the bar diagram as shown in Fig. 7b. ERL + PEG demonstrated higher OH radical scavenging activity with lowest IC₅₀ value of 200 μg/mL. In addition, the hydroxyl scavenging activity of different concentrations of base polymers PVP K-30 and PEG 4000 was evaluated and depicted in Fig. 4S in the supplementary material. Both polymers have negligible activity, even at higher concentrations. As observed in the DPPH test, all the developed ASDs showed higher antioxidant activity when compared to neat ERL. IC₅₀ for ERL is > 300 µg/mL. ERL + PEG and ERL + PVP + PEG formulations demonstrated superior activity compared to ERL alone, particularly at higher concentrations (200–300 µg/mL), with p < 0.01 indicating strong significance. It is interesting to note that the activity is enhanced in ERL + PEG and ERL + PVP + PEG ASDs, though the solubility of the ERL was enhanced in all formulations, here we suspect the enhancement in activity of ERL + PEG and ERL + PVP + PEG may be due to the presence of hydroxyl group in the PEG. It can easily donate hydrogen atoms than PVP.

Anti-cancer activities

1. Cell viability

The in vitro anti-cancer activity of the neat and ASDs of ERL was dissolved in DMSO vehicle against MCF-7 and HCT-116 cells is compared in Fig. 8a–d respectively for relative inhibition percentage after 48 h. The cell inhibition had a positive linear dose-dependent response. It is fascinating to note that cells treated by ERL showed recrystallization behavior when they interacted with the DMEM medium in the culture well. This may be due to its low solubility and recrystallization tendency when exposed to water molecules. It is evidently observed in the phase contrast microscopic images (Fig. 8a,b). However, the ASDs exhibited a significant cytotoxic effect compared to pristine ERL. The dose–response curves for the cytotoxicity against MCF-7 and HCT-116 are shown in Fig. 8c,d. ERL + PEG demonstrated the highest cytotoxic effect against MCF-7 with an IC₅₀ of 19 μM, while ERL + PEG + PVP showed the highest effect against HCT-116 with an IC₅₀ of 19.5 μM.

• (b)Antiproliferative activity

Fig. 8.

Microscopic images of (a) MCF-7, (b) HCT-116 cell lines after drug treatment with various concentrations of neat and ASDs of ERL with polymers. Dose–response curve of (c) MCF-7 and (d) HCT-116 cell lines after drug treatment with various concentrations of neat and ASDs of ERL with polymers. The results are expressed as mean ± SD, with n = 3. Statistical comparisons were conducted using one-way ANOVA, followed by Dunnett’s multiple comparison test. Statistically significant probabilities are denoted as *p < 0.05 , **p < 0.01 ***p < 0.001 and ****p < 0.0001.

Typically, MCF 7 and HCT cells formed a relatively high number of large colonies in DMEM medium under control conditions with and without DMSO vehicle (Fig. 9a,b). In the case of MCF-7, although treatment with ERL alone resulted in significant reductions of colonies, it was notable that cells treated with the prepared formulations of ERL in PVP and PEG polymer matrices formed markedly smaller colonies. Besides, concomitant treatment with the formulated ASD in a blend of PVP/PEG almost completely suppressed anchorage-independent growth of MCF-7cells (Fig. 9a). But in the case of HCT-116 cells, the prepared formulations of ERL in PVP and PEG and their blend polymer completely suppressed their growth, resulting in the death of utmost cells (Fig. 9b).

Fig. 9.

The prepared formulations of ERL reduced (a) MCF-7 and (b) HCT-116 colony formation as analyzed by a colony-forming assay stained with crystal violet.

Toxicity testing

1. Acute toxicity

Though the literature disclosed that there was neither no mortality nor any significant behavior change observed in animals treated with oral administration of 1–320 mg/kg of ERL³². Mice given a 1000 mg/kg dose had reduced spontaneous locomotor activity in their cubicles at the 30-min time point but had normal activity levels when transferred to the open field. In this work, we have carried out a test for potential acute toxicity, which was performed in female Swiss albino mice dosed orally with (50 mg/kg) ERL, and the results were tabulated in table 1. In all tested mice, there was neither no mortality nor any significant behavior change observed during the 14 days of the acute toxicity experimental period, after administering a single oral dose of the testing suspension of 50 mg/kg ERL. After 14 days of treatment, the animals were subjected to gross necropsy with no abnormality of organs observed in the macroscopic observation. This result suggests that neither pristine nor the formulations were not toxic, after an acute exposure.

• (b)Solid-tumor model

Table 1.

General physiological and behavioral observations in mice following treatment with ERL and its ASDs.

Observation	Normal (3 mice)	ERL (3 mice)	ERL + PEG (3 mice)	ERL + PVP + PEG (3 mice)
Food intake	Normal	Normal	Normal	Normal
Water intake	Normal	Normal	Normal	Normal
Body weight	No change	No change	No change	No change
Drowsiness	Not present	Not present	Not present	Not present
Changes in skin	No change	No change	No change	No change
Diarrhea	Not present	Not present	Not present	Not present
Sedation	No effect	No effect	No effect	No effect
General physique	Normal	Normal	Normal	Normal
Death	Alive	Alive	Alive	Alive

A tumor formed due to inoculation of DLA cells was found to be prominent on day-33 in the DLA-treated control mice. The drugs and prepared formulations of ERL treatments prevented the growth in tumor volume. The effects of neat and prepared formulations of ERL on solid tumor development are shown in Fig. 10a–c, and table 2. ERL + PEG was found to be the most successful treatment when administered at a dosage of 10 mg/kg ERL.

Fig. 10.

Effect of neat and formulated ASDs of ERL on DLA-induced solid tumors: (a) Tumor reduction in treated groups vs. DLA control and cyclophosphamide (mean ± S.E.M., n = 6). (b) Gross appearance of mice post-treatment after 33 days. (c) Tumor mass reduction. Statistical comparisons were conducted using one-way ANOVA, followed by Dunnett’s multiple comparison test. Statistically significant probabilities are denoted as *p < 0.05 , **p < 0.01 ***p < 0.001 and ****p < 0.0001.

Table 2.

Effect of ERL and its ASDs on solid tumor weight and tumor inhibition percentage in an experimental model. The table presents the mean tumor weight (grams) and the corresponding percentage of tumor inhibition for different treatment groups.

Groups	Treatment	Weight of solid tumor (gram)	% of tumor inhibition
(a)	Control	5.15 ± 0.001	0.00 ± 0.1
(b)	Standard	0.329 ± 0.003	93.61 ± 0.2
(c)	ERL—low dose	1.290 ± 0.006	74.95 ± 0.3
(d)	ERL—high dose	1.286 ± 0.002	75.03 ± 0.1
(e)	ERL + PEG—low dose	0.167 ± 0.002	96.76 ± 0.4
(f)	ERL + PEG—high dose	0.063 ± 0.004	98.78 ± 0.3
(g)	ERL + PVP + PEG—low dose	0.800 ± 0.009	84.47 ± 0.2
(h)	ERL + PVP + PEG—high dose	3.055 ± 0.008	40.68 ± 0.3

In the control animals, the solid tumor volume induced by DLA cells was found to be about 25 times increased from day 0 to day 33. However, the tumor volume was found to increase to 20 times from day 0 to day 15 in all animals, then decreased to 3 times on day 33 in standard (cyclophosphamide), 9 times, and 8 times on day 33 for ERL-treated groups 5 and 10 mg/kg body weight, 7 times and 0.5 times on day 33 for ERL + PEG treated groups 5 and 10 ERL/kg body weight and 11 times and 14 times on day 33 for ERL + PVP + PEG treated groups 5 and 10 ERL/kg body weight. Gross appearance of mice following treatment with neat and prepared formulations of ERL on DLA-induced solid tumor-bearing mice in comparison with control and treated with a single dose of cyclophosphamide after 33 days of tumour inoculation were shown in Fig. 10b^24,33.

The animals injected with DLA cancer cells alone produced a solid tumour volume of 5.15 ± 0.001 gm on the 31^st day. The standard cyclophosphamide (10 mg/kg b wt) treated mice showed a reduced tumour volume of 0.329 ± 0.003 gm on the same day. The ERL-treated groups 5 and 10 mg/kg body weight doses exhibited a decreased tumour volume to 1.290 ± 0.006 and 1.286 ± 0.002 gm, respectively. While the low and high doses of ERL + PEG at 5 and 10 mg of ERL/kg body weight doses significantly decreased the tumour volume to 0.167 ± 0.002 and 0.063 ± 0.004 gm. But the low and high dose of ERL + PVP + PEG at 5 and 10 mg of ERL/kg body weight doses showed the tumour volume to be 0.800 ± 0.009and 3.055 ± 0.008 gm, respectively (Figure 10c and Table 2).

• (c)Histopathology of tumor tissue in solid tumor models

Experimental DLA-bearing mice in the intramuscular region, which formed solid tumors, revealed substantial pathological alterations distributed throughout the muscular region, as represented in Fig. 11. Examination of tumor sections obtained from the muscular region of the control mice group showed tumours composed of pleomorphic, polyhedral, or oval cells having dense nuclei. The cells are arranged in small groups in glandular patterns and trabecular patterns. The cells have hyperchromatic vesicular nuclei. There are extensive areas of hemorrhage, necrosis, and dense inflammatory cell infiltration. Degenerating tumor cells are seen amidst the necrotic material. The group treated with the standard cyclophosphamide drug revealed tumour composed of pleomorphic, round, oval, or polyhedral cells having hyperchromatic vesicular nuclei. The cells in such groups are arranged in a granular pattern and as sheets of cells. Extensive areas of necrosis and dense inflammatory cell infiltration are seen in most areas. Fibromuscular tissue and tumour composed of pleomorphic, round, and spindle-shaped cells having hyperchromatic vesicular nuclei were found in the DLA solid tumor section in ERL low-dose treated mice group. While inflammation and necrosis are comparatively lesser in ERL high-dose treated group. At the same time, the tumour section of the mice group treated with the developed formulation ERL + PEG low dose showed fibro-collagenous tissue and striated muscle tissue with carcinomatous cells which are pleomorphic, round or oval polyhydral cells having dense nuclei with a few mitotic cells. But its high oral dosage showed fibrofatty tissue and striated muscle tissue with a few areas of necrosis and inflammation with a lack of tumor. The other formulation, ERL + PVP + PEG, low and high doses, revealed no alteration from the control groups with more necrosis and inflammation in high-dose therapy. This may be due to the dissimilarity between the invitro dissolution study and the invivo tumor inhibition results, which can be attributed to the inherent differences in these experimental setups. The dissolution study primarily reflects the drug release kinetics in a controlled environment, while the in vivo model involves complex biological processes such as absorption, distribution, metabolism, and excretion. This variation can significantly impact the observed outcomes. Moreover, the tumour in a mouse model can exhibit significant heterogeneity in terms of blood supply, cellular composition and other factors. This may influence the drug distribution and response, leading to variations in tumour inhibition not fully captured by in vitro dissolution studies. In light of all these considerations, we are committed to further investigating and understanding of these discrepancies.²⁵.

Fig. 11.

Histopathology changes in DLA-induced solid tumor tissues treated with standard-cyclophosphamide, ERL, and developed formulation of ERL (magnification 10 ×).

Conclusion

In this study, we investigated the formulation of stable SDs to enhance the bioavailability of ERL. By utilizing non-toxic, biocompatible polymers (PVP K-30, PEG 4000, and a 1:1 ratio blend), we improved the solubility of ERL and, thereby, its bioavailability and therapeutic efficacy. The successful amorphization of ERL within the polymer matrices was confirmed by the disappearance of characteristic crystalline peaks in XRD patterns, indicating effective dispersion. FTIR and UV–visible spectroscopy analysis confirmed that changes indicate a physical modification rather than a chemical one by a shift in characteristic peaks of ERL towards the lower frequency side. Evaluations of solubility and kinetic solubility revealed that while all formulated, ASDs showed an improvement in kinetic solubility compared to their crystalline counterpart and attained maximum for ERL + PEG. Under antioxidant activity, ERL-based formulations demonstrated varying degrees of radical scavenging. ERL + PVP exhibited the strongest DPPH radical scavenging, while ERL + PEG showed enhanced OH radical scavenging. Cytotoxicity evaluation against MCF-7 and HCT-116 cells showed high activity for ERL + PEG and ERL + PEG + PVP formulations, respectively. In the antitumor model, ERL + PEG demonstrated high efficacy without exhibiting any toxicity. These findings suggest that the solubility patterns of ERL in polymer solutions depend on the type of polymer used. PEG exhibited an excellent dissolution rate and higher anticancer efficacy among the selected polymers, likely due to slight hydrogen bonding. The limitation of the study was that only a 10 wt.% loading was achieved. Alternative solutions, such as adding surfactants or other strategies, need to be explored to enhance the loading capacity. In addition the mechanistic contributions of each polymer were studied uisng temperature-dependent differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS) across a broad frequency and temperature range. The DSC and PXRD results confirmed complete amorphization of ERL, with glass transition temperatures (Tg) of 252 K (ERL + PEG), 373 K (ERL + PVP), and 272 K (ERL + PEG + PVP). BDS revealed distinct relaxation processes: ERL + PEG showed α (331 K) and β relaxations, ERL + PVP exhibited a single α-relaxation (373 K), and ERL + PEG + PVP demonstrated two α-relaxations and one β-relaxation. Fragility indices were 148 (ERL + PEG), 62.1 (ERL + PVP), and 45.5 (ERL + PEG + PVP). These findings ?³⁴ that provide deeper insights into the physical stability and molecular mobility of the formulations, extending beyond chemical structure considerations. In conclusion, the formulation ERL + PEG, can be considered as a viable option to improve the solubility and bioavailability of poorly soluble ERL. However, to validate further and strengthen the translational potential of formulated ASDs we plan to conduct detailed pharmacokinetic (PK) studies, including the evaluation of parameters such as Cmax, Tmax, and AUC, to establish a clear correlation between enhanced solubility and improved systemic exposure. Then, the given enhanced oral absorption of this formulation, it holds promise for industrial application, potentially improving patient outcomes by providing more effective treatments for ovarian cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, and other tyrosine kinase inhibitor-responsive diseases.

Author contributions

K.P. Safna Hussan: .Conceived and designed the analysis: Initiated the concept of using amorphous solid dispersions to enhance ERL bioavailability. Collected and cordinated the physiochemical and biological data. Performed the analysis of FTIR, PXRD, UV–Vis, and biological assay and interpreted the data. Drafted the paper with focus on scientific accuracy and translational relevance. Thekkekara D Babu. Supervised and coordinated the data and helped in interpretation. Provided critical feedback during manuscript revision and data validation. M. Shahin Thayyil: Participated in data acquisition from spectroscopy-based tools. Provided theoretical background on drug-polymer interactions and contributed to data interpretation. Sreeshma T.S: Helped in invitro studies. Archana A: Assisted in vivo studies.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declarations

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-07692-1.