Enhanced oral bioavailability and in vitro evaluation of cannabidiol camel milk-derived exosome formulation in resistant MDA-MB-231 and MDA-MB-468 breast cancer cells

Abstract

The potential of camel milk-derived exosomes (CMDE) to enhance the bioavailability of Cannabidiol (CBD) was investigated. CBD-CMDE formulation was prepared using an established procedure and its particle size was 138.4 ± 4.37 nm, and CBD entrapment efficiency of 56.56 ± 4.26 %. In-vitro release studies showed release of 78.27 ± 5.37 % and 46.42 ± 4.75 % CBD from CMDE and control CBD formulation respectively in pH 6.8 at 24 hr. The apparent permeability (Papp) of CBD-CMDE was found to be enhanced by 3.95-fold with Papp of 22.9*10⁻⁶ ± 0.34 cm/sec as compared to control CBD formulation with Papp of 5.8*10⁻⁶ ± 0.65 cm/sec in MDCK cells. CBD-CMDE was found to be more potent than CBD in 2D cytotoxicity assay with IC₅₀ values of 3.6 ± 0.54 μM, 3.88 ± 0.54 μM and 7.53 ± 0.59 μM, 7.53 ± 0.59 μM against Doxorubicin (DOX) resistant MDA-MB-231 and Rapamycin (RM) resistant MDA-MB-468 breast cancer cells respectively. Moreover, 3D spheroids assay results demonstrated CBD-CMDE with IC₅₀ values of 14 ± 0.85 μM, 15 ± 0.07 μM as compared to CBD alone with IC₅₀ values of 25 ± 0.93 μM, 34.7 ± 0.08 μM in MDA-MB-231 DOX RT cells and MDA-MB-468 RM RT cells respectively. In-vivo PK studies showed enhanced bioavailability of CBD from CBD-exosomes with AUC_(0–24h) of 1350.56 ± 187.50 h.ng/mL as compared to CBD control formulation with AUC_(0–24h) of 351.95 ± 39.10 h.ng/mL with a single oral dose of 12 mg/kg. The data indicate that CMDE significantly improved the oral bioavailability of CBD. Overall, CMDE can be used to enhance the oral absorption of poorly bioavailable APIs.

1. Introduction

In the United States, breast cancer remains a significant public health concern, claiming the lives of around 42,250 women annually. It is the most common cancer among women, making up 30 % of all new diagnoses, and roughly 1 in 8 women will develop invasive breast cancer in their lifetime (CDC 2024; “Cancer Facts & Figures 2024,” n.d.). The number of instances of breast cancer is predicted to increase globally to 4.4 million by 2070 (Zhang et al. 2023). Even though mortality rates have decreased since the late 1990 s, mostly because of adjuvant systemic medication, early mammography and palpation identification, some breast cancers are still resistant to standard treatments. The quality of life of patients is also significantly impacted by the adverse effects of current medications (Caffarel et al. 2012; Chavda et al. 2023).

Several studies have shown that cannabinoids possess anticancer activities including cell migration inhibition, anti-proliferation, apoptosis and anti-angiogenesis against various cancers including breast, skin, lung, prostate and glioblastomas (Kalvala et al. 2023; Sarsembayeva et al. 2022; Hinz and Ramer 2019; Preet et al. 2011; Peeri and Koltai 2022; Tomko et al. 2020). However, cannabinoids such as CBD have poor bioavailability (13–19 %) when taken orally due to their first pass metabolism, high lipophilicity (log P values of > 5) and low aqueous solubility (2–10 μg/ml in water) (Salau et al. 2022; Millar et al. 2018). As a result, CBD falls under the classification of a Class II drug in the Biopharmaceutics Classification System (BCS), indicating its low water solubility and high permeability (Sitovs et al. 2024; Stasiłowicz-Krzemień, Szulc, and Cielecka-Piontek 2023). Thus, there is a necessity for novel delivery systems to enhance its oral absorption.

Exosomes have attracted a lot of attention lately due to their potential application as drug delivery systems for a variety of diseases including cancer. These exosomes or endogenous nanovesicles (EVs), have been shown to be highly stable and biocompatible drug carriers for hydrophilic (like miRNAs and siRNAs) and hydrophobic drugs (taxol, doxorubicin, cisplatin, CBD, etc) in cancer treatment (N. Patel et al. 2021; Moon and Chang 2022; EL Andaloussi et al. 2013; Fang et al. 2022; Tian et al. 2014; G. Zhou et al. 2022; Gebeyehu et al. 2021). In addition to their remarkable roles in mediating cell–cell communication, numerous studies have reported the use of EVs as drug delivery systems. For instance, Cui et al studied loading of self-assembled micelles formed from tanshinone IIA and glycyrrhizic acid in serum derived EVs (Cui et al. 2023); Degirmenci et al investigated entrapment of lapatinib in EVs obtained from epithelial breast cells (MCF10 A) and Zhao et al explored loading of DTX in M1 macrophage-derived EVs (Zhao et al. 2021). Additionally, other APIs such as paclitaxel (PTX) have also been reported to be delivered using macrophage (Kim et al. 2018) and bovine milk derived EVs (Agrawal et al. 2017, Kaur, Nathani, and Singh 2023). Our laboratory has already demonstrated the successful delivery of CBD-loaded exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) for treating triple-negative breast cancer (TNBC). Further, the effectiveness of CBD EVs in enhancing the sensitivity of doxorubicin (DOX) in both in vitro and in vivo in MDA-MB-231 cells was also demonstrated (N. Patel et al. 2021).

Most biological fluids such as plasma, amniotic and cerebral fluids, urine, saliva, milk, etc. contain exosomes (Fitts et al. 2019; Fontana et al. 2021). The only biological liquid that contains exosomes that is accessible on an industrial scale is milk. One of the most significant signaling molecules that facilitates cellular communication between a mother and her child are milk-derived exosomes (S. Sedykh, Kuleshova, and Nevinsky 2020). Milk exosomes have shown promise as novel therapeutic possibilities for the treatment of several illnesses, including cancer. Nevertheless, the literature has limited information regarding the delivery of physiologically active substances to cells through exosomes (Agrawal et al. 2017). Human (Admyre et al. 2007), bovine (Pieters et al. 2022), porcine (Ting Chen et al. 2014), camel (Yassin et al. 2016), horse (S. E. Sedykh et al. 2017), yak (Gao et al. 2019), sheep (Quan et al. 2020), and goat (Santos-Coquillat et al. 2022) are among the species from which exosomes have been isolated. Exosomes derived from these milk sources have been reported to have anti-inflammatory, antioxidant, anticancer and antimicrobial properties (Rashidi et al. 2022). However, camel milk and its components have been reported to possess anticancer activity against a range of cancer diseases, including human breast cancer (Badawy, El-Magd, and AlSadrah 2018). A study by Badawy et al showed that the progression of breast tumors was significantly slowed down by the free, non-loaded EVs derived from camel milk (Badawy, El-Magd, and AlSadrah 2018). These tiny vesicles play a crucial role in intercellular communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids between cells. Studies suggest that camel milk exosomes can modulate the immune system by enhancing the activity of immune cells like natural killer cells and macrophages, while also possessing anti-inflammatory properties that may curb inflammation associated with cancer progression (Hur et al. 2023; Behrouz et al. 2022; Al-Numair et al. 2022). Furthermore, they have been found to induce apoptosis in cancer cells, inhibit angiogenesis crucial for tumor growth, and serve as effective carriers for delivering therapeutic agents directly to target cells (Rashidi et al. 2022; S. Sedykh, Kuleshova, and Nevinsky 2020). While these findings hold significant potential for cancer treatment, further research is essential to fully elucidate their mechanisms of action and evaluate their clinical efficacy and safety. There are limited reports of use of CMDE as a drug delivery system to deliver APIs orally. Moreover, pharmacokinetic profile of APIs delivered via oral camel milk, or any other source of milk derived exosomes is lacking in the literature.

Cell-permeability studies serve as a cost-effective and high-throughput substitute to in-vivo methods for assessing drug absorption and transporter interactions in the gut. During drug discovery and development, the Caco-2 and Madin-Darby canine kidney epithelial (MDCK) cell lines have been used as in-vitro tools to evaluate permeability and transporter interactions. When grown on semiporous membranes, these cells develop confluent monolayers that mimic the intestinal epithelial barrier for permeability, transport, and drug-interaction studies. These tests are useful in pharmaceutical research for predicting absorption, assessing permeability mechanisms, analyzing formulation impacts on drug permeability, and identifying potential transporter-mediated interactions (Volpe 2011). MDCK cells proliferate and differentiate rapidly, reducing the time needed to carry out in-vitro transport investigations. MDCK permeability experiments are an appealing alternative to Caco-2 cells in terms of boosting throughput and shortening the time necessary to undertake transport investigations of new drugs. In addition to screening and determining passive permeability, MDCK cells can be utilized to investigate drug efflux and active transport (Jin et al. 2014).

Our hypothesis for these studies is that bioavailability of CBD can be enhanced using oral CMDE formulation. The objective of this study was to a) develop and optimize the CBD-CMDE formulation b) its evaluation through in vitro cell permeability and release studies c) in vivo pharmacokinetic investigations conducted in Sprague Dawley rats d) assessing the formulation’s efficacy against DOX-resistant MDA-MB-231 and MDA-MB-468 cells in both 2D and 3D assays in vitro and e) in vivo experiments aimed at demonstrating the uptake of CMDE into tumor using a fluorescent Vybrant^™ DiO labeled CMDE (DiO-CMDE). This study will demonstrate for the first time the potential of CME as oral drug delivery vehicle.

2. Materials and Methods

2.1. Materials

CBD was purchased from Open Book Extracts, NC, USA. Camel milk was procured from Desert Farms, CA, USA. Spectra-Por^® Float-A-Lyzer^® G2, 1 mL was purchased from Avantor, VWR, GA, USA. Kolliphor EL (BASF Pharma, NJ, USA), ethanol (Sigma Aldrich, USA), bovine serum albumin (Genesee Scientific, USA), sucrose (Spectrum, USA), probe sonicator (Sino Sonics, China). MDCK, MDA-MB-231 DOX RT, MDA-MB-468 wt cells were purchased from ATCC (Rockville, MD).

2.2. Animals

BALB/c nude mice were obtained from Envigo (Indianapolis, IN). Sprague Dawley (SD) rats were obtained from Charles River Laboratories in Wilmington, Massachusetts, for use in in-vivo pharmacokinetic (PK) studies. The rats were housed in cages with appropriate bedding with controlled conditions, including a temperature of 22 ± 2 °C, a 12:12 h light–dark cycle, and relative humidity kept at 50 ± 15 %. Following the recommendations in the “Guide for the Care and Use of Laboratory Animals” and the criteria established by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), these rats were housed at Florida A&M University animal facilities. Before commencing any experiments, the rats underwent a one-week acclimatization period in the laboratory conditions. Additionally, Florida A&M University’s Institutional Animal Care and Use Committee (IACUC) approved all animal protocols (022–08, 023–02) used in this study.

2.3. Methods

CMDE Isolation: Differential ultracentrifugation was used to separate the exosomes from camel milk. Fat globules, casein aggregates, and other debris were extracted from the milk sample by centrifuging it at 3000 g for 30 min at 4 °C. The supernatant was then collected and centrifuged at 10,000 g/60 min/4°C. Exosomes were isolated from supernatants by ultracentrifugation at 100,000 g for 120 min at 4 °C. To get rid of big debris and microvesicles, a quick wash with phosphate-buffered saline (PBS) was performed. The exosome pellets were collected, reconstituted in PBS to create a homogenous culture, and then stored at −80 °C until needed again (Vaswani et al. 2019) CBD-CMDE Formulation: Briefly, CBD was solubilized in ethanol at a concentration of 6 mg/mL PBS buffer containing CMDE (particle number of 6.2 * 1011/mL) was then added into it and mixed gently. Subsequently, 10 % Kolliphor EL (solubulizer) was then added and vortexed for 1 min. It was then exposed to 3 cycles of probe sonication (20 % amplitude) of 30 sec each with 2 min resting time between each cycle. The formulation was then incubated at 4 °C overnight on a shaker. Further, the final formulation was then ultracentrifuged at 100,000 g for 120 min to remove the unentrapped drug. The supernatant was discarded, and the pellet was dissolved in PBS and kept at 4 °C overnight on a shaker. This final formulation was used for all the studies”

Control CBD Formulation: Briefly, CBD was solubilized in ethanol. Water was then added into it and mixed gently. Kolliphor EL was then added and vortexed for 1 min. Finally, the formulation was then sonicated in a bath sonicator.

2.4. CMDE Characterization

2.4.1. Entrapment efficiency and nanoparticle tracking analysis (NTA)

For entrapment efficiency, CBD formulation was diluted with methanol (50:50) to extract the CBD. It was then vortexed for 5 min, bath sonicated for 5 min and centrifuged at 14,000 RPM for 10 min. Supernatant was then collected and analyzed using HPLC-MS method. Using NTA and a ZetaView instrument (ZetaView^® TWIN PMX-220) in scatter mode, the average particle size (z-average), concentration, and zeta potential of the exosomes and CBD exosomes formulations were examined using the dynamic light scattering (DLS) approach at 25 °C and a 9°-degree scattering angle to characterize total particles and purity. It was evaluated by staining the exosomes with CD9/CD63/CD81 antibodies that were fluorescently labelled using Particle Metrix’s protocol with Alexa 488 fluorochrome (in fluorescent mode) to characterize the exosome membranous vesicles and the obtained data was then processed by using ZetaView Analysis software. The samples were then diluted using particle free water at 1:1000 dilution and measured in triplicate. The percent purity was measured using the following formula: (N. Patel et al. 2021).

$$\%purity=.\frac{Conc.influorescentmode}{Conc.inscattermode}$$

2.4.2. Western blotting for exosome markers

Exosomes were lysed using the radio-immunoprecipitation assay (RIPA) buffer, which contains 150 mM sodium chloride, 0.5 % sodium deoxycholate, 1.0 % Triton X-100, 50 mM Tris pH 8.0, 0.1 % SDS, and 1X Thermo Scientific Halt Protease Inhibitor Cocktail. The samples were lysed on ice for 20 min, and then they were spun down for 20 min at 14,000 rpm. Following the supernatant’s collection, the protein concentration was determined using the Smith test (Bicinchoninic acid assay). The protein lysate’s concentration was calibrated, and it was denatured in 2X Laemmli Sample buffer for 5 min at 100 °C. Protein was electrophoretically transferred from an SDS PAGE gel to a nitrocellulose membrane at an identical concentration using the Trans-Blot^® TurboTM Transfer System (Bio-Rad, Hercules, CA, USA). After that, the membrane was blocked for an additional hour at room temperature using PBS containing 0.1 % Tween 20 and 3 % w/v BSA. Primary antibodies were diluted 1:1000 in blocking solution and added to the membrane. The membrane was then incubated at 4 °C overnight. After one hour of room temperature incubation with secondary antibodies conjugated with horseradish peroxidase, the blots were washed three times in PBST (PBS containing 0.1 % Tween-20) for five minutes apiece. The blots were incubated using the Super Signal West Pico Chemiluminescent substrate, and pictures were captured using the Chemidoc imaging system (Bio-Rad). Densitometry scanning was used to measure the immunoblots using NIH ImageJ software (version 1.54) (Nathani et al. 2024; Hedrick et al. 2016).

2.4.3. Transmission electron microscopy (TEM)

TEM analysis was done to study the morphology of CMDE and CBD-CMDE. Briefly, exosomes were diluted 1:10 using PBS after freeze thawing from −80 °C. Diluted exosomes were placed on coated copper grid and stained with 1 % v/v uranyl acetate in double distilled water. The stained grids were observed by using a Hitachi HT 7800 TEM (Muller et al. 2014).

2.5. HPLC-MS method

HPLC-MS analysis was conducted with a Waters e2695 separation module, QDa detector, Aquity QDa and Waters 2998 photodiode array detector (PDA) (Waters Technology Corporation, USA). Mobile phase containing 90 % ACN (0.1 % formic acid) and 10 % water (0.1 % formic acid) was used at a flow rate of 0.5 ml/min with injection volume of 20 μL. Column temperature was set to 40 °C. A reverse phase C18 column (Nova-Pak^® 3.5 μm, 3.9 × 150 mm; Waters Technology Corporation, USA) with a guard column (Symmetry^®, reversed phase, C18) was used for the elution of samples. QDa was set to mass range: 250 to 350 Da, positive scan, cone voltage 22 V, capillary voltage (positive: 1.5 kV and Negative: 0.8 kV), SIR with mass 315 Da positive polarity and cone voltage of 22 V.

2.6. Linearity

Linearity was established by analyzing five CBD standard concentration in the range of 12.5, 25, 50, 100 and 200 ng/ml. All the standards were processed using solid phase extraction method using Oasis PRiME HLB 96-well μElution Plate (Waters Technology Corporation, USA) with established protocol by the vendor (“Taking the Complexity out of SPE Method Development,” n.d.). Briefly, CBD standards with rat plasma were first diluted with 4 % phosphoric acid and run in 96-well μElution well plate. Further, wells consisting of CBD standards were washed with 200 μL of methanol: water (5:95) solvent and finally eluted with 100 μL of Acetonitrile (CAN): Methanol (90:10) solvent. Collected eluent were then analyzed using HPLC-MS method. Our results showed that the calibration curve (peak intensity area vs concentration) was linear with a correlation coefficient of 0.99. The retention time was found to be 4.44 min.

2.7. Accuracy

Accuracy study was conducted at three concentration levels (25, 50, 100 ng/ml) of CBD standards and the recovery percentage was calculated. The percent recoveries were found to be 97.49 ± 1.34, 98.68 ± 1.07 and 98.31 ± 1.45 % for 25, 50, 100 ng/ml CBD standards respectively.

2.8. Precision

The method precision was validated through intra-day and inter-day testing of CBD standards (25, 50, 100 ng/ml concentration). The method’s intra-day precision was assessed by conducting six independent assays of CBD test samples against a reference standard on the same day. Furthermore, these assessments were replicated over six consecutive days to ascertain inter-day precision. The percentage of the relative standard deviations (%RSD) of 25, 50, 100 ng/ml CBD standards were found to be below 3.9 % and 4.5 % for intra-day and inter-day, respectively.

2.8.1. Detection and quantitation limits

Limit of detection (LOD) and limit of quantification (LOQ) were calculated using following equations based on the standard deviation of the response and the slope as per the ICH guidelines (Abraham 2010).

LOD = 3.3 * (SD of intercept/slope) and LOQ = 10 * (SD of intercept/slope).

LOD and LOQ for the CBD standards were found to be 5.26 ng/ml and 15.95 ng/ml respectively.

2.9. In-vitro release Study

In-vitro drug release studies were performed by using Spectra-Por^® Float-A-Lyzer^® G2, 1 mL dialysis devices with molecular weight cut off (MWCO) of 12 kDa. In summary, 40 mL of release fluids (1.2 and 6.8 pH buffers) containing 20 % ethanol and 0.5 % Kolliphor EL) were added to the device’s receiving compartment along with 1 mL of each CBD exosomes and control formulation in the donor compartment. The glass tube was closed, and the temperature was kept at 37 ± 0.5 °C and 120 rpm. A one milliliter sample was taken out at certain intervals, replaced with fresh medium to maintain the sink condition, and CBD content was measured using LC-MS. (N. Patel et al. 2021; Kutlehria et al. 2018).

2.10. In-Vitro permeability studies

MDCK cells were grown on EMEM media that had 10 % FBS and 1 % antibiotic (Penicillin Streptomycin Neomycin-PSN) added to it. After 80 % confluency, the cells were trypsinized after washing with PBS. In the apical compartment of a Costar^® Trans well^® permeable support with a polyester membrane (3.0 μm pore size, 6.5 mm diameter inserts), the cells were seeded at a density of 33,000 cells/well in 100 μL cell solution in a 24-well plate format. The plates were incubated at 37 °C and 5 % CO₂ in a humidified room. Using a millicell ERS-2 Epithelial volt-ohm meter (Millipore, Billerica, MA), the transepithelial electrical resistance (teer) was measured to continually monitor the integrity of the monolayer and the establishment of tight junctions. After the Trans-well plate was fully confluent, HBSS was used to wash the apical compartment. Using HBSS, the contents of the basolateral compartment were removed and duly replaced. The plates were let to acclimate at 37 °C for ten minutes. The appropriate drug suspension or formulation (6 mg/mL CBD control and CBD exosomes) was then added to the apical compartment buffer, and the basolateral compartment was then replenished with HBSS buffer to conduct an absorptive permeability study. The plates were continuously shaken while being incubated at 37 °C with 5 % CO₂. At 1, 2, and 24-hour intervals, samples (0.2 mL) were taken from the basolateral compartment and replaced with new buffer. Using the previously mentioned in-house developed HPLC method, the amount of drug in the samples was estimated. The following formula was used to determine the apparent permeability (Nottingham et al. 2020; Kaiser et al. 2015).

$$\text{PAPP}(\text{cm}/\text{s})=\frac{dQ}{\cdot C{i}^{\ast }d{T}^{\ast }A}$$

where dQ = amount of drug in receiving compartment (nmole), Ci = the initial concentration of drug (nmole/mL), T = time (sec), and A = area of insert (cm²).

2.11. Cytotoxicity assay

2.11.1. 2D cytotoxicity assay

A concentration dependent cytotoxicity assay was carried out using MDA-MB-231 doxorubicin resistant cells. The cells were seeded at a density of 7000 cell/well in a 96-well plate and maintained in an incubator at 37 °C and CO₂ (5 %). After 24 h, the media was replaced with experimental media having CBD solution and CBD exosomes and incubated for 48 h. The cells were then treated with MTT (0.5 mg/mL) solution for 3 h post which the formazan crystals formed were dissolved in DMSO. The absorbance was measured using Tecan Infinite 200 PRO M Plex multimode microplate reader at 570 nm (Rarokar et al. 2019).

2.11.2. 3D cytotoxicity assay

3D spheroids were cultured by the established protocols in the laboratory using a magnetic nanoshuttle system (Nathani et al. 2024). Briefly, MDA-MB-231 DOX RT cells were tagged with nanoshuttle (10 μL for 10,000 cells) solution (Greiner-Freickenhausen, Germany) by repeated centrifugation at 800 RPM for 7 min thrice. 15,000 cells/well were seeded in cells repellent surface 96-well plates and the plate was kept on magnetic drive to allow the formation of 3D spheroids. The plate was maintained in an incubator at 37 °C and 5 % CO₂ for 5 days with intermediate media changing on 3rd day. The cells were treated with CBD, CBD exosomes for 48 h. After 48 h the media was replaced with 0.5 mg/mL MTT solution and incubated for 3 h. The crystals formed after 3 h were dissolved in DMSO and the absorbance was measured at 570 nm.

2.12. Internalization of oral DiO labelled CMDE into MDA-MB-321 DOX RT tumor

Vybrant^™ DiO labeling solution (5 μL, 1 mM) was added per mL of exosomes suspension and mixed well by gentle pipetting to dilute it to a final concentration of 5 μM. The mixture was sonicated using 3 sec on/off cycles for 2 min at 20 % amplitude. The mixture was then allowed for incubation for 45–60 min at 37°C followed by ultracentrifugation to isolate Vybrant^™ DiO labelled CMDE (DiO-CMDE). It was aliquoted as required and stored at −80°C and protected from light for further use.

In-vivo experiments were conducted to show uptake of exosomes into tumor cells. All experiments were carried out with approval from the Institutional Animal Care and Use Committee (IACUC Protocol number – 023–02), Florida A&M University, Tallahassee, FL, USA as a pilot study on a small number of mice. We obtained female BALB/c-nu/nu mice from Jackson Labs. MDA-MB-231 cells (4 × 10⁶ cells) were mixed with Matrigel (1:1) and were then subcutaneously injected into the right flank of each mouse. The mice developed solid tumors in 7 days and treatment began as soon as the tumor volume reached 100 mm³. The treatment was initiated 10 days after the tumor cells were implanted. Various treatments included: oral administration of PBS as a negative control, DiO alone, and DiO-CMDE. All mice were sacrificed for additional tissue analysis at 4 and 24 hr after oral administration. Subsequently, a Nikon Eclipse Ti 100 inverted fluorescent microscope (Nikon Instruments, Inc., Melville, NY, USA) was used to directly view the fluorescence of the tumor cryosections (40-μm) of mice that had been sacrificed. The microscope was set to operate with a fluorescent filter at 488 nm (Figarol et al. 2018).

2.13. In-vivo pharmacokinetic (PK) study

The animal experiments performed in this study were approved by Institutional Animal Care and Use Committee (IACUC), Florida A&M University, Tallahassee, FL, USA. All research was conducted in accordance with this approved protocol (protocol approval no. 019–07 and approval date: May 10, 2021). Male Sprague–Dawley rats, weighing approximately 250 g, were used to assess the in-vivo systemic absorption of CBD. They were kept for overnight fasting before the commencement of study. The animals were then categorized into two groups: Group 1 received a CBD exosome, while Group 2 received control CBD formulation. A single dose of 12 mg/kg was administered to each group (5 animals per group) orally using oral gavage. Blood samples were collected at specified time intervals of 1, 2, 4, 8, 12 and 24 hr. To obtain plasma, the collected whole blood samples were centrifuged at 4000 rpm at 4 °C. The resulting plasma samples were then stored at −80 °C until they underwent analysis (McCrudden et al., 2014). All the samples were then analyzed using HPLC-MS method (Bagde et al. 2021; K. Patel et al. 2019).

2.14. Stability Studies

CBD-exosomes formulation was diluted with pH 1.2 and 6.8 phosphate buffer with ratio of 1:1 (formulation: buffer). Formulation samples of pH 1.2 and 6.8 buffer were then kept at 37 °C in vitro USP dissolution apparatus for 2 and 24 h respectively. Samples were then collected for particle size, zeta potential, protein concentration and drug content analysis. Using NTA and a ZetaView instrument (ZetaView^® TWIN PMX-220) in scatter mode, the average particle size (z-average), concentration, and zeta potential of the CBD exosomes formulations were examined using the dynamic light scattering (DLS) approach at 25 °C and a 90° scattering angle. All the samples were diluted using particle free water at 1:1000 dilution and measured in triplicate in ZetaView instrument (N. Patel et al. 2021).

The exosomes were lysed in RIPA buffer (150 mM sodium chloride, 1.0 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris, pH 8.0, and 1X Thermo Scientific Halt Protease Inhibitor Cocktail). Following a 20-minute ice lysis, the samples were spun down at 14,000 rpm for 20 min. The protein concentration was determined using the Smith assay (bicinchoninic acid assay) after the supernatant had been collected.

Drug content analysis was conducted using HPLC-MS equipment. Briefly, the samples were diluted with methanol with 1:10 (formulation: methanol) ratio. Drug content was then analyzed using standard calibration curve equation.

2.15. Statistical and PK analyses

Based on at least three repetitions, the raw data findings were displayed as the mean ± standard deviation (SD). The t-test with unpaired experimental design with Welch’s correction was used for the statistical analysis of the data. Differences between groups were considered significant at the p < 0.05 level. Statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). PKSolver was utilized for noncompartmental analysis of in vivo pharmacokinetic data.

3. Results

3.1. Exosome and CBD formulation characterization

CMDE NTA analysis showed average particle concentration of 5.2*10¹¹/mL, protein concentration of 5.6 ± 0.5 mg/mL (BCA assay), particle size of 124.9 ± 3.1 nm and zeta potential of −38.81 ± 0.60 mV in scatter mode. The particle number was 4.1*10¹¹/mL with a size of 104.4 ± 1.6 nm and zeta potential of −30.75 ± 0.04 mV in fluorescent mode. By using the formula in methods, percent purity of the particles was 78.84 %. CBD-exosomes were successfully formulated with particle size of 138.4 ± 4.37 nm and zeta potential of −42.07 ± 0.99 mV. (Fig. 1A–1E) Physical appearance of the formulation was translucent with no sedimentation of particles. After exosomes were separated, western blotting was used to quantify known exosome markers. It was verified that Alix, CD81, and CD63 were expressed. (Fig. 1F) Entrapment efficiency was found to be 56.56 ± 4.26 %. TEM images confirmed the presence of intact vesicular membranes and both CMDE and CBD-CMDE were observed to have particle size in the range of 80–100 nm (Fig. 1G–1H).

Fig. 1.

Nanoparticle tracking analysis and Western blotting of exosomes. A) Size of CMDE in scatter and fluorescence mode B) Size of CBD-CMDE C) Zeta potential of CMDE in scatter mode D) Zeta potential of CMDE in fluorescence mode E) Zeta potential of CBD-CMDE. F) Western blots analysis of CMDE shows expression of proteins Alix, CD63 and CD81. G) TEM image of CMDE. H) TEM image of CBD-CMDE. Images were taken under 20X magnification.

3.2. In-vitro release study

Our in-vitro release studies showed CBD release in both stomach (pH 1.2) and intestinal (pH 6.8) buffer. Release studies conducted at pH 1.2 showed 26.51 ± 0.87 and 19.57 ± 0.80 % drug release from CBD CMDE and control CBD formulation respectively. Moreover, release studies at pH 6.8 showed significantly (p < 0.01) enhanced drug release of 78.27 ± 5.37 % at the end of 24 hr from CBD-CMDE as compared to control CBD formulation which showed 46.42 ± 4.75 % of CBD release. It was also observed that CBD-CMDE showed 2.95 times higher drug release (17.42 ± 1.12 %) at 2 hr as compared to control formulation (5.89 ± 0.51 %). (Fig. 2).

Fig. 2.

In-vitro dissolution study at A) pH 1.2 buffer showing cumulative drug release from CBD-CMDE and control CBD formulation; B) pH 6.8 buffer showing significantly enhanced drug release from CBD-CMDE as compared to control CBD formulation. Results are expressed in Mean ± SD (n = 3).

3.3. CMDE improved permeability of CBD

The cell permeability of CBD was significantly increased when loaded in exosomes relative to control formulation. The apparent permeability of CBD exosomes was found to be 22.9*10⁻⁶ ± 0.34 cm/sec at 2 h whereas the apparent permeability of CBD solution was found to be 5.8*10⁻⁶ ± 0.65 cm/sec at 2 h. It was observed that the apparent permeability of CBD from CBD exosomes was improved by 3.95-folds (P < 0.0001) in comparison to CBD control formulation (Fig. 3).

Fig. 3.

Apparent permeability coefficient (Papp) of Control CBD Formulation and CBD-CMDE in MDCK monolayers. Results were expressed as Mean ± SD (n = 3). ****P < 0.0001.

3.4. Cytotoxicity assay

3.4.1. 2D cytotoxicity assay

A dose dependent cytotoxicity was observed when the MDA-MB-231 DOX RT, MDA-MB-468 RM RT cells were treated with CBD, CBD-CMDE and CMDE. CBD-CMDE were more potent with IC₅₀ value of 3.6 ± 0.54 μM than CBD which had IC₅₀ value of 7.53 ± 0.59 μM post-treating the MDA-MB-231 DOX RT cells for 48 hr. CMDE also exhibited anticancer potential such that around 60 % of MDA-MB-231 DOX RT cells were viable and 40 % cells were dead when treated with 1*10¹¹ particles/mL CBD exosomes were more potent with IC₅₀ value of 3.88 ± 0.54 μM than plain CBD which had IC₅₀ value of 8.08 ± 0.17 μM post treating the MDA-MB-468 RM RT cells for 48 hr. CMDE also exhibited anticancer potential and around 60 % of MDA-MB-468 RM RT cells were viable and 40 % cells were dead when treated with 1*10¹¹ particles/mL Surprisingly, these compounds were found to be highly effective since these cells have very highly resistant against Doxorubicin (40 μM ± 0.79 μM). Synergism between CMDE and CBD was evident in the cytotoxicity studies (Fig. 4).

Fig. 4.

In-vitro screening for anticancer potential of CMDE, CBD and CBD-CMDE in 2D. A, B, C represents CMDE, CBD, CBD-CMDE against MDA-MB-231 DOX RT respectively and D, E, F represents CMDE, CBD, CBD-CMDE against MDA-MB-468 RM RT cells respectively. Results were expressed as μM in Mean ± SD (n = 4).

3.4.2. 3D cytotoxicity assay

As expected, the IC₅₀ values were increased in 3D spheroids assay. The microenvironment of 3D spheroids resembled that of tumor and acts as a barrier for drug penetration leading to increase in IC₅₀ values (Nathani et al. 2024). The IC₅₀ value of CBD-CMDE and CBD solution was found to be 14 ± 0.85 μM, 25 ± 0.93 μM respectively in MDA-MB-231 DOX RT cells. The IC₅₀ value of CBD solution and CBD-CMDE was found to be 34.7 ± 0.08 μM and 15 ± 0.07 μM respectively in MDA-MB-468 RM RT cells (Fig. 5).

Fig. 5.

In-vitro screening for anticancer potential of CMDE, CBD and CBD-CMDE in 3D spheroids. A, B, C represents CMDE, CBD, CBD-CMDE against MDA-MB-231 DOX RT respectively and D, E, F represents CMDE, CBD, CBD-CMDE against MDA-MB-468 RM RT cells respectively. Results were expressed as μM in Mean ± SD (n = 4).

3.5. In-vivo internalization of CMDE in tumor

To verify that CMDE was internalized into the tumor, an initial in-vivo investigation was conducted using DiO-CMDE. The tumor in the mouse given DiO alone did not exhibit any fluorescence that could be distinguished from the negative control. Both mouse tumors (given PBS and DiO alone) exhibited no fluorescence. After 4 and 24 hr, the tumor from the animal administered with DiO-CMDE showed extremely bright fluorescence. (Fig. 6).

Fig. 6.

Microscopic (cryosection) fluorescent images of MDA-MB-231 tumors resistant to DOX showing internalization of CMDE by the tumor 4 and 24 hr after oral administration in mice of PBS (A), DiO alone (B), DiO-CMDE (C, D). Brightfield images are on the right side for the same groups PBS (E), DiO alone (F), DiO-CMDE (G, H). The scale bar represents 20 μm. I) Graph showing fluorescence intensity of images A, B, C, D of negative control. DiO alone, DiO-CMDE (4 h) and DiO-CMDE (24 h) respectively.

3.6. In-vivo pharmacokinetic study

Our results revealed that CBD-CMDE showed Cmax of 448.90 ± 78.53 ng/mL CBD absorption in systemic circulation as compared to control CBD formulation which showed Cmax of 163.39 ± 24.97 ng/mL. Tmax was found to be 4.00 ± 0.00 and 2.00 ± 0.00 h for CBD-CMDE and control CBD formulation respectively. Moreover, the plasma concentration was 5.75-fold higher at the end of 6 hr in case of CBD-CMDE as compared to control formulation. Our data also showed significantly enhanced bioavailability of CBD-CMDE with AUC_(0–24h) of 1350.56 ± 187.50 h.ng/mL as compared to control formulation which showed 351.95 ± 39.10 h.ng/mL (Fig. 7).

Fig. 7.

A) Pharmacokinetic study in SD rats showing significantly enhanced AUC (0–24) and Cmax in CBD-CMDE group compared to control CBD formulation group. Results were expressed as Mean ± SD (n = 5) *P < 0.05; B) PK parameters of CBD-CMDE and control CBD formulation group.

3.7. Stability studies

CBD-CMDE formulation diluted with pH 1.2 buffer showed particle size of 124.9 ± 3.1 nm, zeta potential of −38.81 ± 0.60 mV, protein concentration of 5.6 ± 0.5 mg/mL and drug content of 97.45 ± 2.55 %. CBD-CMDE formulation diluted with pH 6.8 phosphate buffer showed particle size of 124.9 ± 3.1 nm, zeta potential of −38.81 ± 0.60 mV, protein concentration of 5.6 ± 0.5 mg/mL and drug content of 98.21 ± 1.86 %. Our results showed no significant differences in particle size, protein concentration and drug content in the formulation before and after the study suggesting that CBD-CMDE formulation is stable in gastrointestinal tract. Despite a slight reduction in the zeta potential of the CBD CMDE formulation, it remained stable, with values falling within the range of −10 to −20 mV (Supplementary Figure 1).

4. Discussion

Breast cancer is the most common cancer worldwide and approximately thirty percent of all newly diagnosed malignancies in women are for breast cancer each year (“Breast Cancer Facts and Statistics 2024,” n. d.). Rather than being a disease in which a small number of genes, proteins, and/or signaling pathways independently, and autonomously contribute to the evolution of the disease; breast cancer is a heterogeneous and complex cancer with distinct morphological and molecular characteristics (Luque-Bolivar et al. 2020). CBD has been extensively studied as a potent anticancer agent against several solid tumors such as breast, prostate, lung, pancreatic and colorectal cancers (N. Patel et al. 2021; Kaur, Nathani, and Singh 2023; Todorova et al. 2021; Lee et al. 2022). However, the poor oral bioavailability of CBD due to high first pass metabolism and excretion rate has limited its clinical application (Millar et al. 2018). Several approaches have been proposed to improve the oral bioavailability of CBD (Knaub et al. 2019; Atsmon, Cherniakov, et al. 2018; Cherniakov et al. 2017; Atsmon, Heffetz, et al. 2018) but no systematic study has been done with its exosome formulation delivery in vivo.

In the present study, we hypothesized that CMDE can improve the bioavailability of CBD. To overcome the drawbacks of using traditional anticancer agents, numerous attempts have been made in the last few years to use nano-drug delivery systems with higher efficacies and lower toxicities (Godugu, Doddapaneni, and Singh 2017; Patel, Doddapaneni, Chowdhury, et al. 2016; Doddapaneni et al. 2016; Griffin et al. 1993). These nano-drug delivery systems−liposomes, nanoparticles, etc. can selectively accumulate in tumor cells and deliver drugs to specific targets while avoiding detrimental effects on non-cancerous cells via various routes of administration because of their increased permeability, active transport intake, and high retention (Patel, Doddapaneni, Sekar, et al. 2016b; Ferdous, Stembridge, and Singh 1998; Patel, Doddapaneni, Sekar, et al. 2016b; Godugu et al. 2016; A. R. Patel, Chougule, and Singh 2014; Udofot et al. 2016; Shaik, Chatterjee, and Singh 2010; Mandip Singh, Ferdous, and Jackson 1999; M. Singh et al. 1991).

Exosomes are naturally produced drug delivery vehicles and have drawn lot of attention because they can cross the blood brain barrier (Schindler et al. 2019). Milk exosomes are affordable, biocompatible nanoparticles that can be prepared in large quantities and are readily available. We were able to isolate 10 mL of exosomes from 200 mL of camel milk, having 5.2*10¹¹/mL particle number in scatter mode and 4.1*10¹¹/mL in fluorescence mode. It was observed that CMDE size was in the range of 120 to 140 nm (138.4 ± 4.37 nm for formulated exosomes and 124.9 ± 3.1 nm for the plain exosomes), which falls in the typical size range for milk-derived exosomes from larger animals (Baddela et al. 2016; Pieters et al. 2015). The exosome membranous vesicles measured in fluorescent mode had a substantially smaller mean diameter (104.4 ± 1.6 nm) than the total particles (124.9 ± 3.1 nm), according to simultaneous size measurements. The larger non vesicular particles (such as protein aggregates, salt particles, or nanobubbles) that affect the size distribution and consequently the mean diameter to a larger size are likely reasons for this change. However, approximately 80 % of the particles were exosomes which indicates high quality of the particles isolated.

Exosome marker proteins Alix, CD81, and CD63 were all expressed by CMDE, according to western blot analysis. These proteins have been used to characterize CMDE before by other researchers (Badawy, El-Magd, and AlSadrah 2018; Matic and Dia 2022) and our results are in good agreement with them. However, we did not see the expression of HSP70, but our recently conducted proteomics results did show the presence of HSP90 and HSP71 (data not shown). TEM analysis demonstrated that both exosomes and CBD exosomes were spherical, intact and were in the size range of 80–100 nm. The results were in accordance with muller et al who reported that the exosomes derived from plasma exhibited the size range of 50–100 nm (Muller et al. 2014).

Our in vitro release studies showed CBD release in both stomach pH 1.2 and intestinal pH 6.8 buffers. Results showed significantly (p < 0.05) enhanced drug release from CBD-CMDE in 24 h in 6.8pH buffer as compared to control formulation. Moreover, pH 1.2 release study showed no significant difference in drug release at the end of 2 h in CBD-CMDE and control formulation. Both CBD exosomes and CBD control formulations contain Kolliphor RH 40 which act as a solubilizer required to solubilize CBD in the formulation. The main constituent of Kolliphor RH 40 is glycerol polyethylene glycol oxystearate, which, along with fatty acid glycerol polyglycol esters, forms the hydrophobic segment responsible for solubilizing hydrophobic APIs such as CBD. The hydrophilic part consists of polyethylene glycols and glycerol ethoxylate, which contribute to particle stability (Wang et al., 2023). Slow release of drug from CBD control formulations in pH 6.8 release media might be due to pH dependent drug release in acidic pH 1.2 and basic 6.8 buffer. These results are in concordance with Cui et al who also showed more than 70 % drug release in 24 h in pH dependent in-vitro release study of endogenous serum derived exosomes loaded with tanshinoneIIA and glycyrrhizic acid nanomicelles (Cui et al. 2023). This behavior has also been shown in nanoparticle formulations and this can be demonstrated by studies conducted by Yu et al who formulated mucoadhesive dexamethasone-glycol chitosan nanoparticles that showed an extended release of 80 % of dexamethasone over 48 h (Yu et al. 2020).

In-vitro permeability studies using MDCK cells demonstrated that CMDE can traverse tight junctions. High drug loading and entrapment efficiency of CBD-exosomes have also contributed to the improved overall bioavailability of CBD. Also, In-vitro permeability studies using MDCK cells showed significant improvement in permeation of CBD exosomes relative to CBD control formulation. It was observed that the apparent permeability of CBD exosomes is three-fold more than the CBD control formulation. Several studies have reported similar approach of using nano formulations to improve the oral bioavailability of poorly water-soluble drugs (Y. Wang et al. 2017; Zhu et al. 2015; H. Zhou et al. 2014). Wang et al have reported that chitosan nanoparticles improved the intestinal permeability of exendin-4 by 4.7 folds (M. Wang et al. 2014). Similarly, Chen et al demonstrated that the intestinal permeability and subsequently oral bioavailability of Schisantherin A increased by approximately 3.1-fold when formulated as nanocrystals (Tongkai Chen et al. 2016). However, there are no reports with in vitro permeability studies with milk exosomes and ours will be the first report.

CMDE offers several advantages over other formulation designs and has anticancer activity thus providing synergistic effect to the anticancer molecules such as tamoxifen and hesperidin which was encapsulated in CMDE (Badawy, Othman, and El-Magd 2021). In-vitro cytotoxicity studies conducted in our laboratory have also shown significant anti-cancer potential of CMDE against two different TNBC cell lines; MDA-MB-231 DOX RT, MDA-MB-468 RA RT. Several studies have already been reported to suggest anticancer activity of CMDE. For instance, Badawy et al have reported that CMDE exhibited synergistic anticancer activity in combination with tamoxifen and hesperidin against different breast cancer cell lines such as MCF-7, MDA-MB-231 as well as in in-vivo in MCF-7 xenograft mouse model through oral route of administration (Badawy, Othman, and El-Magd 2021). Also, Ali et al demonstrated selective anticancer activity of CMDE against pancreatic cancer cells and were observed to be non-toxic to normal pancreatic cells. The authors further studied the mechanism by which CMDE exhibited antitumor selectivity and attributed it to apoptosis with the involvement of bcl-2, bax and caspases (Ali et al. 2022).

The internalization of DiO-CMDE was confirmed by in vivo tumor uptake, which showed that the DiO was present in tissue sections after 4 and 24 hr and could be discriminated from the controls. This clearly shows CMDE translocation across the intestine and biodistribution to the tumor. Several studies have shown biodistribution of milk exosomes to the tumor in-vivo after oral administration. For instance, Betker et al reported iRGD targeted cow milk exosomes successfully traversed the intestinal barriers and showed significant accumulation in the tumor after 4 h of oral administration. Also, targeting with iRGD peptide reduced the accumulation of exosomes in other organs such as lungs, liver, kidneys, and heart (Betker et al. 2019). However, CMDE in our studies were non-targeted and probably reached the tumors through the EPR effect. The possible mechanisms for CMDE are still being investigated in our laboratory.

In-vivo PK study in SD rats demonstrated significantly enhanced bioavailability in case of CBD-CMDE group as compared to control formulation. Moreover, PK results were found to be in concordance with in vitro release study. Like in vitro release study, in vivo PK results showed no significant difference in systemic drug concentration at the end of 2 h from both CBD exosomes and CBD control formulations, suggesting that CBD was absorbed in stomach in acidic pH from both formulations. However, CBD CMDE group showed significantly higher CBD plasma concentration at 4 h as compared to control formulation. This implies that the use of CMDE may have improved and facilitated the absorption of drugs in the gastrointestinal (GI) tract. Dvora Izgelov et al investigated the pharmacokinetic profile of synthetic CBD oral formulations in a group of healthy human volunteers aged 27–35. Their findings revealed that a single oral dose of 90 mg CBD in sesame oil and self-nano-emulsifying drug delivery system (SNEDSS) significantly increased AUC and Cmax values compared to the same dose administered as a powder. The reported AUC values were 61 ± 18, 66 ± 28, and 8 ± 6h.ng/mL for sesame oil, SNEDSS, and powder, respectively. Correspondingly, the Cmax values for sesame oil, SNEDSS, and powder were 18 ± 9, 14 ± 7, and 0.8 ± 0.7 ng/mL in their study (Izgelov et al. 2020). Feng and colleagues also explored the pharmacokinetics of CBD in sesame oil and various lipid-based formulations, including oleic acid and other lipids. They observed that sesame oil exhibited enhanced systemic absorption of CBD, with an AUC of 821 ± 296 h.ng/mL, compared to other lipid-based formulations, following an oral dose of 12 mg/kg in SD rats (Feng et al. 2021).

In our study, in contrast to both Dvora Izgelov et al and Wanshan Feng et al, we noted a significant increase in AUC to 1350.56 ± 187.50 h.ng/mL in the CMDE group (12 mg/kg oral dose) with enhanced Cmax of 448.90 ± 78.53 ng/mL and mean residence time (MRT) compared to the control formulation in SD rats. Interestingly, even though our control micellar formulation had CBD in a completely solubilized form, the CBD-CMDE exhibited enhanced bioavailability. As far as we are aware, this is the first study demonstrating improved bioavailability through the CMDE approach for a poorly bioavailable and water-insoluble active pharmaceutical ingredient (API) in an in-vivo animal study.

CMDE have been reported to have excellent stability under acidic condition of stomach (Rani et al. 2017; Vashisht et al. 2017). A study by Agarwal et al demonstrated that there was no significant difference in either size or PDI of milk derived exosomes and exosomes loaded with paclitaxel indicating that milk derived exosomes were able to withstand the harsh gastrointestinal conditions (Agrawal et al. 2017). Also, Izumi et al have demonstrated that the miRNA and mRNA in bovine milk were resistant to pH as well as RNAse degradation since they were packed in exosomes (Izumi et al. 2012). Our results demonstrated similar observation that these exosomes were unaffected by acidic conditions such as 1.2 pH buffer. Stability studies showed no significant difference in size, zeta potential, protein content and amount of entrapped drug in the formulation in both 1.2 and 6.8 pH buffers indicating good physical and chemical stability of these exosomes throughout the gastrointestinal tract.

The mechanism of absorption of bovine milk derived exosomes has been reported in earlier studies; Betker et al demonstrated that milk bovine exosomes interact with Fc receptors that are highly expressed in human intestinal tract through IgG. Further, administration of bovine IgG along with milk exosomes hindered their absorption through competitive inhibition and the absorption of exosomes was unaffected by other proteins such as erythropoietin since erythropoietin does not interact with Fc receptor. Also, there was no cross reactivity since bovine milk is well tolerated by humans (Betker et al. 2019). Our proteomic analysis of CMDE has revealed presence of lactadherin, Butyrophilin 1, lactotransferrin, β-casein, α-S1-casein, Ig gamma-3 chain C region (Acession ID: S9XBS9 [4]) in significant amounts along with several other proteins (data not shown) which is in accordance with previous literature by He et al. They reported the presence of lactoperoxidase isoform 1 prepro-protein, lactoferrin, FABP domain-containing protein as well as κ-casein, and alpha-1-acid glycoprotein and peptidoglycan-recognition protein along with the above listed proteins from our results (He et al. 2021). Among these Ig gamma-3 chain C region was found to have a role in binding to Fc receptors. Hence, we believe that CMDE was possibly absorbed through Fc Receptor and this aspect needs to be further investigated in the future.

Overall, our study demonstrates the potential of CMDE to significantly enhance the bioavailability of CBD, a potent anticancer agent. With in-vitro and in-vivo evidence supporting improved drug release, permeability, and absorption, the CMDE approach offers a promising solution to overcome the limitations of CBD’s poor oral bioavailability. CMDE has the capability to be a potent oral drug delivery vehicle with inherent anticancer activity.

5. Conclusion

In conclusion, our study focused on addressing the limitations in the oral bioavailability of CBD, a potent anticancer agent against various cancers including breast cancer. Recognizing the challenges posed by high first-pass metabolism and excretion rates, we explored the potential of CMDE to enhance the bioavailability of CBD. CMDE, isolated and purified through differential ultracentrifugation, proved to be a promising carrier due to their high yield and economical scalability compared to other sources. Our in-vitro cytotoxicity studies demonstrated the anticancer potential of CMDE against DOX resistant MDA-MD-231 and RM resistant MDA-MB-468 cell lines. In-vitro permeability studies using MDCK cells demonstrated the ability of CMDE to traverse tight junctions, while high drug loading and entrapment efficiency contributed to improved overall bioavailability of CBD. Specifically, our in-vivo pharmacokinetic study in SD rats revealed a significant enhancement in bioavailability for the CBD-CMDE compared to the control group. Furthermore, our in-vivo tumor uptake studies demonstrated that DiO-CMDE can overcome intestinal barriers and successfully reach tumors after oral administration. Moreover, our stability studies indicated that these exosomes were stable in the acidic environment of gastrointestinal tract. In overcoming the challenges of poor bioavailability, our study introduces a novel and effective method for enhancing the absorption of poorly bioavailable and water-insoluble pharmaceuticals, making it a noteworthy contribution to the field.

Supplementary Material

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2024.124375.