Functionalized Colloidal Hydroxyapatite for Combined Delivery of Cannabidiol and Epirubicin: Characterization, Biomimetic Membrane Interaction, and Cytotoxicity Studies

Abstract

Introduction

A stable colloidal hydroxyapatite (cHAp) platform was successfully developed for the co-delivery of cannabidiol (CBD), a natural bioactive compound, and epirubicin (EPI), a chemotherapeutic agent, to enhance cytotoxicity against ovarian cancer cells.

Methods

Colloidal cHAp was synthesized and functionalized with CBD and EPI to obtain a multifunctional drug delivery system. The resulting composite was characterized using high-performance liquid chromatography, UV–Vis spectrometry, transmission and scanning electron microscopies, Fourier-transform infrared spectroscopy, Turbiscan analysis, and differential scanning calorimetry. Drug release in phosphate-buffered saline (PBS, pH 7) was evaluated by differential pulse voltammetry. Interactions with model lipid monolayers mimicking healthy and cancerous cell membranes were investigated using the Langmuir technique, and cytotoxicity was assessed by MTS assays.

Results

The successful incorporation of both biologically active compounds into the cHAp carrier and its excellent colloidal stability were confirmed. The epirubicin loading content was 0.0605 mg per 1 mg of cHAp. Drug release followed a time-dependent profile, reaching approximately 2 μg mL⁻¹ after 24 h. The functionalized cHAp systems exhibited enhanced affinity toward model lipid monolayers and significantly reduced the viability of ovarian cancer cells compared with the free drug and non-functionalized carrier.

Conclusion

The developed cHAp platform provides a stable and multifunctional carrier for the co-delivery of CBD and epirubicin. Its favorable physicochemical properties, enhanced membrane interactions, and improved anticancer activity highlight its potential as a promising nanocarrier for targeted ovarian cancer therapy.

Introduction

With the advancement of cancer research, scientists continuously seek novel approaches to combat the disease.¹ One of the most prominent areas of exploration is the use of nanotechnology for cancer therapy.² The development of nanotechnology has enabled the fabrication of biocompatible nanomaterials that not only facilitate targeted drug delivery³ but also reduce the adverse side effects associated with conventional chemotherapy.⁴

Chemotherapy remains a standard treatment for advanced and metastatic cancers because of its ability to reduce tumor burden. However, conventional chemotherapeutic agents affect both malignant and healthy tissues and are often associated with severe side effects, including acute kidney injury,⁵ anorexia,⁶ gastrointestinal toxicity,⁷ and bone marrow suppression.⁸ To address these limitations, numerous targeted drug delivery strategies have been developed, including antigen-mediated targeting⁹ and magnetic guidance using magnetically modified nanomaterials.¹⁰ In parallel, a wide range of nanoparticles—including those based on metals,¹¹ metal oxides,¹² and minerals¹³—have been investigated as drug carriers capable of improving therapeutic efficacy while minimizing systemic toxicity.

Among these materials, hydroxyapatite (HAp) has attracted considerable attention in biomedical applications because of its chemical and structural similarity to the mineral component of natural bone tissue.¹⁴ HAp is a calcium phosphate ceramic with the chemical formula Ca₁₀(PO₄)₆(OH)₂.¹⁵ Its excellent biocompatibility,¹⁶ biological activity,¹⁷ and osteoconductivity¹⁸ have led to its widespread use in bone regeneration, implant coatings, and tissue engineering.^19,20 In nanoparticulate form, colloidal hydroxyapatite (cHAp) exhibits a high surface-to-volume ratio and favorable surface chemistry, making it particularly suitable for drug loading and surface functionalization.²¹ Beyond orthopedic applications, increasing evidence suggests that cHAp may also serve as an effective nanocarrier for anticancer therapy, including the treatment of soft tissue malignancies.²²

Due to its well-organized crystal structure and surface chemistry, cHAp can be readily modified by metal ion doping,^23–25 combination with metal oxides,²⁶ or functionalization with a broad spectrum of therapeutic molecules. These include conventional chemotherapeutic drugs,²⁷ naturally occurring bioactive compounds such as myo-inositols,²⁸ Carica papaya extracts,²⁹ curcuminoids,³⁰ and many other biologically active agents.^31,32 Such versatility makes cHAp an attractive multifunctional platform for drug delivery.

Among naturally occurring bioactive compounds, cannabinoids derived from Cannabis sativa have recently gained considerable attention. Cannabinoids are currently defined as molecules capable of interacting with the human endocannabinoid system.³³ They are widely used for the treatment of anxiety,³⁴ neurological disorders,³⁵ and symptom relief owing to their calming properties.³⁶ More recently, cannabidiol (CBD), a non-psychoactive cannabinoid, has been reported to exert anticancer effects through mechanisms including apoptosis induction, inhibition of cell proliferation, and modulation of signaling pathways involved in tumor progression.³⁷ Furthermore, several studies have suggested that CBD may enhance the activity of conventional chemotherapeutic agents, making combination therapy an emerging area of interest in oncology.³⁸

Despite significant advances in nanoparticle-based drug delivery systems, relatively few studies have explored hydroxyapatite-based nanocarriers for the simultaneous delivery of conventional chemotherapeutic agents and bioactive cannabinoids. In particular, the combination of cannabidiol and epirubicin delivered by colloidal hydroxyapatite has not been systematically investigated. Epirubicin is an anthracycline chemotherapeutic with well-established anticancer efficacy, whereas cannabidiol represents a promising natural compound with reported antiproliferative, pro-apoptotic, and potential chemosensitizing properties. Their combination may therefore provide complementary mechanisms of action while enabling localized and controlled drug delivery. Consequently, there remains a need to develop multifunctional, biocompatible nanocarriers capable of co-delivering these agents and to evaluate their therapeutic potential against ovarian cancer cells.

The novelty of the present study lies in the development of a multifunctional colloidal hydroxyapatite (cHAp) platform simultaneously functionalized with cannabidiol and epirubicin for combined anticancer therapy. The objective of this work was to synthesize and characterize the dual-functionalized cHAp system and to investigate its interactions with biomimetic membranes as well as its cytotoxic activity against ovarian cancer cells. The obtained results demonstrate that functionalization enhances interactions with biomimetic membranes and significantly decreases ovarian cancer cell viability, highlighting the potential of the proposed colloidal suspension as an effective platform for combined delivery of natural bioactive compounds and conventional chemotherapeutic agents.

Experimental

Materials, Procedures, and Methods

The nanostructural hydroxyapatite was fabricated from the inorganic salts such as calcium nitrate tetrahydrate, Ca(NO₃)₂*4H₂O, and diammonium hydrogen phosphate (NH₄)₂HPO₄, both with analytical grade, with the use of 25% ammonia aqueous solution as the precipitating agent. All chemicals were supplied by Chempur, Piekary Śląskie, Poland. Water was purified using the HYDROLAB water filtering system, HYDROLAB, Gliwice, Poland. The anticancer drug, epirubicin hydrochloride of ≥99% grade, was supplied by Sigma-Aldrich, Germany. Lipids: DOPC (1,2-dioleoyl-snglycero-3-phosphocholine, DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol, all with purity >99% were purchased from Avanti Polar Lipids, Birmingham, AL, USA.

Synthesis of Colloidal Hydroxyapatite (cHAp)

The colloidal suspension of nanostructured hydroxyapatite (cHAp), used as a carrier for biologically active compounds, was synthesized via a co-precipitation method from aqueous solutions following a previously reported procedure [28]. Briefly, 20 mL of 0.6 M diammonium hydrogen phosphate was stirred, while 20 mL of 0.5 M calcium nitrate and 10 mL of 25% aqueous ammonia were simultaneously added dropwise from separate burettes. The Ca/P molar ratio was 1.67, corresponding to stoichiometric hydroxyapatite. The reaction was carried out under vigorous stirring with pH maintained at 10–11 to promote controlled particle formation. After complete addition of reagents, the mixture was stirred for 120 min at room temperature. The product was then repeatedly centrifuged (5000 rpm), washed with distilled water, and sonicated to remove residual ammonia. The final product was a stable colloidal suspension of cHAp, which was used without drying in subsequent experiments.

Modification of Colloidal Hydroxyapatite

Cannabidiol (CBD) was incorporated into the cHAp structure after removal of water from the aqueous dispersion by centrifugation (125 mg cHAp, 5000 rpm, 5 min). The obtained sediment was combined with 50 mg of CBD dissolved in ethanol and stirred for 3 h. The suspension was then centrifuged under the same conditions to remove ethanol and unbound CBD, and the solid was resuspended in 3 mL of water. Subsequently, epirubicin (13 mg in 2 mL of water) was added to the CBD-functionalized cHAp and incubated for 24 h to allow adsorption of the drug onto the carrier surface. After incubation, excess epirubicin was removed by centrifugation (5000 rpm, 5 min), and the final composite was resuspended in 3 mL of water.

The loading of CBD and epirubicin onto cHAp nanoparticles occurred via surface adsorption rather than incorporation into the crystal lattice, involving non-covalent interactions between the drug molecules and the hydroxyapatite surface. In this way, a composite system consisting of hydroxyapatite, CBD, and epirubicin was obtained.

Methods

The morphology was investigated using Transmission Electron Microscopy (TEM) Zeiss Libra 120 Plus, Stuttgart, Germany, operating at 120 kV, and Scanning Electron Microscopy Crossbeam 350, Zeiss, Stuttgart, Germany. The morphology was investigated for five freshly performed synthesis and after 6 weeks of storage.

The porosity of the cHAp was measured with the Brunauer-Emmett-Teller (BET) method at 77 K, where the nitrogen N₂ was adsorbed-desorbed, using TriStar II 3020, Micromeritics, USA. The porosity of the cHAp was measured with the Brunauer-Emmett-Teller (BET) method at 77 K, where the nitrogen N₂ was adsorbed-desorbed, using TriStar II 3020, Micromeritics, USA. The porosity was measured for two different synthesis.

The characterization of CBD, epirubicin, HAp, cHAp_CBD, and cHAp_CBD_Epi was carried out by Fourier-transform infrared spectroscopy (FT-IR) using the ATR technique (Alpha apparatus, Bruker). The measurements were performed in the range of 4000–400 cm^–1, with 32 scans at a resolution of 4 cm^–1.

In this work, an HPLC in-house validated method for the qualification and quantification of cannabidiol was carried out. Kanuer Azura HPLC system equipped with DAD MWD 2.1L detector, the Azura pump P 6.1L, autosampler 6.1 L, and column thermostat CT 2.1. and the column Eurospher II C18P 100–3, 100x4,6 mm was used. The aqueous eluent was acidified with phosphoric acid to pH 2.2. Acetonitrile eluent was used as a second mobile phase. Absorption line at 228 nm was monitored. HPLC measurements were done from 5 different syntheses.

The stability of water suspension of the obtained composite was carried out using the multiple light scattering method by the Turbiscan Lab apparatus (Formulaction SA, France), where the colloidal suspension of HAp, cHAp_CBD, and cHAp_CBD_Epi in water was placed in glass cylindrical test tubes with a working height of 54 mm. Then, the samples were placed in the apparatus and subjected to scanning with light at a wavelength of about 880 nm. The scans were carried out every 5 min for half an hour at room temperature (23 °C). The amount of transmitted (LT) and backscattered (BS) light was recorded. The mechanism of destabilization of the water suspension was determined on the basis of the obtained results. Then the Turbiscan Stability Index (TSI) was determined. The stability was performed for five different samples.

The interactions between the conjugates and lipid monolayers were investigated using a Langmuir trough (KSV-Nima KN2003, Biolin Scientific, Manchester, UK) controlled by KSV NIMA LB software. Before each experiment, the trough was cleaned with methanol and chloroform, and the Wilhelmy platinum plate was flame-treated. The trough was filled with either pure water or a cHAp suspension as the subphase, and lipid solutions in chloroform were spread onto the surface. After a 10 min solvent evaporation period, compression isotherms were recorded at a barrier speed of 10 mm/min up to a surface pressure of 35 mN/m, corresponding to the physiological range of biological membranes. All measurements were performed in triplicate. Model membranes were prepared from DOPC, DOPE, DOPS, and cholesterol mixed in appropriate molar ratios to mimic natural cell membranes.

The SKOV-3 human ovarian carcinoma cell line was purchased from the ATCC (Manassas, VA, USA) and cultured according to the recommendation of the supplier. Cytotoxicity studies were conducted using dispersions containing hydroxyapatite (cHAp), cannabidiol (CBD), epirubicin (Epi), hydroxyapatite functionalized with CBD (cHAp_CBD), and a dual-functionalized hydroxyapatite with both CBD and Epi (cHAp_CBD_Epi). The concentrations of cHAp, CBD, and Epi ranged from 0.5 to 3 µg/mL. SKOV-3 ovarian cancer cells were seeded in 96-well plates at a density of 2.5 × 10³ cells per well and incubated at 37 °C in a humidified atmosphere with 5% CO₂. After 24 h, the cells were washed with PBS and then treated with varying concentrations of the tested compounds. Incubation continued for an additional 24, 48, or 72 h.

Cell viability was assessed using the MTS-based CellTiter-96^® AQueous One Solution Cell Proliferation Assay. The absorbance of the formazan product was measured at 492 nm using a microplate reader (Berthold Technologies, Bad Wildbad, Germany). The results are expressed as the percentage of viable cells relative to untreated controls.

Statistical analysis of the experimental data was performed using GraphPad Prism software, version 8.4.3 (GraphPad Software Inc., San Diego, CA, USA). Two-way ANOVA was applied for multiple group comparisons, followed by Tukey’s test.

To analyze drug interaction effects, the CompuSyn software (ComboSyn Inc.) was employed. This software utilizes the Chou–Talalay method to calculate the combination index (CI), half-maximal inhibitory concentration (IC₅₀), and dose-reduction index (DRI). The CI indicates the nature of drug interaction: values <1 suggest synergism, ≈1 indicates additivity, and >1 implies antagonism. The DRI quantifies the fold reduction in drug dose required to achieve the same therapeutic effect when used in combination.

Results and Discussion

Morphology Studies

The morphology of the bare HAp and CBD-Epi-modified nanoparticles was characterized using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). SEM images are shown in the left column of Figure 1a and b, while the corresponding TEM images are presented in the right column. Figure 1a presents cHAp at a scale of 100 nm. The material exhibits needle-like agglomerates that are uniformly distributed in the whole volume of the material, sized in diameter ~12 nm ±2 nm and length above 50 nm. The enlarged insets clearly display the needle-like shape of single particles. Similar to bare HAp, the morphology of cHAp_CBD_Epi hybrid remains similar. Importantly, conjugate retains a similar shape and size with no organic clusters formation, what confirms uniform morphology and excludes the separate organic phase and/or aggregates formation in the material. As the cHAp has an irregular shape, the nitrogen adsorption–desorption isotherms were measured to determine the porosity of the bare cHAp. As can be seen in Figure 1, the recorded isotherm exhibits a typical type IV shape, which is characteristic of mesoporous materials, similarly to the HAp obtained in a similar way in previous studies.³⁹ A gradual increase in the adsorbed volume is observed at low relative pressures at about p/p₀ < 0.8). That indicates the monolayer–multilayer adsorption on the surfaces within the pores. At higher relative pressures at p/p₀ ≈ 0.8–1.0, the increased N₂ adsorption is recorded due to its condensation in the pores. The presence of a well-defined hysteresis loop is characteristic to the mesoporous materials, while the surface area is about 74 m² g⁻¹ that is comparable with the data presented in literature.^40,41 Obtained results confirm that the cHAp offers a potential applications for adsorption-based processes. The following Figure 1d displays the uniform pore size distribution with dominant mesopores in the cHAp, calculated using the BJH (Barrett–Joyner–Halenda) method. The sharp peak at ~12 nm indicates the dominant distribution of these mesopores to the total pore area, where such peak is followed by a secondary distribution of larger mesopores sized 20–50 nm. As the material is measured in the dry form, the different pores size relates to the agglomerates formation in the dried powder. The results are in the good agreement with the morphology studies using SEM and TEM, where the pores between the particular grains are formed.

Figure 1.

SEM (left column) and TEM (right column) images of (a) cHAp, (b) cHAp_CBD_Epi, (c) BET isotherm, and (d) pore characteristics for bare cHAp.

FT-IR Spectroscopy

FTIR spectroscopy was used to confirm the presence of organic molecules in the hybrid conjugates. Spectra of pristine CBD, epirubicin, cHAp, cHAp_CBD, and cHAp_CBD_Epi are presented in Figure 2. The spectra of cHAp_CBD and cHAp_CBD_Epi were found to be highly similar. The FTIR spectra of pristine HAp exhibit intense bands between 500–600 cm⁻¹ and 1000–1200 cm⁻¹, which are characteristic of the vibrations of the PO₄^2- group^42,43. The band at about 1620 cm^–1 that appears in CBD and CBD_Epi modified HAp can be ascribed to the bending vibrations of water and/or vibrations of C=C in the aromatic ring,⁴⁴ while the broad band centered at about 3290 cm^–1 is characteristic to intra-layer hydrogen bonds and the stretching of O–H.^45,46 The bands around 2962 and 2267 cm^–1 and minor bands at 2930 and 2837 cm⁻¹ can be ascribed to –CH₃ and –CH₂ vibrations in cannabidiol structure,^47–50 where the ratio between both bands slightly changes in cHAp_CBD_Epi, for the presence of –CH₃ group in the drug molecule.^44,51,52 Following bands at 1585 and 1546 cm^–1 can be ascribed to the C=C vibrations in the aromatic ring, while the band around 891 cm^–1 comes from C–H vibrations in the aromatic ring. Despite the dominant signals from the HAp, the presence of CBD and Epi is clearly seen in the modified nanoparticles.

Figure 2.

FT–IR spectrum for CBD, epirubicin, cHAp, cHAp_CBD, and cHap_CBD_Epi.

Quantitative Analysis of Active Substances in the Conjugate

Cannabidiol - HPLC Technique

The obtained conjugate was characterized qualitatively and quantitatively using HPLC coupled with UV–Vis detection. Comparison of chromatograms recorded before and after adsorption enabled identification of the detected cannabinoids and quantification of cannabidiol (CBD) adsorption. As shown in Figure 3, the chromatogram of the initial CBD solution contained three peaks. The major peak at a retention time of 9.73 min was assigned to CBD, whereas a minor peak at 5.96 min corresponded to CBDV (cannabidivarin), a trace impurity present in the CBD isolate. An additional low-intensity peak at 1.35 min remained unidentified.

Figure 3.

HPLC-UV chromatogram of supernatant: before and after adsorption.

Following adsorption onto colloidal hydroxyapatite, the chromatogram of the supernatant exhibited the same peaks, but with a markedly reduced CBD signal, confirming successful adsorption. Based on the decrease in peak intensity, the amount of adsorbed CBD was calculated to be 0.124 ± 0.08 mg per 1 mg of cHAp, corresponding to 12.4 ±0,8% surface coverage. This loading capacity is comparable to that reported for lipid nanoparticle-based cannabinoid delivery systems.⁵³

Cannabidiol- Differential Scanning Calorimetry (DSC)

To qualitatively and quantitatively confirm the presence of the cannabinoid within the hydroxyapatite structure, Differential Scanning Calorimetry (DSC) was employed. In the initial stage, a measurement was conducted for pure cannabidiol (CBD); the resulting thermogram revealed a distinct endothermic peak at 67.1 °C, corresponding to the melting point of CBD. Figure 4 (left panel) also presents the thermogram of the cHAp_CBD composite. In the case of hydroxyapatite modified with CBD, a minor peak is observed at approximately 57.1 °C. Although this signal is shifted relative to the peak of pure CBD, it confirms the presence of the cannabinoid either within the pores or on the surface of the hydroxyapatite, thereby indicating successful loading of the carrier. Furthermore, quantitative analysis of cannabidiol (CBD) was conducted using the recorded thermograms. By integrating the area under the endothermic peak corresponding to pure CBD, its melting enthalpy was determined to be 20.1 kJ mol⁻¹. Applying the same method to the thermogram of the CBD-loaded hydroxyapatite sample allowed for the estimation of the cannabinoid content in or on the carrier, which was found to be 10.7± 0.5%. However, this value differs by approximately 15% from that obtained via high-performance liquid chromatography (HPLC) analysis. This discrepancy may be attributed to the partial entrapment of CBD within the deeper pores of the hydroxyapatite matrix, potentially limiting its thermal detection.

Figure 4.

Heat flow as a function of time for cHAp_CBD and CBD.

Drug Conjugation and Release

The amount of epirubicin contained in the conjugate was estimated using UV-vis spectrometry. Absorption spectra were recorded before and after the equilibrium of the adsorption process of the drug onto hydroxyapatite was reached, which enabled the determination of the epirubicin content in the hybrid. As a result of adsorption, a decrease in absorbance was observed, with the absorbance maximum for epirubicin occurring in the range of 470–480 nm, see Figure 5a).

Figure 5.

(a) UV-vis spectra of the epirubicin solution before and after adsorption onto the carrier, (b) DPV curves recorded in different times of Epi release from the carrier.

A calibration curve was prepared, presenting the relationship between absorbance values and several standard concentrations of epirubicin. Using the equation of the obtained regression line and the difference in recorded absorbance values, the amount of the compound in the conjugate was calculated. It was determined to be 0.0605±0,003 mg per 1 mg of cHAp. Due to the fact that epirubicin adsorption occurs only on the cHAp surface, much less of it is bound than in the case of classical carriers with epirubicin alone.⁵⁴

Next, the release of the drug was investigated electrochemically using the differential pulse voltammetry (DPV) method. The measurements were performed using a glassy carbon (GC) electrode (2 mm diameter) as the working electrode, with Ag|AgCl|KCl (saturated) and a Pt wire serving as the reference and counter electrodes, respectively. DPV curves were recorded in the potential range from 0.0 to −0.9 V using a modulation amplitude of approximately 25 mV and a potential step of approximately 4.95 mV. Briefly, 56 mg of cHAp_CBD_Epi was suspended in 1.2 mL of PBS (pH 7), incubated for 30 min, and centrifuged at 300 rpm for 2 min to separate the particles from the supernatant containing the released Epi. The collected supernatant was purged with nitrogen for 10 min to remove dissolved oxygen prior to DPV measurements. This procedure was repeated at successive time points to monitor epirubicin release. As shown in Figure 5b, epirubicin exhibited a cathodic peak at approximately −0.72 V, consistent with previous reports obtained using carbon ink-printed electrodes.⁵⁵ The peak current gradually increased with incubation time, indicating continuous release of epirubicin from the cHAp_CBD_Epi conjugate. After 24 h, the concentration of released epirubicin reached approximately 2 μg/mL, corresponding to the concentration range used in the in vitro cytotoxicity experiments (0.5–3 μg/mL). Notably, the cHAp_CBD_Epi conjugate exhibited significantly greater cytotoxicity than free epirubicin, while combination analysis demonstrated a synergistic interaction between epirubicin and cannabidiol. These findings suggest that the amount of epirubicin released within 24 h was sufficient to contribute to the enhanced biological activity observed for the conjugate.

Turbidimetric Analysis

The stability of aqueous suspensions of hydroxyapatite (cHAp) and its nanocomposites was evaluated using the multiple light scattering technique with the Turbiscan Lab instrument. This method, based on measuring changes in the intensity of backscattered (BS) and transmitted light (LT) along the sample height, enables precise identification of destabilization mechanisms in colloidal systems, such as flocculation, aggregation, or sedimentation.^56–59 The results are presented in Figure 6A and B illustrating the temporal evolution of ΔLT and ΔBS for a 1.5 mg·mL⁻¹ cHAp suspension, clearly demonstrate that sedimentation was the predominant destabilization process. The observed increase in transmitted light (ΔLT) in the upper region of the sample (40–54 mm) indicates clarification of the suspension, ie, the settling of particles and the formation of a transparent supernatant. Concurrently, an increase in backscattered light intensity (ΔBS) in the lower part of the sample reflects the accumulation of settling particles. The absence of significant changes in ΔBS within the central measurement zone confirms that no aggregation or agglomeration occurred, and particles retained their individuality throughout the sedimentation process. Variations in ΔBS and ΔLT detected in the upper section correspond to the lowering of the suspension phase boundary and the emergence of a clear liquid layer.

Figure 6.

(A) ΔLT% and (B) ΔBS for bare cHAp, and (C) TSI change in function of time for cHAp_CBD_Epi.

The stability analysis of the functionalized systems revealed the same destabilization mechanism for the aqueous dispersions. The cHAp_CBD system exhibited the lowest stability, whereas the cHAp_CBD_Epi system demonstrated excellent stability, with minimal signal changes across the entire sample height, indicating durable dispersion over time. The Turbiscan Stability Index (TSI), calculated over the 40–54 mm range, ie, the upper part of the sample where destabilization effects emerge most rapidly, quantitatively confirmed these observations (Figure 6C). The lowest TSI values were recorded for the cHAp_CBD_Epi system, reflecting its superior colloidal stability. Conversely, the cHAp_CBD system showed the highest TSI values, while pure cHAp exhibited an intermediate level of stability. The observed differences in behavior among the systems stem from the surface functionalization of hydroxyapatite. Initially, cHAp particles were coated with cannabidiol (CBD), which contains hydrophobic terpenoid fragments and phenolic groups. The reduced surface hydrophilicity and limited ability to form favorable interactions with water likely contributed to decreased stability and promoted coagulation and rapid sedimentation. Although CBD possesses biological activity, it lacks functional groups capable of effective electrostatic or steric stabilization in aqueous media. In the subsequent stage, the cHAp_CBD nanoparticles were further functionalized with epirubicin a hydrophilic anthracycline drug bearing numerous polar groups (–OH, –NH₂, –C=O). These groups facilitated hydrogen bonding with water molecules and introduced additional surface charge, enhancing the electrostatic stability of the nanoparticles. Moreover, the epirubicin layer likely acted as a steric barrier, limiting particle approach and coagulation.

In summary, cannabidiol functionalization of cHAp leads to a reduced suspension stability, whereas subsequent epirubicin coating significantly improves colloidal properties. The cHAp_CBD_Epi system forms a stable dispersion, which is particularly relevant for subsequent biological studies, such as in vitro cytotoxicity assays and investigations of interactions with biomimetic Langmuir layers. The high stability of the epirubicin-containing suspension allows.

Langmuir Trough

The Langmuir technique is widely used to study biomimetic lipid monolayers at the air–water interface, providing a useful model for investigating molecular interactions relevant to biological membranes.

Lipids and Cannabidiol (CBD) in Chloroform Solution

The first important stage of the experiments was to study the interaction between lipids and pure CBD. Due to its hydrophobic nature, it was not possible to add CBD directly to the subphase. This issue was addressed by dissolving both CBD and lipids in a chloroform solution, maintaining a controlled molar ratio of the components and applying such solution onto the Surface of aqueous subphase. The experiments were carried out using lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). DOPC is widely used as a model for healthy cell membranes, whereas DOPS serves as a model for cancerous cell membranes. Employing these distinct lipids in compositions mimicking the healthy and cancerous membrane enables a systematic examination of the structural differences between normal and malignant membrane systems upon the action of CBD.

Measurements performed for pure DOPC lipid, without the addition of CBD, revealed that the resulting layer exhibits a molecular surface area of approximately 55 Ų and a compressibility modulus (Cs⁻¹) of about 116 mN m⁻¹, indicating that the formed membrane is in a liquid-condensed state (at П=30mN/m).

The second lipid, DOPS, shows a comparable surface area of about 54 Ų and a slightly lower compressibility modulus of 105 mN m⁻¹. These values indicate that the molecules forming such monolayers display a high degree of molecular order and organization.

Even a small addition of CBD, corresponding to a mole fraction of 0.1, causes the surface area per molecule (Apm) to decrease by roughly 15%. As the CBD content in the lipid mixture increases, the surface area per molecule decreases irregularly, and the shape of the resulting isotherm changes accordingly.

For DOPC, when the molar fraction of cannabidiol (CBD) exceeds that of the phospholipid, a distinct inflection point appears on the isotherm, characteristic of a phase transition. This situation is not observable for the DOPS lipid.

For both lipids, increasing the CBD content leads to a gradual decrease in the compressibility modulus (Cs⁻¹), reflecting a loss of film rigidity. At a CBD mole fraction of 0.5, the monolayer exhibits features of a liquid-expanded phase, characterized by a reduced degree of molecular packing and lateral cohesion compared with the liquid-condensed phase.

A CBD mole fraction of 0.7 represents the upper threshold for stable monolayer formation; further increases in CBD content result in monolayer destabilization, even at low surface pressures. These observations indicate that the incorporation of CBD disrupts the lateral organization of the lipid matrix. This is consistent with the physicochemical nature of cannabidiol—a strongly hydrophobic compound incapable of forming stable monolayers on its own and exhibiting limited affinity for zwitterionic phospholipids. Notably, CBD promotes monolayer fluidization even at relatively low molar concentrations, underscoring its pronounced perturbative effect on membrane organization.

Conjugate and Its Components in Subphase

As demonstrated by the experimental results presented in Figure 7, CBD has a significant effect on the formation of lipid monolayers. Therefore, further studies were conducted to investigate its interaction with the drug carrier — colloidal hydroxyapatite (cHAp) in lipid monolayers. A series of experiments was performed to determine the impact of the conjugate composed of hydroxyapatite, cannabidiol, and epirubicin (cHap_CBD_Epi), as well as its individual components, on the formation of lipid monolayers.

Figure 7.

Langmuir isotherm (left panels) and compressibility modulus (right panels) for lipids DOPC (A) and DOPS (B) with increasing concentrations of CBD.

For each experiment, two concentrations of the carrier or conjugate (cHAp, cHAp_CBD, and cHAp_CBD_Epi) were tested: 5 mg L⁻¹ (marked as C1 in Figure 8) and 10 mg L⁻¹ (marked as C2). These samples were added to the subphase, which consisted of ultrapure water.

Figure 8.

Langmuir isotherm (left panel) and compressibility modulus (right panel) for lipid DOPC. For clarity, the isotherm for HAp C₁ was not shown.

In the studies, pure DOPC lipid was used, as well as a lipid mixture containing DOPC, DOPE, DOPS, and cholesterol, which was designed to accurately reflect the lipid composition and biophysical properties of biological membranes.

The first lipid analyzed was 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Analysis of the obtained surface pressure isotherms showed that the addition of hydroxyapatite (10 mg in subphase) does not significantly affect the organization of the lipid film (Figure 8, black and red curves). Only minor deviations are visible on the recorded curves, with differences in the area occupied by a single lipid molecule not exceeding 3%. This is also confirmed by the dependence of the compressibility modulus on surface pressure. All the obtained data are consistent and indicate the formation of a liquid-condensed phase, in line with observations for pure DOPC. These results suggest that hydroxyapatite does not significantly affect the structure of the lipid monolayer, confirming its biocompatibility.

The introduction of hydroxyapatite with adsorbed cannabidiol (CBD) significantly altered the character of the isotherms. Compared to the reference system (pure aqueous subphase and pure cHAp in subphase), distinct phase transitions were observed, which shifted toward lower surface pressures as the concentration of the composite increased. The higher the concentration of the composite, the stronger its interaction with the lipid monolayer. Notably, at a concentration of 10 mg L⁻¹, the characteristic plateau in the low surface pressure range, typical of DOPC, disappeared. Transitions from the gas phase to the liquid-expanded phase were also visible in the compressibility modulus versus surface pressure plots. The addition of hydroxyapatite with CBD shifted the system from the liquid-condensed phase to the liquid-expanded phase. Based on the experimental data, it can be concluded that the presence of cannabidiol on cHAp decreases the degree of molecular order in the monolayer. This effect should be attributed directly to CBD, as hydroxyapatite alone does not significantly influence the organization of the lipid monolayer.

In the next stage of the study, the effect of a newly designed conjugate composed of hydroxyapatite, cannabidiol, and epirubicin was assessed. At the lowest conjugate concentration (labeled as C1), the isotherm profile was similar to that observed for the system containing hydroxyapatite with CBD (no epi added). In the low surface pressure range, a sharp increase in pressure was noted, attributed to strong hydrophobic interactions between CBD and the amphiphilic lipid molecule. At this concentration of conjugate, a phase transition to the gas phase occurred, which was also reflected by a decrease in the compressibility modulus to 47.2 mN·m⁻¹. For the higher conjugate concentration (C2), a significantly different isotherm profile was recorded—the phase transition was less pronounced, and measurements above 30 mN m⁻¹ were not possible due to monolayer collapse. Furthermore, the experimental procedure was complicated by film instability induced by the use of higher lipid quantities (necessary for monolayer formation and recording of the isotherm), with disruption occurring even under low surface pressure conditions. The data obtained confirm the strong influence of the active components of the conjugate on the organization of DOPC molecules within the monolayer.

In the subsequent step, the interactions of the conjugate and its components with a model cancer cell membrane were investigated. The lipid mixture consisted of DOPC, DOPE, DOPS, and cholesterol in a molar ratio of 0.5:0.2:0.2:0.1. DOPS and cholesterol are characteristic components of cancer cell membranes.⁶⁰ Phosphatidylserine is widely recognized as a cancer marker due to its translocation to the outer leaflet of the cell membrane in cancerous transformations. Cholesterol plays a key structural role — it stabilizes the membrane and regulates its fluidity. One of the typical features of cancer cells is an increased cholesterol content relative to phospholipids, which may affect the passive transport of anthracyclines, as the hydroxyl group of cholesterol can form hydrogen bonds with the positively charged amine group present in drugs.⁶¹

Considering the potential application of the tested compounds in cancer therapy, an analysis was conducted on their effects on biomimetic model membranes containing DOPS and cholesterol. Based on the analysis of the isotherm of the lipid mixture on water, the estimated surface area per molecule (at π = 35 mN m⁻¹) was 45 Ų. The obtained compressibility modulus (142.1 mN m⁻¹) indicates the presence of a liquid-condensed phase. The addition of cholesterol to the lipid mixture caused a slight decrease in the surface area and an increase in the compressibility modulus, indicating the formation of a more ordered, stiffer, and less fluid monolayer—in line with literature reports.⁶²

Figure 9 shows that the addition of hydroxyapatite (cHAp) to the subphase, even at its highest concentration in the subphase, did not cause a surface area change exceeding 4%, similarly to the pure DOPC monolayer. Adsorption of CBD on the cHAp surface resulted in the appearance of a phase transition from the liquid-expanded to the gas phase, observed in the pressure range of 7–9 mN m⁻¹. At higher concentrations of the composite, the disappearance of the plateau at low surface pressures and a rapid increase in pressure indicate interactions leading to film reorganization. The surface areas per molecule were similar to those observed for the pure lipid mixture with cholesterol, but the compressibility modulus values significantly decreased. This decrease was more pronounced at higher CBD concentrations, confirming its influence on the reorganization of monolayers modeling cancer cell membranes.

Figure 9.

Langmuir isotherm (left panel) and compressibility modulus (right panel) for mixture of lipids: DOPC, DOPE, DOPS, and cholesterol in a molar ratio of 0.5:0.2:0.2:0.1. For clarity, the isotherm for HAp C₁ was not shown.

Interesting results were also obtained for the conjugate containing both the drug and the cannabinoid. At both C1 and C2 concentrations, an increase in the surface area per molecule (at 30 mN/m) to approximately 70 Ų and a significant reduction in the compressibility modulus relative to the pure lipid mixture with cholesterol were observed, pointing towards the strong interactions of the designed conjugate with monolayers.

The results indicate that the designed conjugate affects the organization of lipid monolayers in both healthy and cancer cell membrane models, which may suggest a lack of selectivity toward a specific lipid component. Nevertheless, when combined with targeted therapy, this may be a beneficial effect. Cancer cells overexpress cannabinoid receptors, which may lead to greater accumulation of the carrier in diseased regions. Additionally, the reorganization of lipids in the membrane in the presence of the cannabinoid may facilitate drug penetration into the cell, increasing the effectiveness of chemotherapy. These effects were confirmed in in vitro studies that are presented below.

In vitro Cytotoxicity Evaluation

The experimental data showed (Figure 10) that pristine hydroxyapatite exhibited negligible cytotoxicity, with cell viability exceeding 80% after 72 h of exposure, confirming its suitability as a drug delivery vehicle. Similarly, CBD alone displayed limited cytotoxicity at the tested concentrations. In contrast, significant cytotoxic effects were observed for cHAp_CBD, Epi, and cHAp_CBD_Epi, with clear dependence on both dose and exposure duration. The cHAp_CBD formulation induced greater cytotoxicity than either component alone, with cell viability decreasing to approximately 50% at 3 µg mL⁻¹ after 72 h. Notably, the combined cHAp_CBD_Epi conjugate demonstrated markedly enhanced cytotoxicity compared to free EPI. After just 24 h, cell viability was reduced by 15–30% more in the conjugate-treated groups than in those treated with Epi alone, depending on the concentration. This effect was even more pronounced at 72 h, where the conjugate reduced cell viability by over 80% even at the lowest tested concentration. At the highest concentration (3 µg mL⁻¹), viability dropped to just 8%.

Figure 10.

Comparison of the metabolic activity of SKOV-3 cells after treatment with different concentrations of cHAp, cHAp_CBD, Epi, and cHAp_CBD_Epi for 24, 48, and 72 hours. Untreated cells were used as the control. Data are presented as mean ± standard deviation from at least two independent measurements. Statistical significance was analyzed for the following group comparisons: cHAp vs cHAp_CBD, cHAp vs Epi, and cHAp vs cHAp_CBD_Epi. A p-value of p ≤ 0.05 was considered statistically significant (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).

Using CompuSyn, the IC₅₀ value for the conjugate was calculated as 0.04696 µg mL-1, indicating high potency. The CI at this concentration was 0.03077, confirming a strong synergistic interaction between CBD and Epi in the cHAp-based delivery system. The DRI was calculated as 2.2 for CBD, indicating that the required dose to achieve a given effect could be reduced by more than two-fold when combined. In contrast, the DRI for Epi was 0.38, suggesting that Epi dose reduction was less significant but still relevant. Previous studies have reported synergistic effects of anthracyclines in combination with peptides,⁶³ antibiotics,⁶⁴ and other anticancer drugs.⁶⁵ In most cases, the reported synergistic effects are lower than those achieved with the cannabinoid-containing carrier presented herein.

While the present results demonstrate the potential of cHAp as a carrier for the co-delivery of CBD and epirubicin in ovarian cancer therapy, the fate of the carrier after administration to soft tissues remains to be elucidated. Unlike applications in bone tissue engineering, where hydroxyapatite may integrate with the surrounding mineralized matrix, cHAp used in soft tissues is expected to primarily serve as a drug delivery vehicle. Its biodistribution, biodegradation, and clearance pathways require further investigation in appropriate in vivo models before clinical translation can be considered.

It should be noted that the present study was performed at a single pH value (pH 7), corresponding to physiological conditions. However, hydroxyapatite-based drug delivery systems are known to be sensitive to environmental pH, and both drug adsorption and release behavior may vary significantly under acidic or alkaline conditions. In particular, the acidic tumor microenvironment may enhance drug release, while more alkaline conditions may affect surface interactions and colloidal stability. Therefore, pH-dependent behavior represents an important aspect that should be addressed in future studies to fully understand the performance of the proposed system.

Overall, these findings should be interpreted in the context of an in vitro study, and further in vivo investigations as well as pH-dependent studies are necessary to fully evaluate the therapeutic potential and translational relevance of the proposed cHAp-based drug delivery system.

Conclusions

This study demonstrates that colloidal hydroxyapatite (cHAp) can be effectively used as a multifunctional carrier for the co-delivery of natural and chemotherapeutic agents. The successful conjugation of cannabidiol (CBD) and epirubicin to cHAp was confirmed through detailed physicochemical characterization, indicating stable and well-defined composite formation. The incorporation of CBD-modified carriers with the drug resulted in significant shifts in the Π–A isotherms, indicating a loosening of lipid packing within the monolayer and partial disruption of biomimetic membranes.

The interaction studies clearly show a strong affinity of the CBD-modified carriers toward lipid membranes, although no distinct preference for cancerous over healthy membrane models was observed. The observed biological effects in vitro, including reduced ovarian cancer cell viability, are attributed to the efficient delivery of epirubicin and CBD by the cHAp-based carrier system rather than to intrinsic anticancer activity of hydroxyapatite itself.

These findings highlight cHAp as a promising drug delivery platform for the co-delivery of natural and conventional anticancer agents, offering potential advantages for improved therapeutic efficacy and drug–membrane interactions in cancer treatment.

Disclosure

The authors report no conflicts of interest in this work.