Cannabidiol‐Loaded Poly Lactic‐Co‐Glycolic Acid Nanoparticles with Improved Bioavailability as a Potential for Osteoarthritis Therapeutic

Cannabidiol (CBD) is a non‐intoxicating cannabinoid from cannabis sativa that has demonstrated efficacious against inflammation, which can be considered as a potential drug for arthritis treatment. However, the poor solubility and low bioavailability limit its clinical application. Here, we report an effective strategy to fabricate Cannabidiol‐loaded poly(lactic‐co‐glycolic acid) copolymer (CBD‐PLGA) nanoparticles (NPs), with a spherical morphology and an average diameter of 238 nm. CBD was sustained release from CBD‐PLGA‐NPs, which improved the bioavailability of CBD. The CBD‐PLGA‐NPs effectively protect the damage of LPS to cell viability. We observed that CBD‐PLGA‐NPs significantly suppressed LPS‐induced primary rat chondrocyte expression of inflammatory cytokines, including interleukin 1β (IL‐1β), interleukin 6 (IL‐6), tumor necrosis factor‐α (TNF‐α) and matrix metalloproteinase 13 (MMP‐13). Remarkably, CBD‐PLGA‐NPs also showed better therapeutic effects of inhibiting the degradation of the extracellular matrix of chondrocytes than equivalent CBD solution. In general, the fabrication CBD‐PLGA‐NPs showed good protection of primary chondrocytes in vitro and is a promising system for osteoarthritis treatment.


Introduction
Osteoarthritis (OA) is a chronic degenerative disease of the whole joint that involves structural changes in the clear articular cartilage, subchondral bone, ligaments, joint capsule, synovium, and muscles around the joint. [1,2] About 10 % of the population suffers from symptomatic knee osteoarthritis at the age of 60. [3,4] Oxidative stress has been shown to be related to the pathogenesis of osteoarthritis and has become an important therapeutic target. Reducing oxidative stress in cartilage that caused by inflammation can improve chondrocyte viability, which would contribute to the treatment of osteoarthritis. Hormones and non-steroidal anti-inflammatory drugs are commonly used in the clinical treatment of osteoarthritis. Yet they can only relieve the symptoms, and long-term use of such drugs will cause significant side effects, [5] such as suppression of platelet aggregation, erosions, and ulcerations in upper gastrointestinal tract mucosa, so as it is necessary to explore more effective drugs or materials for the treatment of OA. The intraarticular injection is an effective treatment for OA by delivering the drug to the site of action, thereby minimizing the systemic toxic effects of the drug. However, the synovial membrane of the joint is easily cleared by drugs, new nano-sustained-release drug delivery systems are urgently needed for the treatment of OA. [6,7] A non-psychoactive component of cannabis, [8] CBD, does not cause physiological dependence and is well tolerated. [9,10] Studies have shown that CBD has certain therapeutic effects on nervous system diseases, epilepsy, tumors, inflammation, liver injury, diabetes, pain, and other diseases. [11][12][13][14] In addition, the abilities to inhibit TNF-α and modulate the immune system of CBD were helpful for the treatment of collagen-induced rheumatoid arthritis. In a lipopolysaccharide-induced model of microglial inflammation, CBD treated inflammation by inhibiting ROS/NF-kb dependent signaling and glucose-dependent NADPH production. [15][16][17] For musculoskeletal disease, systemic administration of CBD effectively suppressed the progression of collagen-induced arthritis by reducing inflammatory cytokine production. [18] Since the progression of OA is closely related to the production of inflammatory factors, CBD may show potential therapeutic effects in OA. Taking these pharmacological properties into consideration, CBD represents an attractive therapeutic option for OA. [19] Unfortunately, its bioavailability has been reported to be extremely low when given orally to dogs, presumably due to a high first-pass effect through the liver. [20] Improving the bioavailability of CBD is important for the clinical application of CBD.
PLGA-based drug products have been approved by the FDA and European Medicine Agency for parenteral administration as drug delivery systems. The utilization of PLGA to fabricate nano/microparticles presented a broad application in disease treatment. [21] PLGA has well-described formulations and methods of production that adapt to various types of drugs and contrast agents, such as hydrophilic or hydrophobic small molecules and macromolecules. The drug and contrast agent can be protected by PLGA which avoids degradation. The surface properties of PLGA can be modified to provide "stealth" and/or better interaction with biological materials. Many targeting and imaging moieties conjugated with PLGA particles to enhance their binding affinity and specificity, and achieve amplifying signals at the target region. Moreover, The PLGAbased nanoparticles can be modified to target specific organs or cells for effectively delivering imaging labels, prolonging plasma half-lives, enhancing stability, improving targeting efficiency, and reducing non-specific binding. [22][23][24] PLGA has been widely used in drug delivery systems due to its biocompatible and biodegradable properties, [25,26] while the biocompatible and biodegradable are essential parameters are considered of polymer. On the other hand, the degradation products of PLGA, lactic and glycolic acid, which are natural metabolites of the human body, indicating PLGA was safe enough used in biomaterials fabrication. Considering the multitudinous advantages of PLGA acting as a biomaterial, the bioavailability of CBD may be improved that modified with PLGA, which is conducive to its further application.
Herein, the purpose of this study was to evaluate the efficacy of Cannabidiol-PLGA-NPs, included characterizing the nanospheres, in studying the anti-inflammatory effect of osteoarthritis, providing ideas for a new nanometer drug delivery system. well plate(Corning, USA), 96-well plate(Corning, USA), Cell culture incubator (Forma, USA), fluorescence quantitative PCR apparatus and enzyme label apparatus (Thermo Fisher Scientific, Waltham, MA, USA).

Preparation of PLGA Carriers with or without Cannabidiol
The PLGA nanoparticles loaded with CBD (CBD-PLGA-NPs) were synthesized using the emulsification-diffusion method. [27] The PLGA-NPs and CBD-PLGA NPs experimental design is illustrated in Figure 1. Two different types of nanoparticles were fabricated for the study: (i) poly lactic-co-glycolic acid nanoparticles (PLGA-NPs); (ii) Cannabidiol-encapsulated poly lactic-co-glycolic acid nanoparticles (CBD-PLGA-NPs). For fabricating CBD encapsulated nanoparticles using single emulsion method, 10 mg CBD and 30 mg PLGA was dissolved in 1 mL dichloromethane (DCM) and dispersed by ultrasonic to form an oil phase. The oil phase was added drop by drop into 2 mL 1 % polyvinyl alcohol (PVA) solvent with a 1 mL syringe and stirred in a magnetic agitator for 30 minutes. The ultrasonic probe was used to disperse the oil phase on the ice at 4°C to form colostrum. The colostrum was added into 6 mL 1 % PVA solution, magnetically stirred for 4 hours, and volatilized organic solvents were washed with double steam water, centrifuged three times, were rapidly frozen at À 80°C followed by lyophilization, and stored in a storage container at 4°C for retention. poly lactic-co-glycolic acid nanoparticles (PLGA-NPs) without drug loading were prepared by the same method.

Particle Size and Charge Surface Detection
The freeze-dried nanoparticles were dissolved in double-steaming water, and the samples were evenly dispersed by ultrasound for about 30s. The fully dispersed samples were placed in a 2 mL colorimetric dish, and the average size and polydispersity index (PDI) of the nanoparticles was measured by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). The experiment was repeated three times for each sample to determine the stability and aggregation in NPs dispersion by Zeta potential. The values presented are averages and standard deviations of triplicated measurements.

Transmission Electron Microscopy (TEM) Analysis
Firstly, to study the visualize and characterize particle size, size distribution and morphology of all the NPs, 5 mg PLGA-NPs and 5 mg CBD-PLGA-NPs were dissolved in 1 mL ultrapure water, respectively, followed by ultrasound for 15 min and the samples were added to the carbon net and dried at room temperature, then photographed for observation. The morphology of PLGA-NPs and CBD-PLGA-NPs was determined by using TEM (Bruker, Germany).

Quantification of CBD by HPLC
Quantification of CBD was performed by high-performance liquid chromatography (HPLC). The apparatus consisted of an HPLC Waters chromatograph with a model 510 pump, an Empower Login HPLC System Manager Software (Waters Corporation, Milford, MA, USA). A ZORBAX300SB-C18 column (5 μm, 4.6 mm × 250 mm) (Teknokroma S. Coop., Barcelona, Spain) was used. the mobile phase was acetonitrile and methanol (65 : 35, V/V). The 0.05 M phosphate buffer was prepared from monobasic potassium phosphate (KH 2 PO4) and adjusted to pH 2 with phosphoric acid. Before use, the mobile phase was filtered through 0.45 m filters and degassed. The flow rate was 2.0 mL/min and the sample volume was 20 μL. For analysis, a wavelength of 220 nm was used with the sensitivity adjusted to 0.250 aufs. All analyses were performed at 25.0 � 0.5°C. The analytical balance was used to accurately the concentration of CBD (1.25, 2.5, 5, 10, 20, 40, 80, and 160 μg/mL).

In Vitro Drug Release Study
The release behavior of CBD from the CBD-PLGA-NPs was monitored at 37°C.8 mg CBD-PLGA-NPs were placed in a dialysis bag (MW3500, SolarBio), placed in 8 mL PBS solution containing 1 % Tween-20 (TW, pH = 7.4), and placed in a thermostatic gas bath vibrator at 37°C (100 rpm/min). 1 mL sample was withdrawn at predetermined time intervals from each dissolution medium and was replenished to sustain the condition of the sink. The released CBD concentration was analyzed by using HPLC. All the studies were performed three times and the mean value was calculated. [28] Encapsulation Efficiency and Drug Loading 1 mg CBD-PLGA-NPs were dissolved in 1 ml dichloromethane and 4 ml methanol, and the nanoparticles were destroyed by repeated vortices using a vortex instrument to fully release the drug. Centrifugation was conducted at 10000 rpm/min, and the supernatants were collected and estimated using High-Pressure Liquid Chromatography (HPLC). [29] The percentage drug loading and encapsulation efficiency of the nanoparticles were calculated using the following equations (Eqs. 1 and 2): The viability of CBD-PLGA-NPs on the proliferation of LPSinduced chondrocytes was measured by CCK-8 assay. (Keygen, Nanjing, China). Briefly, the chondrocytes were seeded in a 96well plate at a density of 5000 cells overnight. Thereafter, various concentration of CBD-PLGA-NPs was added and further incubated for 48 h. After treatment, 10 μL of CCK-8 were added to each well and cultivated for 4 h. Finally, the absorbance at 450 nm was measured using a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer).

Live/Dead Tests
The Live/dead cells assay was performed using a live/dead viability assay kit (Invitrogen, USA). The cells were washed with PBS and incubated with the solution containing 2 μM of Calcein AM and 4 μM of Ethidium homodimer-1 for 30 min at room temperature in the dark. Then the cells were washed with PBS before being observed using a fluorescent microscope (Olympus BX53, Tokyo, Japan). As described in the manufacturer's protocol, live cells were stained green and dead cells were stained red.

qRT-PCR Detection
RNA separation kits (Magen, China) were used to extract total RNA. The reverse transcription is done by a reverse transcription kit (Fermentas Company, USA). [30] All qRT-PCR reactions were performed by a light Cycle 96 system (Roche, Switzerland) for 10 min at 95°C, followed by 40 cycles with 10 s duration at 95°C and then 60 s duration at 60°C. Each experiment was measured in triplicate, and 2 À ΔΔCt method was used to calculate the relative gene expression of each sample. The primers were used as follows in the Table 1.

Hematoxylin and Eosin (H&E) Staining
For in vitro study, the cells in all groups were fixed with 95 % alcohol for 30 min. After washing with PBS, the slides were fixed and cleaned, dyed with hematoxylin for 2-3 min, washed with PBS, soaked with Eosin for 1 min, and washed with PBS. After the slides were dried, the slides were sealed with neutral resin, and cell morphology was observed under a microscope and photographed.

Safranin O Staining Assays
For in vitro study, Safranin O staining was used to evaluate the deposition of glycosaminoglycans (GAGs). Cells in all groups were fixed with 95 % alcohol for 30 min. After washing with PBS, the cell slides were soaked with Safranin O dye for 15 min, then cleaned with PBS. After natural drying, the slides were sealed with neutral resin. Cell morphology was observed under a microscope and photos were taken.

Immunofluorescence Staining Assay
The chondrocytes with a density of 1 × 10 4 cells/mL were cultured in the 24-well plates with sterile cover glasses, treated with LPS (5 nM) for 12 h, and then co-cultured with 1.5 mg/mL of the above CBD-PLGA-NPs solutions. After co-culturing for 12 h, the chondrocytes were fixed with 4 % paraformaldehyde for 10 min, and then permeabilized with 0.1 % Triton X-100 (Aladdin Bio-Chem Technology Co, Ltd., Shanghai, China) and blocked with 3 % bovine serum albumin/phosphate-buffered saline (BSA/PBS, Aladdin Bio-Chem Technology Co, Ltd, Shanghai, China) at 25°C for 15 min and 30 min, respectively. After washing with PBS, the cells were stained against the specific rat primary antibody of MMP-13 and IL-6 (at 1 : 200 dilution, Boster, Wuhan, China) overnight at 4°C. Subsequently, the cells were gently washed with PBS and reacted against the specific Alexa Fluor-coupled secondary anti-rabbit of IgG (1 : 100 dilution, Boster, Wuhan, China) at 25°C for 1 h in darkness. Meanwhile, the cell nuclei were counterstained using 4, 6-diamidino-2phenyindole dilactate (DAPI, Life Tech, USA) in darkness for 15 min, and the cell actin was labeled by Alexa Fluor 594 phalloidin (Life Tech, USA). Finally, the staining images were captured using a laser scanning confocal microscope (LSM800, ZEISS, Germany), and the expression of MMP-13 and IL-6 was investigated by the Image J software.

Statistical Analysis
All data were reported as mean � standard deviation of at least three experiments. The two-sided student t-test was used for statistical analysis. P value less than or equal to 0.05 was considered statistically significant.

Characterization of PLGA-NPs and CBD-PLGA-NPs
The average size of PLGA-NPs was 220.4 � 2.22 nm and CBD-PLGA-NPs was 212.9 � 2.47 nm in diameter ( Table 2). The sizes of all the NPs are within the range of NPs able of active cellular uptake via endocytosis. [31] The NPs were all negatively charged as determined by Zeta potential. The NPs showed a good stability since the Zeta potential was between À 20 mV and À 10 mV values typically indicative of moderate NPs stability. [32][33][34] TEM was performed to analyze the morphology of PLGA-NPs and CBD-PLGA-NPs. As shown in Figure 2A&2B, PLGA-NPs and CBD-PLGA-NPs had regular spherical morphologies with an average diameter of 200 nm. CBD did not much change the morphology of PLGA-NPs and CBD-PLGA-NPs presented well dispersion. The particle size determination of PLGA-NPs and CBD-PLGA-NPs was reported by Malvin particle size analyzer. The average particle size of PLGA-NPs and CBD-PLGA-NPs is consistent with TEM results.

Drug Release from CBD-PLGA-NPs
The CBD-loading and encapsulation efficiency of CBD-PLGA-NPs were measured using HPLC. The results showed that CBD-PLGA-NPs exhibited high drug-loading and encapsulation efficiency, which were up to 22.1 � 2.87 % and 88.4 � 11.49 %, respectively. The release behavior of CBD from CBD-PLGA-NPs was further tested. CBD concentrations were obtained using an acceptable standard curve (Figure 3A), and the standard curve formula is as Table 1. Primers for RT-PCR performance.

ChemBioChem
Research Article doi.org/10.1002/cbic.202200698  follows: Y = 99093X + 380819, R2 = 0.9961. The good linear relationship between concentration and peak area in various concentration CBD means that CBD released from CBD-PLGA-NPs concentration would be detected accurately. As shown in Figure 3B, CBD was sustained released in the detection period, which is up to 60 days, indicating that the CBD-PLGA-NPs have the function of long-term slow release.

Anti-Inflammatory Assay
CCK8 assessment was performed to detect the anti-inflammatory activity of CBD-PLGA-NPs. As shown in Figure 4, after LPS induction, CBD-PLGA-NPs treated cells viability decreased to 67.92 % at the concentration of 30 μg /mL, while the viability of the cells was not different compared with the control group at concentrations below. Therefore, 20 μg/mL of CBD-PLGA-NPs were applied for the subsequent experiments.

Cell Activity Tests
The live/dead cell staining assay revealed that cell survival was obviously decreased with the stimulation of LPS, as evidenced by more dead cells and less viable cells in the control group ( Figure 5). While the treatment of CBD or CBD-PLGA-NPs showed weaker red fluorescence light than the control group, indicating that the cell survival rate was significantly increased. Particularly, more viable cells were observed from the CBD-PLGA-NPs group, suggesting that CBD-PLGA-NPs can be served as an effective inhibitor against LPS-induced cell dead in chondrocytes.

Detection of IL-1β, IL-6, TNF-α and MMP-13 Gene Expression
Gene expression levels of cartilage-specific markers including IL-1β, IL-6, TNF-α, and MMP-13 were analyzed by qRT-PCR to  explore the effect of CBD-PLGA-NPs on chondrocytes. The levels of IL-1β, IL-6, TNF-α, and MMP-13 were notably increased by the stimulation of LPS ( Figure 6). CBD-PLGA-NPs remarkably decreased the damage in chondrocytes that were stimulated by LPS. Furthermore, among the CBD and CBD-PLGA-NPs treated groups, most minimized changes in phenotype loss of chondrocytes were shown in CBD-PLGA-NPs group. The expression levels of IL-1β, IL-6, TNF-α, and MMP-13 genes were increased when LPS-induced inflammation compared with the control group (P < 0.05). In contrast, the expression levels of IL-1β, IL-6, TNF-α, and MMP-13 genes were significantly decreased after treatment with CBD-PLGA-NPs (P < 0.05), which indicated that CBD-PLGA-NPs could down-regulate the expression of inflammation-related genes.

Hematoxylin and Eosin (H&E) Staining
H&E staining was used to detect the effect of CBD-PLGA-NPs on the morphology of chondrocytes. The morphology of chondrocytes in the control group all showed the perfect condition. While chondrocytes were transformed into elongated and fibroblast-like ones and lost the typically fusiform-like shape when treated with LPS ( Figure 7). CBD-PLGA-NPs treatment reversed the morphology of LPS-treated chondrocytes to polygonal and round shape. CBD can also recover several chondrocytes. Yet, the recovered ratios were much lower than that CBD-PLGA-NPs treatment group, which indicated that CBD-PLGA-NPs showed better chondrocytes protection function.

Safranin O Staining
To further verify the effect of CBD-PLGA-NPs on the extracellular matrix (ECM) protection of chondrocytes, safranin O staining, which stained the glycosaminoglycan (GAG, a main component of ECM of chondrocytes) was detected. As shown in Figure 8, LPS treatment group cells showed less red, indicating that LPS caused obvious GAG loss compared with the control group. The GAG of CBD-PLGA-NPs treatment group was similar to the control group, suggesting that CBD-PLGA-NPs had the most significant potent inhibition on GAG loss. What's more, the chondrocyte morphology was similar to those in the control group, which was consistent with H&E staining results.

IL-6 and MMP-13 Protein of Chondrocytes Analysis
The effect of CBD-PLGA-NPs on the expression of inflammatory factors IL-6 and MMP-13 in chondrocytes was detected by immunofluorescence assay (Figure 9). The secretions of IL-6 and MMP-13 were the features of OA. The results of immunofluorescence staining showed that obvious green fluorescence appeared in the LPS treatment group, indicating the high expression level of IL-6 and MMP-13 in the LPS group. However, the green fluorescence of chondrocytes that were treated with CBD-PLGA-NPs was hardly observed. These results suggested that CBD-PLGA-NPs exhibited a better anti-inflammatory against OA chondrocytes, which would be helpful for OA therapy.

Discussion
OA is a chronic degenerative disease, mainly due to the invasion of inflammatory factors and the imbalance of oxidative stress balance. [35,36] Inhibiting the expression of inflammatory factors is an efficient way to treat OA. [37] CBD has been shown anti-inflammatory properties in previous studies. [38] Thus, it is a promising candidate for treating OA. However, the low solubility and bioavailability limit its further application in OA treatment. Developing a simple and effective drug delivery system that can sustainably release CBD is meaningful for increasing CBD utilization. Takahashi et al. showed that oral administration of a liposome-encapsulated CBD to rats could significantly increase the bioavailability and cellular uptake efficiency of CBD compared to free CBD. [39] The pharmacokinetics in vivo revealed that CBD-entrapped nano-particles demonstrate at least a 9-fold increase in oral bioavailability when compared to CBD administered with piperine as an absorption enhancer. [40][41][42] PLGA is widely used as a drug carrier due to its biodegradable and drug control release characteristic. In this study, CBD and PLGA were used to fabricate CBD nano drug delivery system (CBD-PLGA-NPs) aimed to explore the therapeutic effect of CBD on LPS-induced inflammation of chondrocytes. CBD-PLGA-NPs were synthesized by ultrasonic with uniform particle size and presented slow drug release characteristics. The emulsion-solvent evaporation method was successfully performed to generate PLGA-NPs and CBD-PLGA-NPs, which showed a burst release fashion profile. This method allowed quantification and optimization of drug encapsulation with a concomitant control of the NPs size. the method applied in this study is a promising avenue for the future research in NPs therapy, since the compounds are entrapped into the shell  of the NPs reducing their potential loss in solution. CBD-PLGA-NPs achieved continuous release, which may avoid the CBD rapid clearance in the joint cavity, thus resulting improve CBD treatment effect.
Furthermore, the anti-inflammatory activity detection showed that CBD and CBD-PLGA-NPs significantly inhibited the OA development-related gene expression, such as IL-1β, IL-6, TNF-α, and MMP-13 genes. It is worth noting that CBD-PLGA-NPs performed a better inhibition effect than free CBD. These studies demonstrated that CBD-PLGA-NPs could enhance CBD chondroprotective effects against LPS-induced inflammation of rat chondrocytes. The enhancement of chondroprotective effects of CBD encapsulated in PLGA may relate to an increase in CBD bioavailability and stability.
Further detecting the anti-inflammatory of CBD-PLGA-NPs, the results revealed that CBD-PLGA-NPs could reverse hypocellularity in LPS-induced chondrocytes, which was shown better than the free CBD. Reducing cellularity is a characteristic feature of OA cartilage. Recent studies have shown a positive correlation between the degree of severity of OA and chondrocyte loss in both experimentally induced OA in rabbit cartilage and human OA cartilage. Actually, saving the plausible number of cartilage cells in the joint articular structure is important in OA pathology and progression, because chondrocytes are the only component capable of controlling vital activities of the articular cartilage.
As for cartilage tissue engineering, extracellular matrix (ECM) proteins in cartilage are of great significance for the regulation of cell behavior, proliferation, differentiation, and morphogenesis. [43][44][45] Earlier studies have reported that LPS has an inhibitory effect on the activity of glyceraldehydes-3 phosphate dehydrogenase in chondrocytes resulting in disruption of glycolysis, hydration of the extracellular matrix, increased extractability as well as reduced quantity and synthesis of proteoglycans, and eventually leads to cell death. In addition to the change of cellularity, the ECM was observed reduced in LPS-treated cells, which suggested that the cells were damaged by LPS. Proteoglycan depletion may be secondary to cell loss due to the OA process. CBD-PLGA-NPs treated cells not only increase cellularity but also efficiently prevent LPS-induced ECM degradation.
It is a possibility that CBD-PLGA-NPs penetrate the chondrocytes and stimulates glycosaminoglycan and proteoglycan synthesis. Study has revealed that CBD can stimulate matrix synthesis by restoring glycosaminoglycan synthesis. [46] Histological examination using Mankin scoring showed that intraarticular injection of LPS in rat knee joints induced cartilage structural changes, matrix degradation, and chondrocyte disorganization. [47,48] What's more, MMP-13, a key enzyme of cartilage matrix degradation in osteoarthritis, expression was also inhibited.

Conclusion
In summary, CBD-PLGA-NPs were successfully developed with an upcoming and promising methodology to demonstrate the efficiency in drug delivery for treatment of osteoarthritis. This study aimed at characterizing and testing the effects of CBD-PLGA-NPs in OA nanotherapy. The results from in vitro experiments indicate that CBD-PLGA-NPs released the content loaded in a stable release fashion. Additionally, CBD-PLGA-NPs was not toxic to chondrocyte cells. The fabricated CBD-PLGA-NPs could effectively enhance the chondroprotective effects of CBD by inhibiting the expression of inflammatory factors, increasing cellularity, and improving structural changes, which can be regarded as a potential system to treat OA.