Effects of Combined CBGA and Cannabis-derived terpene nanoformulations on TRPV1 Activation: Implications for Enhanced Pain Management

Effects of combined CBGA and cannabis-derived terpene nanoformulations on TRPV1 activation: Implications for enhanced pain management Mazen M. El-Hammadi a,* , Andrea L. Small-Howard b , Mercedes Fern´ andez-Ar´ evalo a , Helen Turner c , Lucía Martín-Banderas a,d,* a Departamento de Farmacia y Tecnología Farmac´ eutica, Facultad de Farmacia, Universidad de Sevilla, c/Prof. García Gonz´ alez, n◦2, 41012 Sevilla, Spain b GB Sciences, Inc. (OTCQB:GBLX), 9205 W. Russell Road, Suite 240 Las Vegas, Nevada 89148, United States c Laboratory of Pharmacology and Analytics, School of Natural Sciences and Mathematics, Chaminade University of Honolulu, Honolulu, HI, United States d Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Seville, Spain ARTICLE INFO Keywords: Cannabigerolic acid Cannabis-based terpenes PLGA polymeric nanoparticles Beta-myrcene Nerolidol Beta-caryophyllene Nanomedicine Chronic pain ABSTRACT Cannabinoids and terpenes, key bioactive components of cannabis, are increasingly studied for their individual and combined contributions to the therapeutic potential of cannabis-based treatments, with ongoing research exploring their distinct and interactive effects. This study aimed to encapsulate cannabigerolic acid (CBGA) in poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (PEG-PLGA NPs) and investigate the effects of combining CBGA NPs with cannabis-derived terpene-loaded NPs (myrcene [MC], nerolidol [NL], and caryophyllene [CPh]) for potential applications in pain management. CBGA NPs (152 nm) and terpene-loaded NPs (233–297 nm) were prepared via nanoprecipitation and emulsion-solvent evaporation, respectively, exhibiting a polydispersity index <0.3 and negative zeta potentials (−23 to −26 mV). Encapsulation efficiency was 98.6 % for CBGA and 13–33 % for terpenes. CBGA release followed a biphasic profile, with ~ 20 % released within 4 h and sustained release over 72 h. In vitro evaluation used HEK293 cells expressing the nociceptive transient receptor potential vanilloid-1 (TRPV1) channel, a key mediator of pain perception. TRPV1 activation was assessed via calcium influx kinetics (Fluo-4 indicator). The EC50 values were 23.8 µg/mL (CBGA NPs), 8.0 µg/mL (MC NPs), 6.7 µg/mL (NL NPs), and 13.3 µg/mL (CPh NPs). Combinatorial treatments of CBGA NPs with terpene NPs at their respective EC50 concentrations revealed significantly enhanced calcium influx compared to individual NPs, with the strongest interaction observed for CBGA/NL and moderate effects for CBGA/MC. Fluorescence imaging further corroborated these findings. These results suggest that combining CBGA NPs with terpeneloaded NPs could potentiate pain-relief efficacy, offering a promising strategy for advanced therapeutic formulations. 1. Introduction Cannabis sativa has long been valued for its diverse medicinal properties. This versatile plant produces more than 500 bioactive compounds, including cannabinoids, terpenes, flavonoids, and other phytochemicals, which collectively shape its complex pharmacological profile. Modern research increasingly highlights its therapeutic potential for managing chronic pain, inflammation, anxiety, and neurological disorders (Russo and Marcu, 2017). The plant’s broad range of bioactive compounds offers a multi-faceted approach to treatment by targeting various pathways and mechanisms within the body (Bridgeman and Abazia, 2017). However, clinical utilization of Cannabis sativa faces challenges, including standardization of formulations, consistency in dosing, and gaps in understanding long-term effects. Further research is critical to harness its full potential for developing safe, effective, and targeted therapies derived from this ancient plant. Cannabinoids, a pharmacologically active class of compounds in Cannabis sativa, modulate calcium signaling through interactions with transient receptor potential (TRP) channels, such as TRPV1 (Jansen et al., 2019; Starkus et al., 2019; De Petrocellis et al., 2011; Muller et al., 2018; Storozhuk and Zholos, 2018), and calcium release-activated calcium (CRAC) channels (Faouzi et al., 2022). The TRPV1 channel, a * Corresponding authors at: Departamento de Farmacia y Tecnología Farmac´ eutica, Facultad de Farmacia, Universidad de Sevilla, c/Prof. García Gonz´ alez, n◦2, 41012 Sevilla, Spain. E-mail addresses: [email protected] (M.M. El-Hammadi), [email protected] (L. Martín-Banderas). Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm https://doi.org/10.1016/j.ijpharm.2025.125766 Received 5 March 2025; Received in revised form 22 May 2025; Accepted 23 May 2025 International Journal of Pharmaceutics 679 (2025) 125766 Available online 24 May 2025 0378-5173/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). major pain modulation target, is expressed in nociceptive neurons and other cell types, where it acts as a sensor for noxious stimuli (e.g., heat, acidic pH) (Storozhuk et al., 2019). Cannabinoid-TRPV1 interactions trigger calcium influx, a key mechanism in pain signal modulation (De Petrocellis et al., 2013). Cannabigerolic acid (CBGA) (Fig. 1E), a non-psychoactive “mother cannabinoid,” serves as the biosynthetic precursor to major cannabinoids such as tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA) (Anderson et al., 2021). Produced in the cannabis plant via enzymatic condensation of geranyl pyrophosphate and olivetolic acid, CBGA is the central intermediate in cannabinoid biosynthesis (ElSohly, 2017). Emerging evidence underscores CBGA’s therapeutic potential, particularly for pain and inflammatory management (Calapai et al., 2022; Ruhaak et al., 2011). Mechanistically, CBGA inhibits store-operated calcium entry (SOCE), downregulates IL-2 production in T cells, and attenuates kidney inflammation (Faouzi et al., 2022; Suzuki et al., 2023). However, the therapeutic potential of CBGA is limited by its poor solubility, stability, and bioavailability (Zirpel et al., 2015). Nanoparticle encapsulation has emerged as a promising strategy to address these limitations, by enhancing CBGA’s pharmacokinetic profile while improving its stability and enabling targeted delivery (Stella et al., 2021; Lazzarotto Rebelatto et al., 2023). In addition, CBGA has been identified as the most potent cannabinoid for suppressing TRPM7 activity, suggesting therapeutic potential for TRPM7-mediated pathologies including cancer, stroke, and kidney disease (Suzuki et al., 2024). The plant’s terpene profile significantly influences cannabinoid activity through the “entourage effect”—a synergistic relationship between cannabinoids and terpenes that potentiates therapeutic efficacy (Anand et al., 2021). Specific terpenes such as β-myrcene (MC), β-caryophyllene (CPh), and nerolidol (NL) (Fig. 1A-A-D) have been shown to enhance analgesic, anti-inflammatory, and anxiolytic effects when combined with cannabinoids, surpassing the effects of individual compounds (Russo, 2011; Ferber et al., 2020; Christensen et al., 2023). For example, Jansen et al. demonstrated that a Cannabis sativa-derived terpene mixture potentiated intracellular calcium influx in TRPV1expressing HEK cells, with MC showing the strongest activity and NL exhibiting moderate effects. Notably, MC-induced calcium influx was TRPV1-dependent, as evidenced by complete inhibition with the TRPV1 antagonist capsazepine. Molecular docking studies suggested MC binds TRPV1 via hydrophobic, non-covalent interactions (Jansen et al., 2019). Our research group recently developed nanofomulations of MC, CPh, and NL encapsulated in poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (PEG-PLGA NPs) and evaluated their efficacy in HEK TRPV1 cells. An in-depth study was conducted on the activity of free and encapsulated terpenes, assessing both individual and combinational effects. Through calcium influx assays (Fluo-4 indicator, 1-hour observation), we demonstrated that the terpene-loaded NPs and their combinations significantly enhanced fluorescence intensity compared to free terpenes, confirming NP-mediated improvement in cellular uptake and bioactivity (Small-Howard et al., 2020; El-Hammadi et al., 2021; ElHammadi et al., 2022). These PLGA-based nanosystems provide distinct advantages for drug delivery, including FDA-approved biocompatibility, tunable biodegradability, and sustained release kinetics (El-Hammadi et al., 2015; El-Hammadi and Arias, 2022; Kumar et al., 2024). The synergistic effects (entourage effect) observed between cannabinoids and terpenes highlight the potential for optimized therapeutic formulations. By exploiting the entourage effect, new treatments can be developed to enhance efficacy while reducing adverse effects (Ferber et al., 2020), offering new opportunities for managing complex conditions including chronic pain, epilepsy, and mental health disorders (Petzke et al., 2022; Leinen et al., 2023; Hoch et al., 2024). Based on this premise, our study evaluated the therapeutic potential of CBGA-loaded PLGA-PEG NPs in combined with terpene-loaded NPs for pain management using in vitro experiments. Three key cannabis-based terpenes were utilized in this work, namely β–myrcene, β–caryophyllene, and nerolidol (Fig. 1). 2. Materials and methods 2.1. Materials MC (ref. W276200), PEG-PLGA (PEG average Mn 2,000, PLGA average Mn 11,500; lactide:glycolide 50:50], Pluronic® F-68, Span® 60, and trehalose were purchased from Sigma-Merck (Germany). CPh (≥80 % purity, food grade) and NL (a mixture of cisand trans-isomers, ≥97 % purity) were provided by GB Sciences (NV, USA). Polyvinyl alcohol (PVA) 72,000 g/mol (polymerization grade 1600) and HPLC-grade solvents (ethyl acetate, and dichloromethane) were obtained from Panreac Química (Spain). All other chemicals were analytical-grade and sourced from Merck (Germany). Ultrapure water was prepared using a Milli-Q Advantage A10 system (Millipore, Spain). 2.2. Preparation of nanoparticles 2.2.1. Preparation CBGA-loaded nanoparticles CBGA-loaded NPs were prepared using a nanoprecipitation technique following our established protocol (Duran-Lobato et al., 2014; Berrocoso et al., 2017). Briefly, CBGA (3.75 mg), PLGA (22.5 mg) and Span® 60 (7.5 mg) were dissolved in 1.5 mL of acetone. The organic phase was then slowly introduced dropwise into 4.5 mL of an aqueous Pluronic® F-68 solution (0.5 % w/v) using a syringe pump at a controlled rate of 5 mL/h under continuous stirring (500 rpm). Acetone was subsequently evaporated over 1 h in a chemical hood at room Fig. 1. Chemical structures of (A) β–myrcene, (B) β–caryophyllene, (C) trans-nerolidol, (D) cis-nerolidol, and (E) cannabigerolic acid (CBGA). M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 2 temperature. The concentrations of Span® 60 and Pluronic® F-68 were selected based on previously optimized conditions developed by our group for cannabinoid nanoencapsulation, ensuring consistent particle size, stability, and encapsulation efficiency across batches (DuranLobato et al., 2014; Berrocoso et al., 2017; Alvarez-Fuentes et al., 2012; Martin-Banderas et al., 2014). Process parameters for nanoprecipitation, including stirring speed, solvent-to-nonsolvent ratio, and injection rate. were chosen based on prior optimizations to achieve minimal polydispersity index (PDI) and consistent particle size. To eliminate unencapsulated compounds and excess surfactant, the resulting suspension was transferred to an Amicon® tube and spun at 4,000 ×g at 12 ◦C. For lyophilization, trehalose (1:5 w/w PLGA-PEG/trehalose) was added to the concentrated NPs, followed by freeze-drying (48 h, −80.0 ±0.5 ◦C and 0.057 mbar; Cryodos freeze-drier, Telstar Industrial S.L., Spain). Post-lyophilization stability was assessed by DLS analysis of reconstituted NPs for size and zeta potential, with measurements performed under identical conditions to pre-lyophilization characterization. 2.2.2. Terpene-loaded nanoparticles Terpene-loaded PEG-PLGA NPs were prepared via emulsion-solvent evaporation (Small-Howard et al., 2020; El-Hammadi et al., 2021). Briefly, 10 mg of terpene (MC, CPh, or NL) and 40 mg of PEG-PLGA were dissolved in 1 mL of ethyl acetate t(organic phase), which was then added dropwise to 5 mL of a 0.5 % (w/v) PVA aqueous solution under high-speed homogenization (Polytron® PT 2500 E, Kinematica AG, Switzerland) for 1 min. The 1:5 organic-to-aqueous phase ratio promoted optimal nanoparticle characteristics by ensuring rapid solvent diffusion and stable emulsion formation (El-Hammadi et al., 2021). Following solvent removal (rotary evaporation), NPs were purified via ultrafiltration (Amicon® tubes with Ultracel-100 kDa regenerated cellulose membranes, 15 mL capacity) at 4,000 ×g and 12 ◦C. The resulting NPs were resuspended in a trehalose solution (1:5; PLGA-PEG/ trehalose), and lyophilized (−80 ◦C, <0.100 mbar; Cryodos freezedrier, Telstar Industrial S.L., Spain) to obtain a white, cotton-like powder. 2.3. Nanoparticle characterization Particle size distribution and zeta potential were determined by dynamic light scattering (DLS) and laser Doppler electrophoresis, respectively, using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). For morphological analysis, nanoparticle suspensions were applied to carbon-coated copper grids, stained with 2 % uranyl acetate, and airdried prior to imaging by transmission electron microscopy (TEM; Zeiss Libra 120, Carl Zeiss Microscopy, Germany). 2.4. Measurement of CBGA load capacity CBGA loading into the PLGA-PEG NPs was determined using a reverse phase-high performance liquid chromatography (RP-HPLC) method, as previously described for cannabinoids with some modifications (Alvarez-Fuentes et al., 2012). The RP-HPLC analysis was performed using a Hitachi LaChrom Elite® HPLC System, equipped with a quaternary pump L-2130, a diode array detector L-2455, and an automatic injector L-2200. A C18 Waters Atlantis T3 column (3 µm, 4.6 × 100 mm) maintained at 25.0 ±0.1 ◦C (L-2350 column oven, Elite LaChrom1) was used in this analysis. The mobile phase was methanol: acetonitrile:water (52:30:18 v/v) at pH 4.5. The mobile phase was maintained at 1.8 mL/min flow rate. The detection wavelength was 227 nm and the injection volume was 20 µL. CBGA standard solutions (33.75–500 µg/mL in methanol) yielded a sharp, symmetric peak (retention time: 3.7 ±0.1 min) within an 8-min run time. For analysis, NPs were dissolved in acetonitrile, and data were processed using EZChrom Elite Software. Encapsulation efficiency (EE%) and drug loading (DL%) were calculated using Eqs. (1) and (2): EE(%) = massofincorporatedCBGA(mg) initialmassofCBGA(mg)×100 (1) DL(%) = massofincorporatedCBGA(mg) massofnanocarrier(mg)×100 (2) 2.5. Measurement of terpene load capacity The terpene content in nanoparticles was quantified by gas chromatography-mass spectrometry (GC–MS) using a TRACE GC system coupled with a DELTA V™ mass spectrometer (Thermo Scientific, USA) following established methods (El-Hammadi et al., 2021; El-Hammadi et al., 2022). Lyophilized NPs were dissolved in dichloromethane (DCM) and analyzed using an SPB-1 capillary column (30 m ×0.25 mm ×1.5 µm) with a 15-minute run time, showing retention times of 6.92 min (MC), 5.73 min (CPh), 6.77 min (cis-NL), and 7.16 min (trans-NL). The mass spectrometer operated in electron impact (EI) mode (70 eV) with full scan acquisition (m/z 20–300) and an ion source temperature of 250 ◦C. Quantification employed selected ion monitoring (SIM) of characteristic fragment ions at m/z 69 (MC), 93 (CPh), and 133 (NL). Data were processed using Xcalibur 2.0.7 software (Thermo Scientific, USA), and terpene content was calculated as encapsulation efficiency (EE%) and drug loading (DL%) according to Eqs. (3) and (4). EE(%) = massofterpeneincoporated(mg) initialmassofterpene(mg)×100 (3) DL(%) = massofterpeneincoporated(mg) massofnanoparticles(mg)×100 (4) 2.6. In vitro release of CBGA from nanoparticles The in vitro release profile of CBGA from nanoparticles was evaluated using a dialysis method under sink conditions. CBGA-loaded NPs (1 mL, 1.6 mg/mL CBGA equivalent) were placed in Visking dialysis tubing (12–14 kDa MWCO, Medicell International, UK) and immersed in 4 mL of PBS (pH 7.4 ±0.1) containing 0.5 % (w/v%) Tween 80. The system was incubated at 37.0 ±0.5 ◦C in a thermostatic shaker (500 rpm; Titramax 1000, Heidolph, Germany), with free (non-encapsulated) CBGA solution, treated under the same conditions as the nanoparticle formulations, serving as control. Aliquots (350 µL) were collected at predetermined intervals (0.5–80 h) and immediately replaced with fresh buffer to maintain constant volume. CBGA concentration in the release medium was quantified by HPLC, with results expressed as cumulative drug release percentage over time. % Cumulative drug release =amount of drug loaded in NPs [mg] − amount of drug remained in NPs [mg] amount of drug loaded in NPs [mg]×100 (5) M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 3 2.7. In vitro experiments 2.7.1. Cell maintenance HEK293 cells stably expressing TRPV1 channels (kindly provided by Dr. Helen Turner, Chaminade University of Honolulu) were cultured in Minimum Essential Medium (MEM; Corning, NY, USA) supplemented with 10 % fetal bovine serum (FBS), 50 U/mL penicillin, 50 µg/mL streptomycin (Sigma-Aldrich), and 0.6 mg/mL geneticin (Gibco, Switzerland). Cells were maintained at 37 ◦C with 5 % CO 2 humidified atmosphere. 2.7.2. Reconstitution and preparation of nanoparticles for in vitro assays For biological testing, lyophilized NPs were aseptically reconstituted in sterile phosphate-buffered saline (PBS) to their original volume using a laminar flow hood. The resulting nanoparticle suspensions were then diluted in either assay buffer or complete culture medium to achieve the desired treatment concentrations immediately prior to experimentation. 2.7.3. Determination of EC50 of CBGAand terpene-loaded nanoparticles The half-maximal effective concentration (EC50), defined as the concentration needed to achieve 50 % of the maximum effect, was determined for each NP formulation by evaluating calcium influx in HEK-TRPV1 cells. Cells were exposed to serial dilutions of CBGA-, MC-, NL-, or CPh-loaded NPs for 60 min while monitoring intracellular calcium levels using fluorescence. Parallel cytotoxicity assessments were conducted under identical exposure conditions to ensure the selected concentration ranges maintained cell viability >80 %. 2.7.3.1. Calcium signaling assay. The calcium signaling assay can be used to measure the cytosolic concentration of calcium ions. In this method, the non-fluorescent Fluo-4 acetoxymethyl ester (Fluo-4 AM; Thermo Fisher Scientific) is cleaved by intracellular esterases to release the free, fluorescent calcium indicator Fluo-4. Upon binding to calcium ions, Fluo-4 emits a strong green fluorescence when excited by light at a wavelength of 488 nm, allowing for the detection and visualization of calcium levels within the cell. 2.7.3.1.1. Assay procedure. HEK TRPV1 cells were harvested using trypsin, followed by deactivation with media and centrifugation at 1,000 rpm for 5 min at room temperature. The cells were then counted, washed twice with 1 mM calcium assay buffer (composed of Na Ringer’s solution [140 mM NaCl, 2.8 mM KCl, 2 mM MgCl 2 , 11 mM glucose, 10 mM HEPES], 2 mM probenecid, 1 mM CaCl 2 ; pH 7.4), and collected by centrifugation under the same conditions. The cells were resuspended in a 1 μ M Fluo-4 AM solution (prepared by mixing 1 μ L of 5 mM Fluo-4 AM with 1 μ L of 20 % Pluronic F-127 in DMSO and the addition of 5 mL of 1 mM calcium assay buffer, followed by incubation for 10 min at 37 ◦C in the dark). The suspended cells were then further incubated for 30 min at 37 ◦C in the dark. Following incubation, the cells were washed twice with the calcium assay buffer, resuspended in the buffer, and seeded into opaque-walled 96-well plates at a density of 15 ×10 4 cells per 180 μ L/ well. Fluorescence was measured using a plate reader (Synergy HTX, BioTek, USA) with excitation and emission wavelengths of 485 nm and 528 nm, respectively. After establishing a baseline (three measurements), 20 μ L of the stimulant solution/suspension (free CBGA, CBGAloaded NPs, terpene-loaded NPs, or controls) was added to each well, and measurements continued for 1 h at a rate of one read every 26 sec. Control responses were subtracted, and the resulting data were averaged to generate calcium signal profiles. The area under the curve (AUC) for each concentration of CBGA/terpene NPs was also calculated for further analysis. 2.7.3.1.2. Preparation of stimulant dispersions. All NP dispersions were prepared in 1 mM Ca Assay Buffer immediately prior to experimentation. The NPs were diluted directly using the assay buffer, and blank NPs were also prepared at equivalent concentrations. CBGAand terpene-loaded NPs were prepared at concentrations of 10, 40, 100, 200, 400, and 1000 μ g/mL, yielding final well concentrations of 1, 4, 10, 20, 40, and 100 μ g/mL after 1:10 dilution in cell suspensions. Ionomycin (4 μ M), a calcium ionophore that increases the intracellular concentration of Ca 2+ , was used as a positive control. All nanoparticle suspensions were vortexed (30 sec) and sonicated (5 min, 37 ◦C) immediately before use to ensure monodispersion. 2.7.3.2. In vitro cytotoxicity. To determine the effective and non-toxic concentrations of the NPs, the cytotoxicity of the CBGA and terpeneloaded NPs was assessed using the MTT assay (3-(4,5-dimethylthiazol2-yl)-3,5-diphenyl tetrazolium bromide) after a short 60 min incubation period. As demonstrated in our prior work (El-Hammadi et al., 2022), blank NPs showed no cytotoxicity (cell viability >95 % at all tested concentrations) and were consequently excluded from this assay. Cells were seeded in 96-well plates at a density of 45 ×10 3 cells per well and cultured for 48 h at 37 ◦C in a 5 % CO 2 atmosphere. The cells were then treated with various concentrations of CBGAor terpene-loaded NPs (1, 4, 10, 20, 40, 100 µg/mL; corresponding to the concentration of encapsulated compound) in culture medium. After 60 min of incubation at 37.0 ±0.5 ◦C, the medium was removed and replaced with 200 μ L of MTT solution (0.5 mg/mL in culture medium). Following a 4-hour incubation at the same temperature, the medium was discarded, and 200 μ L of DMSO was added to each well to solubilize the formazan crystals. Untreated cells and cells treated with 1 % Triton X-100 were used as controls. The optical density (OD) at 570 nm was measured using a microplate reader (Synergy HT, BioTek Instruments, Inc., Vermont, USA). Relative cell viability (%) was computed using the following formula: Relative cell viability(%) = OD treated cells OD control (untreated)cells ×100 (6) Concentrations resulting in a viability decrease below 80 % were considered cytotoxic adhering to ISO 10993-5:2009 standards. 2.7.3.3. Calculation of EC50. The EC50 for CBGA and terpene-loaded NPs were determined based on the AUC values, calculated from fluorescence responses measured over 60 min in HEK cells incubated with various concentrations of the NPs under investigation. EC50 values were derived from nonlinear regression of log-transformed concentration–response data (GraphPad Prism 8), with curve fits constrained to baseline (0 %) and maximal response (100 % ionomycin control). 2.7.4. Investigating combined effects of CBGA NPs with terpene NPs To evaluate the combined effects of CBGA NPs and terpene NPs on calcium influx in HEK TRPV1 cells, a calcium signaling assay was conducted, followed by fluorescence imaging and analysis 30 min after treatment. 2.7.4.1. Calcium signaling assay. This calcium signaling assay was employed to investigate the impact of combining CBGA NPs with terpene NPs on the calcium influx in HEK TRPV1 cells. 2.7.4.1.1. Assay procedure. The procedure outlined in Section 2.7.3.1.1 was followed, with the AUC calculated for each plot corresponding to the specific treatments. 2.7.4.1.2. Preparation of stimulant dispersions. CBGA, MC, NL, and CPh NPs, along with combinations of CBGA NPs with either MC, NL, or CPh NPs, were dispersed in 1 mM calcium assay buffer. Nanoparticle dispersions were prepared at 10 times their EC50 concentration to achieve the desired EC50 concentration upon addition to the cells. In combination formulations, the final concentrations of both CBGA NPs and terpene NPs were adjusted to match their respective EC50 values. Additionally, blank NPs were prepared at corresponding concentrations. A four μ M ionomycin solution was used as a positive control. M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 4 2.7.4.2. Fluorescence imaging – Image acquiring and analysis 2.7.4.2.1. Assay procedure. The ability of the nanoformulations to modulate calcium influx in HEK TRPV1 cells was further assessed by measuring intercellular fluorescence intensity using Fluo-4 AM under a fluorescence microscope. In this set of experiments, HEK TRPV1 cells were seeded in 96-well cell culture plates a density of 3 ×10 4 per well in 200 µL of cell suspension. The plates were incubated for 48 h at 37 ◦C a humidified atmosphere with 5 % CO 2 . On the day of treatment, the cells were washed twice with 1 mM Ca Assay Buffer. Fluo-4 AM (1 μ M) was added to each well (100 μ L per well), and the plates were incubated for 30 min at 37 ◦C in the dark. After incubation, the cells were washed twice with 1 mM Ca Assay Buffer, followed by the addition of 180 μ L of 1 mM Ca Assay Buffer per well prior to treatment. 2.7.4.2.2. Preparation of stimulant solutions/dispersions. Stimulant dispersions were prepared following the same procedure described in Section 2.7.3.1.2. 2.7.4.2.3. Treatment and imaging. A volume of 20 μ L of the previously prepared stimulants or controls was added to each well, bringing the total volume to 200 μ L per well (20 μ L stimulant/control +180 μ L buffer). The final concentrations of CBGA and terpene-loaded NPs, both individually and in combination, were adjusted to match the EC50 values of each respective nanoparticle formulation. For example, in the CBGA/MC combination, CBGA NPs and MC NPs were applied at their respective EC50 concentrations during incubation with the cells. The intensity of intracellular green fluorescence was observed using Nikon inverted microscope Eclipse Ti (Japan). Live-cell images were captured 30 min post-treatment with the following exposure settings: 1second exposure time, 7.6 ×gain, and 470 nm emission wavelength, using a FITC filter. 2.7.4.2.4. Image analysis. The acquired images were analyzed using ImageJ software (Version 1.52r; NIH, USA). The fluorescence intensity was calculated using the following equation: cellfluoresence =IT− (AT×IB AB) N(7) where: I T , total intensity of the image; A T : total area of the image; I B , background intensity; A B , background area; and N, number of cells. Subsequently, the relative fluorescence intensity was expressed as a percentage using the following equation: I%=ITreat −INegC IIono −INegC ×100 (8) where: I%, relative intensity percentage; I Treat , fluorescence intensity of treated cells; I Iono , fluorescence intensity of cells treated with ionomycin; and I Neg C , fluorescence intensity of the negative control. 2.8. Statistical analysis All data are presented as the mean ±standard deviation (SD). All experiments were conducted in at least three independent assays. Group comparisons were performed using one-way ANOVA with LSD post hoc testing. Data analysis was conducted using SPSS Statistics version 26. Statistical significance was set at p <0.05 for all analyses. 3. Results 3.1. Nanoparticle characterization The physicochemical characterization of nanoparticles revealed well-defined formulations suitable for biological evaluation (Table 1). CBGA-loaded NPs displayed a mean hydrodynamic diameter of 152 nm, while terpene-loaded NPs (MC, NL, CPh) showed slightly larger sizes of 233 nm, 279 nm, and 297 nm, respectively. All formulations exhibited excellent monodispersity (PdI <0.3) and negative zeta potentials ranging from −23 to −25 mV, indicating stable colloidal suspensions. Encapsulation efficiency was markedly higher for CBGA (98.6 ±0.9 %) compared to terpenes (13.2 ±1.2 % for MC, 28.4 ±2.2 % for NL, and 32.9 ±2.3 % for CPh). Following lyophilization and reconstitution, NPs maintained their original characteristics with less than 10 % variation in size, PdI, and zeta potential, confirming the stability of the formulations under processing conditions. Transmission electron microscopy further verified the absence of particle aggregation post-lyophilization (Fig. 2). 3.2. CBGA release kinetics The in vitro release of CBGA from the PLGA-PEG NPs was assessed in PBS (pH 7.4 ±0.1) containing 0.5 % (w/v%) Tween® 80 at 37.0 ± 0.5 ◦C. The CBGA NPs exhibited biphasic release kinetics, characterized by an initial burst release phase (~20 % cumulative release within 4 h) followed by sustained drug release over 72 h (Fig. 3). In contrast, free CBGA (control) rapidly diffused through the dialysis membrane, reaching equilibrium within 2 h. Finally, in vitro release profile was analyzed using KinetDS software Table 1 Characteristics of CBGA-, MC-, NLand CPh-loaded nanoparticles (values are the mean ±SD). Formula Preparation method Mean diameter (nm) PdI Zeta potential (mV) EE% DL% CBGA NPs Nanoprecipitation 152.0 ±8.3 0.262 ±0.018 −25.6 ±1.5 98.6 ±0.9 12.1 ±0.3 MC NPs Emulsion-solvent evaporation 233.4 ±6.4 0.238 ±0.007 −24.2 ±1.1 13.2 ±1.2 3.4 ±0.3 NL NPs Emulsion-solvent evaporation 279.3 ±10.9 0.242 ±0.021 −24.9 ±2.2 28.1 ±2.2 7.0 ±0.5 CPh NPs Emulsion-solvent evaporation 296.9 ±9.7 0.224 ±0.027 −23.4 ±0.9 32.9 ±2.3 7.8 ±0.5 Fig. 2. TEM images of CBGA-loaded PEG-PLGA NPs. Scale bar: 100 nm. M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 5 to identify the dominant release mechanisms. The best linear correlation was obtained when percentage of released drug was fitted to traditional methods, such as the Korsmeyer-Peppas (K-P) and Weibull models, both widely used to describe in vitro drug release kinetics from PLGA-based systems (Table 2). The K–P model describes drug release using a power-law equation, while the Weibull model is often preferred for its greater flexibility in capturing both Fickian and non-Fickian diffusion behaviors. In the present study, the best fit was achieved with the Weibull model (R 2 = 0.984), highlighting its suitability for characterizing the release profile of CBGA from PLGA-based nanoparticles. In contrast, the K–P model showed a lower correlation (R 2 =0.861), reflecting its more limited applicability in complex, biphasic release systems. The superior fit of the Weibull model supports the biphasic release behavior observed experimentally, characterized by an initial burst followed by sustained release. 3.3. Determination of EC50 values The EC50 values for CBGA, MC, NL, and CPh NPs were determined by evaluating a range of NP concentrations and monitoring the calcium signaling/fluorescence response in TRPV1 HEK cells (Fig. 4). The effective concentration range was established by comparing the AUC values of the fluorescence response plots (Fig. 5A). In addition, the MTT cytotoxicity assay was performed to identify the non-cytotoxic concentration range of the developed NPs (Fig. 5B). As a result, the maximum effective concentrations (i.e., concentrations beyond which no statistically significant increase in effect was observed) were determined to be 40 µg/mL for CBGA NPs, 20 µg/mL for MC NPs, 10 µg/mL for NL NPs, and 20 µg/mL for CPh NPs. Next, the EC50 for each NP formulation was calculated from the concentration–response curves generated by plotting normalized AUC values of calcium flux against log-transformed NP concentrations (Fig. 6). The AUCs of the fluorescence response of cells treated with NPs were normalized against the AUC of the ionomycin fluorescence response. The resulting EC50 values are presented in Table 3. 3.4. Investigating the combined effects of CBGA NPs with terpene NPs The calcium responses of HEK TRPV1 cells to co-treatment with CBGA NPs with MC, NL, and CPh NPs were investigated (Fig. 7). Overall, all combinations resulted in a significant increase in calcium influx compared to the effects of the cannabinoid or individual terpenes alone (Fig. 7B). Fig. 7C presents a comparison between the calculated and experimental AUC values of calcium response plots for cells treated with Fig. 3. Cumulative release profile of CBGA from PLGA-PEG NPs. The nanoparticles were enclosed in a dialysis bag, and the release was monitored in phosphate-buffered saline (PBS, pH 7.4) containing 0.5 % (w/v) Tween 80 at 37 ◦C. Free CBGA, subjected to the same conditions, was used as a reference for comparison. Table 2 Correlation coefficients (R 2 ), rate constants (K) and release exponent (A) obtained from fitting release data to Weibull and Korsmeyer-Peppas models. Model Mathematicla equation R 2 A K Weibull y=100⎛ ⎜ ⎝1−e− (t−lag k)A⎞ ⎟ ⎠ 0.984 0.831 10.407 KorsmeyerPeppas ln(y) = a×ln(x) + b; Which is equal to y=K×xa 0.861 0.998 3.052 Fig. 4. Relative fluorescence units (RFU) measured using a calcium signaling assay. HEK TRPV1 cells (15 ×10 4 cells/well, 180 μ l) were pre-treated with 1 μ M Fluo-4 AM and suspended in 1 mM Ca Assay Buffer prior to the addition of stimulants (CBGA NPs or terpene-loaded NPs) at various concentrations. Ionomycin (4 μ M) was used as a positive control. Fluorescence was measured over a 1-hour period, with readings taken every 26 s at an excitation/emission wavelengths of 485/528 nm. (A) Calcium responses induced by free and encapsulated CBGA and (B) corresponding AUCs (p <0.05); (C) calcium responses by CBGA NPs; (D) calcium responses induced by MC NPs; (E) calcium responses induced by NL NPs; and (F) calcium responses induced by CPh NPs. The experiment was conducted in triplicate (n =3), and the calcium response results are presented as the mean values. M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 6 combinations of CBGA NPs and terpene NPs. The calculated values were obtained by summing the individual AUCs of CBGA NPs and terpene NPs presented in Fig. 6B. The combination of CBGA/NL NPs demonstrated a significantly higher experimental value compared to the calculated value, whereas no statistically significant differences were observed for the CBGA/MC and CBGA/NL combinational formulations. 3.5. Fluorescence imaging – Image acquisition and analysis Fig. 8 shows fluorescence images of HEK TRPV1 cells acquired 30 min after treatment with (Fig. 8A) assay buffer, (Fig. 8B) blank NPs, (Fig. 8C) ionomycin, terpene-loaded NPs (Fig. 8D-F), CBGA NPs (Fig. 8G), and combinations of CBGA NPs with terpene-loaded NPs (Fig. 8H-J). Fluorescence intensity from these images was quantified and expressed as a relative percentage by comparing the fluorescence of cells treated with NPs to that of cells treated with the positive control, ionomycin (Equation (8)). Overall, at the tested concentrations (EC50), all combinations of CBGA NPs with terpene-loaded NPs showed significantly higher fluorescence intensity compared to individual NPs (p <0.05; Fig. 8K). Furthermore, the experimental relative fluorescence intensities (RFI) of CBGA/terpene combinations were generally higher than calculated values, with statistically significant differences observed for CBGA/NL and CBGA/CPh combinational nanoformulations (p <0.05; Fig. 8L). 4. Discussion This study highlights the potential of nanoparticle-based delivery systems to enhance the bioactivity of cannabinoids and terpenes for targeted therapeutic applications. PEG–PLGA NPs encapsulating CBGA and three cannabis-derived terpenes, including MC, NL, and CPh were successfully formulated and characterized, and their effects were subsequently evaluated in HEK cells overexpressing the TRPV1 receptor. To ensure reproducibility and consistency in nanoparticle formulation, we employed standardized and validated preparation protocols for both CBGAand terpene-loaded NPs, resulting in minimal batch-tobatch variability (<10 %) in size and zeta potential. Consistent drug loading and encapsulation efficiencies were confirmed using HPLC and GC–MS, ensuring accurate dosing across experiments. The lower encapsulation efficiency observed for terpenes, compared to CBGA, is likely attributable to their volatile and liquid nature, which makes them more susceptible to diffusion losses during formulation (El-Hammadi et al., 2021); unlike the more stable, solid-state CBGA. In addition, TRPV1 activation assays were conducted using EC50 concentrations derived from independently validated dose–response curves, enabling Fig. 5. Determination of non-cytotoxic and effective concentrations of CBGAand terpene-loaded NPs. (A) In vitro viability of HEK TRPV1 after a 1-hour exposure to various concentrations of nanoparticles. Untreated cells served as the control for calculating relative cell viability (%). (B) Areas under the curve (AUCs) of fluorescence intensity for HEK TRPV1 cells incubated with different concentrations of NPs, measured over a 1-hour period (corresponding to the plots in Fig. 3). Data are represented as means ±SD from triplicate cultures (n =6). Fig. 6. Area under the curve (AUC) of fluorescence responses in HEK cells when incubated with varying concentrations of CBGAand terpene-loaded NPs for 1 h. Results were normalized to the fluorescence responses of cells treated with ionomycin. Data are presented as means ±SD from triplicate experiments. Table 3 Half-maximal effective concentration (EC50) values calculated for CBGA, MC, NL, and CPh NPs. Data are represented as means ±SD from triplicate cultures. Formula EC50 Corresponding response (%AUC normalized to ionomycin)µg/mL µM CBGA NPs 23.8 ±2.4 65.9 ±6.5 45.8 ±2.0 MC NPs 8.0 ±0.6 61.5 ±4.1 14.1 ±1.6 NL NPs 6.7 ±0.9 29.9 ±4.2 21.9 ±5.4 CPh NPs 13.3 ±0.5 65.3 ±2.4 3.3 ±0.7 M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 7 Fig. 7. Fluorescence changes measured using a calcium signaling assay. HEK TRPV1 cells (15 ×10 4 cells/well; 180 μ L) were pre-treated with 1 μ M Fluo-4 AM and suspended in 1 mM Ca Assay Buffer before the addition of stimulants (CBGA NPs, terpene-loaded NPs, or their combination) at a final concentration corresponding to the EC50 for each type of nanoparticle. Ionomycin (4 μ M) was used as a positive control. Fluorescence was monitored over 1 h, with readings taken every 40 s at an excitation/emission wavelength of 485/528 nm. (A) Calcium response plots; (B) corresponding AUCs, and (C) comparisons of calculated and experimental AUC values corresponding to combinations of CBGA NPs and terpene NPs. The experiment was conducted in triplicate (n =3). Calcium responses and AUC results are presented as mean values and mean ±SD, respectively. Statistically significant differences are indicated as follows: * for combination formula vs. CBGA NPs (p < 0.05); †for combination formula vs. respective terpene NPs (p <0.05); # for experimental combination formula vs. calculated combination formula (p <0.05). M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 8 biologically relevant and consistent comparisons. While large-scale production and long-term stability require further development, our current methodology offers a robust and reproducible platform for preclinical applications. The physicochemical properties of the nanoformulations (such as size, surface charge, and composition) play a key role in modulating biological interactions. All NPs exhibited uniform particle sizes within the optimal sub-300 range with a narrow size distribution (PdI <0.3), which are generally favorable for cellular uptake and interaction with membrane-localized receptors like TRPV1. Although uptake is typically more efficient for particles under 200 nm, several studies have demonstrated that nanoparticles approaching 300 nm can also be internalized by cells through alternative endocytic pathways, depending on their surface properties and the cell type involved (L´ opez-Viota et al., 2023; Gratton et al., 2008; Petithory et al., 2021). Nevertheless, significant differences were observed between CBGA and terpene-loaded NPs in Fig. 8. Fluorescence images of HEK TRPV1 cells, pre-incubated with Fluo-4, captured 30 min after the addition of: (A) buffer, (B) blank NPs, (C) ionomycin, (D) CPh NPs, (E) MC NPs, (F) NL NPs, (G) CBGA NPs, (H) CBGA NPs/CPh NPs, (I) CBGA NPs/MC NPs, and (J) CBGA NPs/NL NPs. The concentrations of CBGAand terpeneloaded NPs in both individual and combination formulations were set at the EC50 for each respective nanoparticle. The images shown are representative of three independent experiments. Scale bar: 50 μ m. (K) corresponding Relative fluorescence intensity (RFI) of cell images, and (L) comparisons of calculated and experimental RFI values corresponding to combinations of CBGA NPs and terpene NPs. The experiment was conducted in triplicate (n =3). RFI values are presented as mean ±SD, respectively. Statistically significant differences are indicated as follows: * for combination formula vs. CBGA NPs (p <0.05); †for combination formula vs. respective terpene NPs (p <0.05); # for experimental combination formula vs. calculated combination formula (p <0.05). M.M. El-Hammadi et al. International Journal of Pharmaceutics 679 (2025) 125766 9