Poly(cannabinoid)s: Hemp-Derived Biocompatible Thermoplastic Polyesters with Inherent Antioxidant Properties

Abstract

The legalization of hemp cultivation in the United States has caused the price of hemp-derived cannabinoids to decrease 10-fold within 2 years. Cannabidiol (CBD), one of many naturally occurring diols found in hemp, can be purified in high yield for low cost, making it an interesting candidate for polymer feedstock. In this study, two polyesters were synthesized from the condensation of either CBD or cannabigerol (CBG) with adipoyl chloride. Poly(CBD-Adipate) was cast into free-standing films and subjected to thermal, mechanical, and biological characterization. Poly(CBD-Adipate) films exhibited a lack of cytotoxicity toward adipose-derived stem cells while displaying an inherent antioxidant activity compared to poly(lactide) films. Additionally, this material was found to be semi-crystalline and able to be melt-processed into a plastic hemp leaf using a silicone baking mold.

Graphical Abstract

INTRODUCTION

Demand for polymers derived from renewable resources has increased over the last few decades due to concerns over resource availability and an increased understanding of plastic pollution.^1–4 Currently, poly(lactic acid) (PLLA) offers the best potential for polymers derived from renewable sources due to the ease of industrial scale production. PLLA accounts for nearly 25% of globally produced bioplastics, finding application in textiles, packaging, food storage, and biomedical devices.^5–8 In addition to its good processability and mechanical properties, PLLA stands out from other bioplastics as lactic acid is produced at low cost through the fermentation of corn and sugarcane.⁹

Hemp (Cannabis sativa L.), like corn and sugarcane, is a robust plant that has historically been cultivated for its high fiber content and nutrient-rich seeds.¹⁰ Recently, hemp has generated global interest for its ability to produce nearly 20% by weight of cannabidiol (CBD), in addition to numerous trace cannabinoids.¹¹ CBD is well studied and has been shown to therapeutically benefit a wide range of diseases and conditions including nausea, inflammation, and infection, while lacking the psychoactive side effects associated with tetrahydrocannabinol, another well-known cannabinoid.^12–16 Many of CBD’s medicinal benefits have been attributed to its natural antioxidative, anti-inflammatory, and anti-nociceptive properties.^17–20

Research on cannabis has historically focused on the medicinal benefits of its isolated compounds and the use of its fibers. Cannabinoids have, unfortunately, not been considered as potential polymer feedstock, likely due to the complex legal status of cannabis and the high cost of isolating these compounds. Since the passing of the 2018 Farm Bill, the price of pure cannabinoids derived from hemp has dropped dramatically as industrial cultivation has become federally legal.²¹ CBD, and other cannabinoids, offers the necessary chemical functionality to produce aliphatic polyesters similar to PLLA. Polymers made from cannabinoids, therefore, have the potential to replicate the commercial success of PLLA due to the high biomass yield of the hemp crop and high concentration of cannabinoids within that biomass.^10,11,22 Herein, the synthesis and material characterization of polyesters made from a polycondensation reaction of CBD and cannabigerol (CBG) with adipoyl chloride to produce poly(CBD-Adipate) and poly(CBG-Adipate) are reported. The adipate linking group was selected for these polymers as it is readily available, and polyaliphatic esters have been studied extensively for their biocompatibility.^23–25 Additionally, free-standing films of PLLA, poly(CBD-Adipate) (CBD100), and a 50:50 blend (CBD50) were cast and subjected to several biological assays to examine the activity of poly(CBD-Adipate) in a dose-dependent manner. The assays utilized include cytotoxicity, antioxidant activity, and anti-inflammatory activity. PLLA was selected as the control for its ability to produce uniform free-standing films and its well-studied biocompatibility.²⁶

RESULTS AND DISCUSSION

CBD and CBG were polymerized with adipoyl chloride (Scheme 1) from a simple condensation reaction to produce polyesters of moderate molecular weights, ca. 21 kDa. Glass transition temperatures (T_g) were found to be ca. 38 °C for poly(CBD-Adipate) and ‒30 °C for poly(CBG-Adipate), as measured by DSC and are provided in the Supporting Information. To our knowledge, this is the first reported polymerization reaction involving cannabinoids. Previously, CBD has been reported for its use as a capping agent for the synthesis of metal nanoparticles, as well as its incorporation into different polymer systems as an additive for drug delivery and increasing antioxidant capabilities.^27–29 In each case, CBD was incorporated directly as a small molecule and not as part of the main material structure. Free-standing films of poly(CBD-Adipate) could be cast from solutions of 1,4 dioxane; however, poly(CBG-Adipate) was unable to produce free-standing films due to its low T_g. Figure 1a represents the real-time weight, thickness (microns), temperature (°C), and in-plane and outof-plane birefringence data as a function of time for poly(CBD-Adipate) films undergoing drying from dioxane solutions. Weight and thickness values decrease and level off beyond a critical value (time ~ 10 min) as the solvent evaporates. Throughout the experiment, surface temperature values remain constant. In-plane birefringence (Δn₁₂) remains constant at zero during drying, indicating that there is in-plane isotropy. More important data on the anisotropy development come from out-of-plane birefringence (Δn₂₃) measurements. In the initial as-cast state, it is zero as the chain axes in the solution are completely random. Beyond a critical drying level, it rapidly increases to a high value around 10 min. These results on in plane and out-of-plane birefringence indicate that the initially randomly oriented polymer chain axes lay down in the plane randomly as the thickness decreases during solvent evaporation.³⁰ Thus, dried films attain uniaxial symmetry with optic axes oriented in the normal direction.

Scheme 1.

Synthesis of Poly(CBD-Adipate) and Poly(CBG-Adipate)

Figure 1.

(A) Real-time drying data of 18 wt % poly(CBD-Adipate)−dioxane solution (temperature: room temperature ~ 23 °C, air speed of 0.33 m/s). (B) DSC of the poly(CBD-Adipate) film showing the isothermal crystallization (1st heating) and subsequent melting of crystalline domains (2nd heating). (C) Wide-angle X-ray scattering patterns and peak positions of the annealed sample in DSC. (D) Stretching of the poly(CBD-Adipate) film at T_g + 15 (43 °C). (E) Hemp leaf molded by heating finely ground poly(CBD-Adipate) in a common silicone baking mold at 110 °C for 30 min with an approximate size of 5 cm by 3 cm, weighing 6.3 g.

The films were found to be amorphous as the aromatic groups and side groups designed in this polymer make it easy to vitrify even when solidified from highly mobile solutions. The DSC curves of the films (Figure 1B) exhibit an exotherm, indicating crystallization, with a broad melting temperature (100–260 °C). The poly(CBD-Adipate) film was sealed in an aluminum pan and heated to 170 °C, held for 40 min, cooled to 0 °C, and heated back up to 300 °C at 10 °C/min, as is shown in Figure 1B. During the second heating, a broad melting peak was detected, indicating the development of a small amount of crystallinity. The wide breadth of the endotherm suggests that there is a high degree of distortion and/or size distribution in these ordered regions. The sample’s wide-angle X-ray diffraction pattern of this sample (Figure 1C) does not show distinct sharp crystalline peaks, likely caused by high distortions in the crystalline domains. Poly(CBD-Adipate) films were studied using a custom-designed real-time mechano-optical measurement instrument, as described in the Experimental Section. The stretching was conducted at T_g + 15 °C (43 °C) (Figure 1d). At this temperature, Young’s modulus was found to be 83.15 MPa with a yield strength of 1.42 MPa. Strain hardening occurs when the true strain reaches 1.244, after which the slope of the true strain−stress curve increases rapidly. The photoelastic constant, Regime I, and strain optical constant were calculated as 8.31 × 10^–5 MPa^–1, 5.55 × 10^–3 MPa^–1, and 1.16 × 10^–2, respectively (Figure S1). Remarkably, poly(CBD-Adipate) exhibits exceedingly high stretchability despite its moderate molecular weight. To demonstrate melt processability, a hemp leaf was made from a common silicone baking mold by heating finely ground poly(CBD-Adipate) in a convection oven at 110 °C for 30 min. The resulting leaf measured approximately 5 cm by 3 cm, weighed 6.3 g (96.9% yield), and retained the fine detail of the baking mold, as shown in Figure 1E. To investigate the possibility of heat-induced cross-linking of CBD’s two alkene bonds, ¹H NMR spectra were taken before and after heating the polymer samples. Poly(CBD-Adipate) remained readily soluble after heating without significant differences between NMR spectra, as shown in Figure 2, which is consistent with crystallinity induced during heat treatment rather than cross-linking.

Figure 2.

¹H NMR spectra of poly(CBD-Adipate) (A) before heating and (B) after heating at 170 °C for 40 min in air. All three alkene hydrogen peaks (4.25–5.25 ppm) remain unchanged after heating, indicating a lack of cross-link formation.

Given PLLA’s extensive use as a biomedical material, we sought to investigate the potential for poly(CBD-Adipate) to be used in similar applications. Films of PLLA, a 50:50 blend of PLLA and poly(CBD-Adipate) (CBD50), and pure poly(CBD-Adipate) (CBD100) were subjected to several biological assays. The cytotoxicity of poly(CBD-Adipate) was assessed by a LIVE/DEAD staining of rabbit adipose-derived stem cells (ADSCs) cultured on tissue culture plastic (TCP), CBD50, CBD100, and PLLA films after 24 h, shown in Figure 3A. The cells on TCP and all three polymer films showed strong green fluorescence (live cells) with no detectable red fluorescence (dead cells). ADSCs showed characteristic elongated fibroblastic morphology when grown on TCP and polymer films, irrespective of CBD content. This was corroborated by the metabolic activity assay (Figure 3B), which showed no significant differences in the metabolic activities of ADSCs cultured on PLLA and CBD films when compared to cells cultured on TCP.

Figure 3.

(A) LIVE/DEAD staining of ADSCs 24 h post culture. Cells cultured on TCP, PLLA, CBD 50 film, and CBD 100 film. Live cells were stained green, and dead cells were stained red. (B) Percentage metabolic activity of ADSCs on CBD and PLLA films compared to cells on TCP. Metabolic activity of ADSCs did not show any statistically significant differences between the test groups. n = 6; one way ANOVA with Tukey’s multiple comparisons post hoc test.

The radical scavenging activities of CBD films were compared to PLLA films using an oxygen radical absorbance assay (ORAC), Figure 4. CBD100 films retained more than 90% of fluorescence at 5 min post addition of reactive oxygen species (ROS), whereas CBD50 and PLLA films retained 85% activity at that time. By 15 min, only CBD100 showed more than 50% fluorescence, while the activities of CBD50 and PLLA films decreased to ~35%. After 30 min, the CBD100 films maintained statistically higher fluorescence when compared to CBD50 and PLLA (Figure 4a). In fact, CBD100 films showed statistically higher antioxidant activity compared to CBD50 and PLLA films at all points in time over a 1 h period post ROS addition, as shown in Figure 4b,c. These results support material tunability through a concentration-dependent antioxidant activity of poly(CBD-Adipate). To our knowledge, poly(CBD-Adipate) appears to be the only nonphenolic synthetic antioxidant polymer reported. The most commonly reported polymers exhibiting antioxidant properties tend to be biopolymers including PLA, PEO, lignan, and other polysaccharides that have been processed to incorporate phenols through physical mixing or chemical modification.^26,31,32 We expect that the antioxidant ability of poly(CBD-Adipate) could be further improved using the same phenol incorporating techniques that have been reported for other biopolymers.

Figure 4.

(A) Antioxidant activity of polymer films expressed as average fluorescence units as a function of time (0–30 min). The CBD100 film showed significantly higher antioxidant activity when compared to PLLA films at all the time points. (n = 5); two-way ANOVA with Tukey’s multiple comparisons post hoc test. (B) Fluorescence decay curves as a function of time (0–1 h). (C) Bar graph showing the area under the decay curves. (n = 6); one-way ANOVA with Tukey’s multiple comparisons hoc test.

Considering the conservation of CBD antioxidant properties when polymerized, poly(CBD-Adipate) was further examined for potential anti-inflammatory activity, as well as the ability to modulate cannabinoid (CB) receptors. Figure 5a–c shows gene expression profiles of inflammatory cytokines in primed immortalized bone marrow macrophages (BMMs), 24 h post culture onto polymer films. Significant changes in gene expression levels of pro-inflammatory cytokines were not observed when cultured on CBD films as compared to PLLA films. Figure 5d,e shows expression profiles of cannabinoid receptors in BMMs after culture on polymer films. Significant changes in expression profiles of cannabinoid receptors were not observed when compared to PLLA films. The lack of modulation of CB1 and CB2 receptors in BMMs is likely caused by the sequestering of CBD into high molecular weight of the polymer chains. As the anti-inflammatory effects of CBD are known to be mediated through receptor binding, the availability of bindable CBD will be limited when polymerized.^18,20,33,34

Figure 5.

Gene expression profile of inflammatory cytokines by BMMs upon culture on CBD100, CBD50, and PLLA films (A–C). No significant changes observed between groups. Modulation of cannabinoid receptors in BMMs upon culture on CBD100, CBD50, and PLLA films (D–E). No significant changes in CBD receptor expression compared to the PLLA film. (n = 3); one-way ANOVA with Tukey’s multiple comparisons post hoc test.

A lack of cytotoxicity shown by poly(CBD-Adipate) films suggest its potential as a biomaterial for a variety of applications. Intrinsic antioxidant properties of the material make it specifically attractive for applications such as wound healing and tissue regeneration.^35,36 The lack of CB receptor modulation would likely prevent typical side effects of CBD if consumed, suggesting safe use in products currently made with PLLA. Future investigations will seek to increase the antioxidant activity and introduce anti-inflammatory activity by blending low-molecular-weight poly(CBD-Adipate) oligomers into the films, increasing the amount of CBD available for receptor binding.

DISCUSSION

In summary, polyesters of moderate molecular weight were synthesized from the condensation reaction of CBD and CBG with adipoyl chloride. Poly(CBD-Adipate) was thermally molded at mild temperatures using a common silicone baking mold, with no chemical changes detected after heating. The apparent ease by which this material can be thermally molded suggests its viability for large-scale melt processability. Films of poly(CBD-Adipate) lacked a cytotoxic response while exhibiting antioxidant capability. Compared to PLLA, films made from poly(CBD-Adipate) maintained statistically higher antioxidant activities for extended periods of time. With the cost of pure CBD isolate having decreased 10-fold in the last 2 years and the ability for melt processing, the cost to produce poly(cannabinoid)s at scale could soon rival that of PLLA. Poly(CBD-Adipate) could find application for various biomedical materials, food storage, and any commodity-type materials currently made from PLLA without the need for additional antioxidant additives. CBD is only one of more than 100 naturally occurring cannabinoids and one of 75 with a diol structure that could be incorporated into polyesters. Poly-(cannabinoid)s offer an interesting alternative to produce new materials from renewable sources.

EXPERIMENTAL SECTION

Materials.

CBD was purchased from EcoGen BioSciences and used as received. CBG was purchased from Mile High Labs, Inc and used as received. All other chemicals were purchased from Sigma-Aldrich and used without further purification unless otherwise noted. ¹H NMR spectra were collected using a Bruker AVANCE 500 MHz instrument. TGA was conducted using a TA Instruments TGA Q-500, and DSC was collected using a TA Instruments DSC Q-20. GPC was taken using a WATERS GPC equipped with a 1515 HPLC Pump and Waters 717Plus Autoinjector.

Preparation of Poly(CBD-Adipate).

A mixture containing 80 mL of anhydrous methylene chloride (DCM) and 40 mL of anhydrous pyridine was added to a flame-dried 250 mL three-neck round bottom flask. CBD (10 g, 0.0318 mol) was added to the solution and allowed to dissolve under stirring. The solution was then chilled in an ice-water bath. Adipoyl chloride (4.66 mL, 0.0318 mol) was added dropwise over 60 min, and the reactants were stirred for 4 days. The solution was concentrated under vacuum and precipitated into cold methanol, yielding white polymer strands (12.15 g, 90% yield). Mn 21k, PDI 1.63. ¹H NMR (500 MHz, CDCl₃): δ (ppm):6.75 (s, 2H), 5.25 (s, 1H), 4.59 (s, 1H), 4.51 (s, 1H), 3.54 (d, 1H), 2.58 (m, 7H), 2.18 (m, 1H), 2.09 (m, 1H), 1.86‒1.63 (m, 14 H), 1.35 (m, 4H), 0.92 (t, 3H).

Preparation of Poly(CBG-Adipate).

A solution containing 80 mL of anhydrous methylene chloride (DCM) and 40 mL of anhydrous pyridine was added to a dried 250 mL three-neck round bottom flask. CBG (10 g, 0.0316 mol) was added to the solution and allowed to dissolve while stirring. The solution was then chilled in an ice-water bath, and 4.62 mL (0.0316 mol) of adipoyl chloride was added dropwise over 60 min. The reactants were allowed to stir for 4 days. The solution was concentrated under vacuum and precipitated into cold methanol, yielding white polymer strands (11.73 g, 87.6% yield). Mn 21k, PDI 1.61. ¹H NMR (500 MHz, CDCl₃): δ (ppm): 6.82 (s, 2H), 5.07 (m, 2H), 3.17 (m, 2H), 2.68–2.50 (m 6H), 2.07 (m, 2H), 1.99 (m, 2H) 1.90 (m, 4H), 1.77–1.57 (m, 11H), 1.36 (m, 4H), 0.93 (t, 3H).

Preparation of Polymer Films.

18 wt % of polymer [PLLA, poly(CBD-Adipate), and 50:50 wt % blend] was dissolved in 1,4-dioxane solution and prepared using a Thinky planetary centrifugal mixer for 1 h, for improved dissolution, uniformity, and degassing. Films were cast on the glass substrate using a motorized drawdown coater. The doctor blade was set to an initial casting thickness of 203 μm.

Crystallization Experiment.

A solution-cast poly(CBD-Adipate) film containing ~3 wt % dioxane was sealed in an aluminum pan, heated to 170 °C, held for 40 min, cooled to 0 °C, and heated back up to 300 °C. The heating and cooling rates for this experiment were 10 °C/min. Scans were run under a nitrogen atmosphere.

Real-Time Weight, Thickness, Temperature, and Birefringence Measurements.

Solution-cast films were dried in the chamber of a real-time measurement system described in detail elsewhere.^30,37 This system measures weight, thickness, and in/out-of-plane birefringence of cast solution on a glass substrate with controlled airspeed and temperature. The air temperature can be adjusted from room temperature to 200 °C, and airspeed can vary from 0 to 4.5 m/s. In this study, the solutions were dried at room temperature with an airspeed of 0.33 m/s. Both the airspeed and the temperature were kept constant during drying. Relative humidity within the chamber was 50%. The final thicknesses of the peeled films were measured to be 17 μm using a micrometer. The cast film was 12 cm long and 10 cm wide. Real-time weight measurements incorporated both global and local weight change. The former was directly acquired from precision balance, while the latter was calculated using real-time thickness data and density of the coatings.³⁸ In-plane (Δn₁₂) and out-of-plane (Δn₂₃) birefringences are calculated using the following equations

$$\Delta n(12)=\frac{R_0(t)}{d(t)}$$ (1)

$$\Delta n(23)=-\frac{1}{\text{d}}\left[\frac{R_0(\text{t})-R_{45}(t)\sqrt{1-\frac{{\text{sin}}^2\Phi }{{\overline{n}}^2}}}{\frac{{\text{sin}}^2\Phi }{{\overline{n}}^2}}\right]$$ (2)

where d(t) is the instantaneous thickness. R₀(t) and R₄₅(t) are the instantaneous 0° retardations and 45° retardations, respectively, at 546 nm. $\overline{n}$ is the average refractive index of the drying solution, and it is estimated linearly through the concentration change

$$\overline{n}=n_{\text{polymer }}\cdot {\chi }_{\text{polymer }}+n_{\text{solvent }}\cdot \left(1-{\chi }_{\text{polymer }}\right)$$ (3)

where n_polymer is the average refractive index of the polymer, n_solvent is the refractive index of the solvent, and χ_polymer is the solid content in solution.

Instrumented Uniaxial Stretcher and Mechano-Optical Behavior.

Dumbbell-shaped CBD samples were cut from the solution-cast films. A custom-designed real-time mechano-optical measurement instrument was used for this study.^39–42 This method is based on the light intensity method.⁴³ White light is used to determine the optical retardation while simultaneously measuring width W_t and axial force F_t during stretching. With the assumption of simple uniaxial extension and incompressibility, the time variation of the local thickness is calculated. It allows the calculation of local true stresses and local true strains using

$$\text{ true strain }=\frac{\text{ elongation }}{\text{ inital length }}=\frac{L_t}{L_0}-1={\left(\frac{W_0}{W_{\text{t}}}\right)}^2-1$$ (4)

$$\text{ true stress }=\frac{F_{\text{t}}}{W_{\text{t}}\cdot D_{\text{t}}}=\frac{F_{\text{t}}}{\left(W_{\text{t}}^2/W_0\right)\cdot D_0}$$ (5)

where W_t is the real-time width, W₀ is the initial width, L₀ is the initial length, L_t is the real-time length of the film, F_t is the time variation of force, D₀ is the initial film thickness, and D_t is the real-time film thickness calculated using uniaxial symmetry

$$D_{\text{t}}=\left(\frac{W_{\text{t}}}{W_0}\right)\cdot D_0$$ (6)

Samples were clamped and fixed in the arms of the uniaxial stretching machine inside an environmental oven. A thermocouple inside the oven was used to ensure that the film had reached the desired temperature before initiating stretching. CBD samples were uniaxially stretched at 43 °C (T_g + 15 °C after solution casting) with 20 mm/min stretching speed. In our experiment, the photoelastic constant and strain optical constant are obtained from the initial linear slopes of the birefringence-true stress and birefringence-true strain curves, respectively. The experimental stress−strain plot can be found in the Supporting Information.

Wide-Angle X-ray Scattering.

A Bruker AXS Generator equipped with a copper target tube and a two-dimensional detector was used to obtain the WAXD patterns of the stretched samples. Monochromatized Cu Kα radiation was obtained by operating the generator at 40 kV and 40 mA. A typical exposure time of 10 min was used.

Cell Culture.

Rabbit ADSCs were isolated as reported previously.⁴⁴ ADSCs were grown on TCP and maintained in a humidified incubator at 37 °C, 5% CO₂. Cells were passaged every 3–4 days or upon reaching 80% confluency using 0.25% trypsin−EDTA (Gibco 25200056). Cells were kept in DMEM/F12 (1:1) (Invitrogen 11330‒032) supplemented with 10% fetal bovine serum (FBS, Gibco 16000036) and 1% penicillin−streptomycin (Gibco 15140122). Cells passaged up to passage 6 were used for the studies. Immortalized BMMs were grown on TCP and maintained in a humidified incubator at 37 °C, 5% CO₂. Cells were passaged every 3–4 days or upon reaching 80% confluency using 0.25% trypsin−EDTA (Gibco 25200056). Cells were kept in DMEM (Gibco 10313021) supplemented with 10% FBS (Gibco 16000036), and 1% penicillin−streptomycin (Gibco 15140122). Cells passaged up to passage 20 were used for the studies.

LIVE/DEAD Assay.

LIVE/DEAD staining was carried out using the Invitrogen LIVE/DEAD Viability/Cytotoxicity kit (Cat# L3224). After 24 h post seeding, culture media was removed, and cells were washed with phosphate-buffered saline (PBS). A working solution of calcein-AM (0.5 μL/1 mL PBS) and ethedium (2 μL/1 mL PBS) was prepared and directly added to the cells. Following a short 5 min incubation at 37 °C, cells were imaged at 485 nm excitation and 520 nm emission using a Leica DMi8 microscope.

MTS Assay.

ADSCs were seeded on TCP, CBD50, CBD100, or PLLA films and incubated for 24 h. At that time, the media was removed, and 1:10 diluted MTS reagent (CellTiter 96 AQueous One Solution Reagent, Promega G3582) in culture media was added to the wells, incubated for an additional 2 h to allow color change and read at 490 nm.

Antioxidant Assay.

Antioxidant activity was evaluated using the well-established ORAC assay (Abcam ab233473). Assay was carried out as per the manufacturer’s protocol. In short, circular discs of CBD50, CBD100, or PLLA films were placed inside black-walled 96-well plates. Fluorescein solution was then added to the wells and incubated for 30 min at 37 °C. Upon addition of free radical initiator solution, the plate was transferred to a prewarmed (37 °C) microplate reader with Ex/Em set at 480/520 nm. Wells were read once every minute between 1 and 60 min.

Anti-Inflammatory Assay.

Immortalized bone marrow macrophages were seeded on TCP and treated with 20 ng/mL lipopolysaccharide to induce an inflammatory response. After a 2 h incubation at 37 °C, primed macrophages were trypsinized and seeded directly on top of CBD50, CBD100, or PLLA films and incubated at 37 °C for a further 24 h. Upon completion of 24 h incubation, cells were processed for RNA isolation and PCR analysis.

RNA Isolation and cDNA Preparation.

RNA from BMM cells (described above) was isolated using the Qiagen RNeasy Plus mini kit (74134) and Qiagen QIAshredder kit (79654) following the manufacturer’s protocol without modifications. RNA content was quantified using nanodrop, and cDNA library was synthesized (Takara 639549) as per manufacturer’s instructions. cDNA content was quantified using nanodrop and stored at ‒20 °C until further use.

qRT-PCR Analysis.

Taqman primers used for PCR analysis of inflammatory cytokines (TNFa, IL1B, IL6) were purchased from Thermo Fisher. qRT-PCR was performed using the iTaqMan Universal Probes Supermix (Biorad 1725131) as per manufacturer’s instructions. GAPDH was used to normalize samples. Samples were tested in triplicates. SYBR cannabinoid receptor1 and cannabinoid receptor 2 primers were designed in house using the NCBI primer design tool and synthesized by Integrated DNA Technologies (Coralville, IA). qRT-PCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Biorad 1725270) following the manufacturer’s protocol. GAPDH was used to normalize samples. Samples were tested in triplicates.

Taqman Primers:

GAPDH: Mm99999915_g1, TNFa: Mm00443258_m1, IL6: Mm00446190_m1, and IL1B: Mm01336189_m1.

Sybr Primers:

• GAPDH

  • F: ATG-AAT-ACG-GCT-ACA-GCA-ACA-GG
  • R: CTC-TTG-CTC-AGT-GTC-CTT-GCT-G
• CB1r

  • F: GTA-CCA-TCA-CCA-CAG-ACC-TCC-TC
  • R: GGA-TTC-AGA-ATC-ATG-AAG-CAC-TCC-A
• CB2r

  • F: CAG-GAC-AAG-GCT-TCA-CAA-GAC
  • R: GAC-AGG-CTT-TGG-CTG-CTT-CTA-C

Statistics.

Ordinary one-way ANOVA and two-way ANOVA were used for data analysis using Graphpad Prism 9.3. Error bars represent SD. Significance is designated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤0.001, and ****P ≤ 0.0001.