Poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles as a platform for the improved oral delivery of cannabidiol

Cannabidiol (CBD), a non-psychoactive constituent of Cannabis, has proven neuroprotective, anti-inflammatory and antioxidant properties though his therapeutic use, especially by the oral route, is still challenged by the poor aqueous solubility that results in low oral bioavailability. In this work, we investigate the encapsulation of CBD within nanoparticles of a highly hydrophobic poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer produced by a simple and reproducible nanoprecipitation method. The encapsulation efficiency is ~ 100% and the CBD loading 11% w/w (high performance liquid chromatography). CBD-loaded nanoparticles show a monomodal size distribution with sizes of up to 100 nm (dynamic light scattering), a spherical morphology, and the absence of CBD crystals (high resolution-scanning electron microscopy and cryogenic-transmission electron microscopy) which is in line with a very efficient nanoencapsulation. Then, the CBD release profile from the nanoparticles is assessed under gastric- and intestine-like conditions. At pH 1.2, only 10% of the payload is released after 1 h. Conversely, at pH 6.8, a release of 80% is recorded after 2 h. Finally, the oral pharmacokinetics is investigated in rats and compared to a free CBD suspension. CBD-loaded nanoparticles lead to a statistically significant ~ 20-fold increase of the maximum drug concentration in plasma (Cmax) and a shortening of the time to the Cmax (tmax) from 4 to 0.3 h, indicating a more complete and faster absorption than in free form. Moreover, the area-under-the-curve (AUC), a measure of oral bioavailability, increased by 14 times. Overall results highlight the promise of this simple, reproducible, and scalable nanotechnology strategy to improve the oral performance of CBD with respect to common oily formulations and/or lipid-based drug delivery systems associated with systemic adverse effects.


Introduction
Cannabinoids are being explored as active pharmaceutical ingredients with potential therapeutic applications in a broad spectrum of medical conditions that include cancer, diseases of the central nervous system (e.g., epilepsy), inflammation and neurodevelopmental disorders (e.g., autism spectrum disorder) [1][2][3][4][5].More than 140 different cannabinoids have been reported in the literature, the most abundant and most extensively studied of which are (−)-Δ 9 tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD) [6,7].While Δ 9 -THC is best known for giving the cannabis plant its psychotropic effects, over the past few years, CBD, a non-psychoactive cannabinoid [8][9][10], has been gaining considerable attention for its potential therapeutic benefits as anti-cancer, anti-inflammatory and anti-seizure agent with antioxidant properties [2,11,12].Despite its promising clinical potential, CBD displays high lipophilicity (logP = 6.3) and low aqueous solubility (~ 10 mg/L) [13] and it is classified into Class II of the Biopharmaceutic Classification System [14].These physicochemical properties challenge the development of pharmaceutical formulations and leads to low and variable oral bioavailability in the 6-19% range [10,15].The oral bioavailability is also reduced due to extensive hepatic first-pass metabolism [8,16].The oral administration of CBD is commonly carried out in oil or ethanolic formulations (tinctures) for swallowing, sublingual drops, or as an oromucosal spray [12].However, CBD lipid-based formulations often undergo digestion in the gastrointestinal tract, which can result in CBD precipitation and limited gastrointestinal absorption [17].In addition, CBD solutions in edible oils have shown variable and delayed absorption [18,19], which results in unpredictable oral pharmacokinetics (PK) [20,21].Some oils have also shown adverse effects such allergy [19].Currently, only a few CBD formulations are available on the market.Sativex ® (GW Pharmaceuticals), a CBD and Δ 9 -THC (1:1 ratio) sublingual spray, has been approved for use in the UK, Spain, Canada, New Zealand, and Israel [22,23].It contains ethanol (EtOH) and propylene glycol as solubilizing agents, which may cause irritation in the administration site, and, over a long-period treatment may be harmful to the oral mucosa.Epidiolex ® (GW Pharmaceuticals) is an oral CBD solution in sesame oil as the primary vehicle, with dehydrated EtOH, strawberry flavoring, and sucralose excipients and it is the first and only Food and Drug Administration (FDA)-approved CBD drug product in the USA [24,25].It is prescribed only for Lennox-Gastaut syndrome, Dravet syndrome, or tuberous sclerosis, rare and severe syndromes characterized by seizures.Yet, as mentioned before, the FDA is concerned about allergies caused by sesame oil, the main excipient in this product [21].
To overcome these limitations, other administration strategies and oral delivery formulations are being explored [26,27].Nanomedicine demonstrated the ability to improve the performance of drugs with different physicochemical properties [28][29][30].Polymeric nanoparticles are one of the most versatile nanotechnology platforms due to the ability to tailor their features (e.g., composition, hydrophilic-lipophilic balance, size, degree of crystallinity/amorphousness and nanostructure) to fit the cargo of interest [31] and synthesize them by various conventional and advanced methods such as self-assembly, electrospray and spray-drying [32][33][34].
Poly(epsilon-caprolactone) (PCL) is one of the most wellinvestigated synthetic aliphatic biomedical polyesters for the development of drug delivery systems, owing to biodegradability, biocompatibility, and good encapsulation capacity and release, especially of hydrophobic payloads [35,36].PCL nanoparticles can be produced by a simple and reproducible nanoprecipitation method [36,37].We previously reported on the encapsulation of the antiretroviral efavirenz within PCL nanoparticles which leads to a statistically significant increase of its oral bioavailability with respect to an aqueous drug suspension [38].Moreover, the synthesis of nanoparticles made of block copolymers of PCL and a relatively small content (up to ~ 20% w/w) of poly(ethylene glycol) (PEG) by a nanoprecipitation method preserves their encapsulation capacity for hydrophobic molecules, and results in a better control of the size and size distribution and very good physical stability in suspension when compared to pure PCL counterparts [39].The steric stabilization mechanism relies on the formation of a hydrated surface due to the migration of PEG blocks to the nanoparticle surface during its formation by nanoprecipitation in water [40].Poly(ethylene glycol)-b-poly(epsilon-caprolactone) (PEG-b-PCL) nanoparticles have been widely investigated in the last decades as drug nanocarriers owing to their very good biocompatibility and have been approved by FDA [41,42].
Aiming to investigate simple, reproducible, and scalable nanotechnology strategies to improve the oral bioavailability of CBD and overcome the drawbacks of oily formulations that are often associated with adverse effects, in this work, we synthesized CBD-loaded PEG-b-PCL nanoparticles by a nanoprecipitation method and after a through characterization in vitro, the oral PK was compared to a CBD suspension in rat.Overall, our results highlight the promise of this nanodrug delivery platform for the improved oral administration of hydrophobic cannabinoids.

Materials
All the reagents and solvents were used as received and without further purification, if not otherwise stated.

Synthesis and characterization of poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer
A PEG-b-PCL triblock copolymer was synthesized via a ring opening polymerization (ROP) reaction of epsiloncaprolactone (CL, Sigma-Aldrich, St. Louis, MO, USA) initiated by the terminal hydroxyl group of the monofunctional methoxy PEG (mPEG4000, molecular weight -MW -4000 g/mol, Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) in the presence of tin(II) 2-ethylhexanoate (Sn(Oct) 2 , Sigma-Aldrich) as catalyst.Both CL and Sn(Oct) 2 were dried prior to use with molecular sieves 3A (Alfa Aesar, Ward Hill, MA, USA) pre-activated at 150 °C (4 h) at least 24 h before use.Feed weights of the reactants were calculated based on the desired MW of the PCL block (~ 30,000 g/ mol).For this, mPEG (0.5 g) was weighed, poured into a round-bottom flask with a magnetic stirring bar, melted at 100 °C, dried under vacuum for 2 h, allowed to cool at 25 °C and the flask was sealed with a septum under dry nitrogen environment conditions, which were maintained throughout the reaction.Next, CL (3.75 g) was added to the liquid by injection through the septum, the reaction mixture heated to 145 °C, Sn(Oct) 2 (1:200 molar ratio to CL) added to the reaction mixture and the reaction allowed to proceed at 145 °C for 2.5 h.The reaction mixture was cooled to 25 °C, dissolved in dichloromethane (20 mL, Sigma-Aldrich) and precipitated in an excess of diethyl ether (500 mL).The precipitated PEG-b-PCL copolymer was filtered in a Büchner funnel using filter paper (Whatman™ 1, Sigma Aldrich) to remove remaining unreacted reagents, washed several times with diethyl ether, vacuum-dried at 25 °C (Vacuum Oven Lab-Line Instruments Inc., Dubuque, IL, USA) until constant weight and stored at -24 °C until use.
The weight-average molecular weight (M w ), the numberaverage molecular weight (M n ) and the dispersity (Ð, M w / M n ) of the PEG-b-PCL copolymer were measured by gel permeation chromatography (GPC) in a Viscotek 270max GPC system (Malvern Instruments, Malvern, UK) equipped with refractive index, viscometer, and light scattering detectors.The copolymer was dissolved in tetrahydrofuran (THF, 5 mg/mL) for 24 h and a THF solution containing 200 ppm of butylated hydroxytoluene was used as the mobile phase.The injection volume was 100 μL and the chromatographic separation was performed using three LT4000L-Mixed bed (300 × 8.0 mm) columns at a flow rate of 1.0 mL/min and a temperature of 35 °C.The instrument calibration was done with 105,000 and 245,000 g/mol polystyrene standards (Malvern Instruments).
The proton-nuclear magnetic resonance ( 1 H-NMR) spectrum of the copolymer was recorded in a 1% w/v solution of deuterated acetone (acetone-d 6 , Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA).Chemical shifts are reported in ppm using the signal of acetone at 2.05 ppm as internal standard.

Synthesis and characterization of poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles
PEG-b-PCL nanoparticles were synthesized using a nanoprecipitation method with homogenization.Briefly, PEG-b-PCL (50 mg) was dissolved in acetone (10 mL, Bio-Lab Ltd., Jerusalem, Israel), a beaker with deionized water (50 mL, the anti-solvent) was put in a water bath with controlled temperature (23 °C, Hei-Tec Magnetic Stirrer, 600 rpm, Heidolph Instruments, Schwabach, Germany) and homogenized at 12,100 rpm with a homogenizer (POLYTRON ® PT 2500 E, Kinematica AG, Luzern, Switzerland).Then, the acetone solution of the copolymer was added at once, homogenized for 16 min and kept under constant magnetic stirring (600 rpm) for 6 h to allow complete acetone evaporation.The nanoparticle suspension was kept at 4 °C until use.To produce CBD-loaded nanoparticles, CBD (6 mg) was added to the copolymer solution in acetone (50 mg in 10 mL), allowed to dissolve overnight protected from light and the process conducted as described above.
To quantify the CBD encapsulation efficiency (%EE) and loading (%DL) in the nanoparticles, we used analytical reversed phase ultra-high performance liquid chromatographyultraviolet detector (HPLC-UV, UltiMate 3000, Thermo Fisher Scientific UHPLC, Bremen, Germany) equipped with a photodiode array (PDA) detector with a Kinetex C18 core-shell column (2.6 μm, 150 mm × 2.1 mm i.d.) and a guard column (0.5 μm depth filter × 0.1 mm, Phenomenex, Torrance, CA, USA) and an A/B isocratic method (solvent A: 0.1% acetic acid in water, solvent B: 0.1% acetic acid in acetonitrile).The isocratic program conditions were: 15% A and 85% B for 5 min.A flow rate of 0.25 mL/min was used, the column temperature was held at 30 °C and the injection volume was 1-4 μL.Liquid chromatography/mass spectrometry (LC/MS) grade acetonitrile, water for the mobile phase and HPLC grade EtOH for sample preparation were obtained from Mercury Scientific and Industrial Products Ltd. (Rosh Ha'ayin, Israel).LC/MS grade acetic acid was purchased from Sigma-Aldrich (Rehovot, Israel).The CBD content quantified by HPLC-UV was used to calculate %EE according to Eq. 1 and the %DL according to Eq. 2 Each nanoparticle suspension was injected to the HPLC-UV in duplicates and results expressed as mean ± S.D of quintuplicates (n = 5).
The hydrodynamic diameter (D h ) and polydispersity index (PDI) of CBD-free and CBD-loaded PEG-b-PCL nanoparticles (0.1% w/v of nanoparticles in suspensions) were measured at 25 and 37 °C by dynamic light scattering (DLS, Zetasizer Nanoseries ZS90, Malvern Instruments) at a scattering angle of 173°.Results are expressed as mean ± S.D. of at least three measurements.DLS data were analyzed using CONTIN algorithms (Malvern Instruments).Zeta-potential (Z-potential) measurements were carried out by using laser Doppler micro-electrophoresis in the same instrument.The morphology of CBD-loaded nanoparticles was visualized by high resolution-scanning electron microscopy (HR-SEM, 0.02-30 kV Zeiss Ultra-Plus FEG-SEM, Carl Zeiss NTS GmbH, Oberkochen, Germany).Samples were prepared by spraying 20 μL of a sample on a silicon wafer (CZ polished silicon wafers < 100 > oriented, highly doped N/Arsenic, SEH Europe Ltd., Livingston, UK) by using nitrogen gas flow to cast small droplets over the wafer, and by doing so, ensuring a thin layer of particles distributed on it.The suspension was used immediately after nanoprecipitation and did not undergo freeze-drying.This process was repeated several times, and the sample was allowed to dry for at least 24 h at 25 °C and atmospheric pressure.The acceleration voltage was 1.5 kV, and the images were acquired using in-lens secondary electrons at 2-3 mm working distance.The size of 25 nanoparticles was measured using ImageJ 1.8 software (National Institutes of Health, Bethesda, MD, USA), and expressed as mean ± S.D.
Imaging was also performed by high resolution-cryogenictransmission electron microscopy (HR-cryo-TEM) in a FEI T12 G2 electron microscope (FEI, Hillsboro, Oregon, US) operated at 120 kV.Images were recorded digitally by a Gatan US 1000 high-resolution CCD camera (Tecnai T12 G2, FEI), using the DigitalMicrograph ® software.A drop (3 µL) of each sample was placed on a carbon-coated perforated polymer film, supported on a 200 mesh TEM grid (Ted Pella, Inc., Redding, CA, USA), and mounted on a tweezer.The drop was turned into a thin film (preferably less than 300 nm) by blotting away excess solution with a filter paper-covered metal strip.The grid was then plunged quickly into liquid ethane at its freezing point (183 °C).Prior to the specimen preparation, grids were plasma etched in a PELCO EasiGlow glowdischarger (Ted Pella, Inc.) to increase their hydrophilicity.The size of 25 nanoparticles was measured using ImageJ 1.8 software, and expressed as mean ± S.D.

CBD release in vitro
Since the CBD-loaded nanoparticles are envisioned for oral administration, the CBD release in vitro was studied under gastric-like (pH = 1.2, 2 h, n = 3) and intestine-like conditions (pH 6.8, 24 h, n = 3).
For gastric-like conditions media of 0.1N HCl (37%, 0.1 M pH = 1.2, Merck Chemicals GmbH) containing 0.1% w/v TWEEN ® 80 (Merck Chemicals GmbH).The solution was prepared as follows: 4 mL of HCl was added to 125 mL of distilled water and the final volume adjusted to 500 mL with distilled water; intestine-like conditions were obtained by using 0.1 M PBS (pH = 6.8) containing 0.1% w/v TWEEN ® 80 for 0-24 h.The PBS was prepared as follows: 87 g of potassium phosphate dibasic (K 2 HPO 4 , Spectrum Chemical MFG Corp., Gardena, CA, USA) and 68 g of potassium phosphate mono basic (KH 2 PO 4 , Merck Chemicals GmbH) were dissolved separately in 0.5 L of distilled water and mixed until complete dissolution.Then, 50.3 mL of KH 2 PO 4 and 49.7 mL of K 2 HPO 4 were added to a 1 L measuring flask and distilled water was added to complete 1 L.
For both the gastric-and intestine-like release tests, 1.35 mL of nanoparticle suspension (CBD concentration of 0.12 mg/mL) was added to 250 mL of the release medium and the suspension were shaken (150 rpm) in an Excella E25 shaker incubator (New Brunswick Scientific, Edison, NJ, USA) at 37 °C.At different timepoints, aliquots were taken (1 mL, was done in duplicates) and pre-heated fresh release medium was added to maintain a constant volume.Aliquots were ultracentrifuged at 180,000 G, at 4 °C, for 30 min (Sorvall Discovery M120 SE Micro-Ultracentrifuge, Thermo Fisher Scientific).Then, two aliquots of 300 μL were taken at each timepoint, frozen and dried by SpeedVac for 13 h, followed by CBD extraction from each with 300 μL of EtOH.Each CBD solution in EtOH obtained from the extraction of one release aliquot was injected into the HPLC-UV (as detailed above) in duplicates for CBD quantification, and the results expressed as mean ± S.D.
First, the CBD release rate profile data were fitted using Higuchi, first-order, and Hixson-Crowell models; our results did not fit them.The Korsmeyer-Peppas model is very popular, though only release data up to 60% can be used, which was not possible in this work due to the relatively fast CBD release.Thus, we modeled our data to a hyperbolic tangent (tanh) function model proposed by Eltayeb et al. [43] that is based on the diffusive release model by Peppas and collaborators, but approximates the release to 100%, as expressed by Eq. 3 where Q ∞ is the total fraction of CBD released from the nanoparticles, Q t is the fraction released in time t and α is a constant related to the particle size and diffusion constant.The model fitting was done with Python.

Cryo/lyoprotection of the CBD-loaded nanoparticles
To stabilize the CBD-loaded nanoparticles during freezedrying and enable redispersion towards in vivo studies, 2-hydroxy-propyl-beta-cyclodextrin (Hpβ-CD, Glentham Life Sciences, Corsham, UK) was tested as a cryo/lyoprotectant.For this, Hpβ-CD was added to the CBD-loaded nanosuspension (DL% = 11%, CBD concentration of 0.012% w/v and nanoparticle concentration of 0.1% w/v) and their ability to preserve the nanoparticles was assessed in a threestep experiment.After each step, the D h (measured by intensity and number) and the PDI were measured by DLS at 25 °C.The first step comprised the addition of the cryo/ lyoprotectant at concentrations of 1-15% w/v.In the second, the cryo/lyoprotection efficiency was assessed in a freezingthawing cycle at -196 and 24 °C, respectively.For this, the cryo/lyo-protectant was added to the CBD-loaded nanoparticle suspensions, the system frozen in liquid nitrogen, thawed at 25 °C and the D h and PDI of the nanoparticles before and after the freeze-thawing cycle compared.The cryo/lyoprotectant concentration that preserved the nanoparticle properties was used for the third step that was the freeze-drying of the suspension for 72 h (Labconco Free Zone 4.5 plus L Benchtop Freeze Dry System, Labconco Corp., Kansas City, MO, USA), and its resuspension.Based on that assay, Hpβ-CD concentrations of 2.5%, 4.5% and 6% w/v were chosen to cryo/lyoprotect and concentrate the CBD-loaded nanoparticle suspension upon freeze-drying and redispersion by 6.5 and 12.5-fold for the pharmacokinetic (PK) study.Nanoparticles were characterized with DLS at 25 °C.

Comparative oral CBD pharmacokinetics in rat
An oral PK study was conducted at "Science in Action" (Ness Ziona, Israel) and following the ethical guidelines of animal experimentation.Male rats (Sprague Dawley, ~ 200 g, 7-8 weeks old) were allowed to acclimatize for one week, then fasted for 4 h with free access to water, and randomly divided into two groups (n = 5).The control group was administered by gavage 2 mL of a pure CBD suspension prepared as followed: 50 mL water was added to 72.5 mg CBD (i.e., 2.9 mg CBD per gavage) and 9.2 mg of Cremophor EL ® (Merck Chemicals GmbH), vortexed and the suspension was stirred for 30 min with a magnetic stirrer (300 rpm).The treatment group was administered 2 mL of CBD-loaded nanoparticle suspensions prepared as described before ("Synthesis and characterization of poly(ethylene glycol)-b-poly(epsiloncaprolactone) block copolymer" section) with CBD concentration of 0.12 mg/mL.Then, the nanosuspension (24 mL) was added to 1.1 g of Hpβ-CD, vortexed, and freeze-dried (Labconco Free Zone 4.5 plus L Benchtop Freeze Dry System) for 72 h.Prior to the gavage administration, the dry powder of CBD-loaded nanoparticles and cryo/lyoprotectant containing 2.9 mg CBD was re-suspended in water by vortex and magnetic stirring (300 rpm, 30 min).Both rat groups were administered a CBD dose of 14.5 mg/kg.Blood samples (100 μL) were taken from the cheek vein at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h post-administration.Then, rats were euthanized by CO 2 inhalation.
Briefly, blood samples were centrifuged for 30 min at 4 °C, plasma was extracted by a vigorous vortex with a solution consisting of methanol:acetonitrile:acetic acid at a 50:50:0.1 volume ratio and spiked with CBD, 7-OH-CBD and 7-COOH-CBD deuterated internal standards.All samples were centrifuged (14,000 rpm) for 20 min at 4 °C.Volumes of 0.7 mL were collected from plasma supernatants, diluted with 3 mL 0.1% v/v acetic acid in water, and loaded onto C8 solid phase extraction cartridges and loaded onto pre-conditioned Agela Cleanert C8 solid phase extraction cartridges (SPE, 500 mg of sorbent, 50 μm particle size, Agela Technologies, CA, USA) using 5 mL of 0.1% v/v acetic acid in methanol followed by 3 mL of water with 0.1% v/v acetic acid.CBD was eluted from the SPE columns with 2 mL 0.1% v/v acetic acid in methanol, evaporated to dryness by SpeedVac, reconstituted in 100 μL ethanol and filtered through a 0.22 μm PTFE syringe filter (Silicol Scientific Equipment Ltd., Or Yehuda, Israel).
HPLC-MS analyses were performed using a Thermo Fisher Scientific ultra HPLC system coupled with a Q Exactive™ Focus Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Scientific).The chromatographic separation was achieved using a Halo C18 Fused Core column (2.7 μm, 150 mm × 2.1 mm i.d.) with a guard column (2.7 μm, 5 mm × 2.1 mm i.d) (Advanced Materials Technology, Wilmington, DE, USA) and a ternary A/B/C multistep gradient (solvent A: 0.1% acetic acid in water, solvent B: 0.1% acetic acid in acetonitrile, and solvent C: methanol).The multistep gradient program was set as follows: Initial conditions were 50% B raised to 67% B until 3 min, held at 67% B for 5 min, and then raised to 90% B until 12 min, held at 90% B until 15 min, decreased to 50% B over the next min, and held at 50% B until 20 min for re-equilibration of the system prior to the next injection.Solvent C was initially 5% and then lowered to 3% until 3 min, held at 3% until 8 min, raised to 5% until 12 min and then kept constant at 5% throughout the run.A flow rate of 0.25 mL/min was used, the column temperature was 30 °C and the injection volume was 1 μL.MS acquisition was carried out with a heated electro spray ionization ion source operated in switching mode.The source parameters were similar for both negative and positive modes: sheath gas flow rate, auxiliary gas flow rate and sweep gas flow rate: 50, 20 and 0 arbitrary units respectively; capillary temperature: 350 °C; heater temperature: 50 °C; spray voltage: 3.00 kV.Ten-point of analytical standards were prepared in EtOH.The concentration range in CBD calibration curves was 0.1-125 ng/mL.In addition, calibration curves of 7-OH-CBD in the 0.125-100 ng/mL range and 7-COOH-CBD in the 1.25-100 ng/mL range were also prepared.All the calibration curves were determined according to the weighted least-squares linear regression method with a weighting factor of 1/X, R 2 = 0.99.Areaunder-the-curve (AUC) values were calculated directly from the serum concentration-time curve using the linear trapezoidal method for all eight timepoints.

Statistical analysis
Non-compartmental analysis of PK parameters was performed using the PKSolver program (China Pharmaceutical University, Jiangsu, China) [45].Data was determined as mean ± SEM, significant differences among experimental groups were determined using an unpaired student t-test with p < 0.05.

Synthesis and characterization of poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer
To produce physically stable polymeric nanoparticles that display good encapsulation capacity if hydrophobic cargos, we synthesized a PEG-b-PCL copolymer with a relatively low hydrophilic-lipophilic balance that preserves the hydrophobic nature of PCL, which enables the encapsulation of a plethora of hydrophobic cargos.PEG-b-PCL nanoparticles have been widely investigated in drug delivery owing to biodegradability and excellent biocompatibility for administration by different routes.
In this context, we used a monofunctional PEG with a MW of 4000 g/mol (mPEG4000) as initiator of the ROP of CL (Fig. 1A).The feed ratios were calculated to produce a block copolymer with a theoretical MW of 30,000 g/mol and PCL content of ~ 90%.The reaction yield was ~ 63%.The synthesis of PEG-b-PCL copolymers was initially confirmed by GPC where a MW growth with respect to mPEG4000 was observed.The M w was 18,400 g/mol and the dispersity (Đ) 2.03.
The chemical characterization of the copolymer was conducted by 1 H-NMR and ATR-FTIR.The 1 H-NMR spectrum of the block copolymer showed characteristic peaks of CL and mPEG4000 (Fig. 1B).The strong peak at δ = 3.59 ppm arose from the methylene protons of the ethylene oxide repeating unit of the mPEG4000 block and at δ = 3.29 of the terminal methoxy group.The peaks at δ = 1.40 (e), 1.63 (d,f), 2.31 (c), 4.05 (g) were assigned to the protons from methylene groups of the PCL blocks [46].
A shifting of the characteristic peaks of CL was in line with a successful ROP.
ATR-FTIR analysis exhibited the characteristic bands of these copolymers (Fig. 1C).Pure CL shows a typical band at 1726 cm −1 due to the stretching vibrations of the C = O group and the bands at 2936 and 2862 cm −1 are the symmetric and asymmetric stretching vibrations of -CH 2 .mPEG4000 exhibits bands at 1101 and 1242 cm −1 of stretching vibrations of the -OCH 2 CH 2 repeating units of PEG.PEG-b-PCL displays the characteristic bands of both PEG and CL blocks.

Synthesis and characterization of poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles
Nanoparticles were produced a homogenization-assisted nanoprecipitation method that results in good encapsulation of hydrophobic cargos and the fast migration of PEG blocks to the interface between the hydrophobic PCL and the aqueous medium [39,47], as described in Fig. 2A.The D h , size distribution (estimated by the PDI) and surface charge density (estimated by the Z-potential) were analyzed by DLS, at 25 and 37 °C.
CBD-free nanoparticles showed a monomodal size distribution (one size population) with a D h of 76 ± 3 nm by intensity and 31 ± 3 nm by number, with PDI of 0.17 ± 0.01 and Z-potential of -15.0 ± 0.1 mV at 25 °C.At 37 °C, the D h was 75 ± 2 nm by intensity and 40 ± 5 nm by number, with a PDI of 0.17 ± 0.03 and Z-potential of -24.0 ± 0.3 mV.Negative Z-potential values are characteristic of PEG-b-PCL nanoparticles [41,43].The encapsulation capacity of polymeric nanoparticles depends on the polymer and the physicochemical properties of the cargo.To ensure maximum %EE and good %DL that fits preclinical PK studies so that the given nanoparticle dose enables to administer a clinically relevant CBD dose, we tailored the production method to encapsulate 11% w/w CBD.CBD-loaded nanoparticles present D h by intensity and number of 79 ± 1 and 51 ± 1 nm, respectively, and PDI of 0.10 ± 0.01, at 25 °C.The Z-potential was -15.0 ± 0.2 mV.At 37 °C, the properties were similar, D h values by intensity and number being 63 ± 1 and 40 ± 1 nm, respectively, and PDI of 0.15 ± 0.01.The Z-potential was -24.0 ± 0.3 mV.CBD-loaded nanoparticles showed good physical stability at 25 °C for at least five days, as observed for other PCL nanoparticles [41,43].The results strongly suggested that CBD is fully encapsulated within the nanoparticles.Otherwise, other size populations of free CBD particles and greater PDI values would have been measured by DLS.
The spherical morphology of CBD-loaded nanoparticles was visualized by HR-SEM (Fig. 2B).No pure CBD crystals could be observed outside the nanoparticles, in good agreement with DLS data.The average nanoparticle size estimated by HR-SEM was 49 ± 9 nm.In addition, HR-cryo-TEM analysis showed an average size of 30 ± 13 nm and the absence of CBD crystals (Fig. 2C).
To further assess the nanoencapsulation capacity of these nanoparticles, DLS data were compared to a CBD suspension produced by the same nanoprecipitation method though without the addition of the block copolymer.Pure CBD particles showed D h of ~ 200 nm and extensive aggregation with a D h of ~ 8000 nm and a bimodal size distribution after 2 h at 25 °C (data not shown).
Altogether, these results indicated that CBD was successfully nanoencapsulated within these polymeric nanoparticles.Conversely, when the drug was not fully encapsulated, several size populations and greater PDI values were measured by DLS.The CBD content in CBD-loaded nanoparticles was quantified by HPLC-UV.Results indicated %EE and %DL values of 102 ± 2 and 11 ± 1%, respectively.

CBD release in vitro
The oral route is the most patient-compliant, especially for drugs of long-term or chronic administration [48,49].In oral nano-drug delivery, the nanocarrier often serves as a reservoir that releases the drug in free form in the gut, enabling it absorption.In this context, a relatively slow release in the acid gastric medium and a fast one in the neutral intestinal one is required to maximize oral bioavailability.To evaluate the potential of CBD-loaded nanoparticle suspensions to increase its oral bioavailability with respect to pure CBD suspensions, its release profile in vitro was assessed under pH conditions that mimic the stomach (pH = 1.2) and the small intestine (pH = 6.8).The release at pH 1.2 was ~ 10% after 2 h, indicating that the limited release can probably protect CBD from chemical degradation in the stomach (Fig. 3A).
Conversely, the release at pH 6.8 reached 70% after 15 min and 80% after 1 h (Fig. 3A).The CBD release data under intestinal conditions was fitted to different release models, of which the best fitting was the tanh model [43].This model considers a diffusive release from a homogeneous particle that can be applied along the whole release process and approximates a release of ~ 100% (Fig. 3B).In this work, Q t = 0.78tanh (0.38t 0.5 ) was calculated (Fig. S1).

Cryo/lyoprotection of CBD-loaded nanoparticles
Nanosuspensions containing 11% w/w (equivalent to 0.12 mg/mL) CBD did not fit the dose and volume of formulation to administer a clinically relevant dose in vivo.A plethora of cryo/lyoprotectants has been attempted to freeze-dry polymeric nanoparticles and enable their redispersion in liquid medium with variable success [50].Cyclodextrins such as Hpβ-CD have been reported among the most efficient ones.Therefore, Hpβ-CD was used as a cryo/lyoprotectant to freeze-dry, redisperse, and concentrate the nanosuspensions.After the addition of 1%, 2.5%, 5%, 10%, and 15% w/v Hpβ-CD, the D h (by number) increased from 40 ± 1 nm to 43 ± 3, 45 ± 1, 50 ± 1, 58 ± 3 and 70 ± 6 nm, respectively, with a monomodal size distribution and PDI in the 0.11-0.16range.The size growth for the most concentrated cryo/lyoprotectant solution in the nanosuspension was most probably associated with its own self-aggregation, a well-known phenomenon that leads to the formation of nanostructures of several tens of nanometers [51].Then, the cryo/lyoprotected nanosuspension were exposed to a freezing-thawing cycle and the D h and PDI measured again; this method has been used to predict the efficiency of the freeze-drying process of PEG-b-PCLbased nanoparticles [52].The suspension without Hpβ-CD exhibited a sharp D h increase to 134 ± 12 nm (Table S1).Conversely, this cryo/lyoprotectant preserved sizes in the original range (Fig. 2D, Table S1).Finally, nanosuspensions containing different cryo/lyoprotectant concentrations were freeze-dried for 72 h, resuspended in water and the D h measured again.Results indicated that 1% w/v Hpβ-CD did not prevent their agglomeration (Table S2).Conversely, higher concentrations (2.5-10%) enabled their effective redispersion.To concentrate the nanosuspension towards in vivo studies, the nanosuspensions were added 4.5% w/v Hpβ-CD, freeze-dried, redispersed in a 12.5-fold smaller volume than the original system and the size measured again.The D h by intensity and number was 448 ± 16 and 355 ± 12 nm, respectively.

Oral CBD pharmacokinetics in rat
CBD is being pursued as a therapeutic treatment for multiple conditions, usually by oral delivery.Like many lipophilic drugs, the oral bioavailability of CBD is known to be poor due to its low water solubility and limited absorption.To assess whether the nanoencapsulation of CBD improves its oral bioavailability, free CBD and CBD-loaded nanoparticle suspensions were administered by gavage to Sprague-Dawley rats (n = 5) and the PK parameters calculated and compared (Fig. 4).
The CBD suspension resulted in a low maximum concentration (C max ) of 1.1 ± 0.4 ng/mL and the time to the drug peak concentration (t max ) was 4 h (Table 1).In contrast, a CBD-loaded nanoparticle suspension exhibited a significantly higher C max of 21.0 ± 4.1 ng/mL, representing a 19.1-fold increase.In addition, the t max  3.7 ± 1.3 51.9 ± 14.0 c was 0.3 h, indicating a dramatic increase in the intestinal CBD absorption, even when compared to previous works where a t max (in rats) of 1-8 h was reported [20].The AUC represents the total exposure to the drug experienced by the subject, and it is proportional to drug bioavailability.AUC 0.25-24 h indicated a sharp 14-fold increase from 3.7 ± 1.3 to 51.9 ± 14.0 ng/mL × h with the nanoparticles (Table 1).Remarkably, the nanoparticles also showed a bimodal absorption profile with two maxima that has been previously reported for CBD [20].
Of further interest was the detection of CBD metabolites 7-OH-CBD and 7-COOH-CBD as they can provide additional information regarding the CBD plasma profile.CBD undergoes extensive first-pass metabolism in the liver first to 7-OH-CBD and then to 7-COOH-CBD) and its metabolites are mostly excreted via the kidneys [10].Plasma concentrations of these two metabolites followed the expected trend with some delay (Fig. 5).After 0.25 h, the plasma concentration of 7-OH-CBD was higher than 7-COOH-CBD, while over time they almost equaled (2-6 h) and later 7-COOH-CBD exceeded that of 7-OH-CBD.Since both metabolites are formed after the CBD absorption, results are in good agreement with a bimodal CBD absorption profile for the nanoparticles.In the case of the free CBD suspension, metabolites could not be detected.

Conclusions
In this work, we investigated the potential of PEG-b-PCL nanoparticles to increase the oral bioavailability of CBD.Nanoparticles showing a monomodal size distribution and excellent %EE of ~ 100% and %DL of 11% w/w were produced by a simple and reproducible nanoprecipitation method.Release studies in vitro indicated that under gastric-like conditions (pH = 1.2) CBD is released to a very limited extent, which may prevent its acid degradation.Conversely, under intestinal ones (pH = 6.8), the release was fast which would make it available for absorption.Then, we studied a cryo/lyoprotection method to enable their concentration towards in vivo studies using a hydrophilic cyclodextrin of very good biocompatibility with positive results.Finally, the oral PK after the administration of free CBD and CBD-loaded nanoparticle suspensions was assessed in rat.A sharp increase in C max and AUC 0.25-24 and decrease of the t max was observed for the nanoparticles.Overall, our results provide solid background for the use of PEG-b-PCL nanoparticles for the oral delivery of this prominent cannabinoid.

Fig. 1
Fig. 1 Synthesis and characterization of the PEG-b-PCL polymer.A Synthetic pathway.B 1 H-NMR spectra of CL, mPEG4000 and the PEG-b-PCL copolymer recorded in acetone-d6.C ATR-FTIR spectra of CL, mPEG4000 and the PEG-b-PCL copolymer.The characteristic bands are indicated

Fig. 2
Fig. 2 Synthesis and characterization of CBD-loaded PEG-b-PCL nanoparticles.A Schematic representation of the synthetic method.B Representative HR-SEM micrograph of CBD-loaded nanoparticles.C,D Representative HR-cryo-TEM micrograph of C CBD-loaded

Table 1
Pharmacokinetics parameters after oral administration (gavage) of a free CBD suspension and 11% w/w CBD-loaded nanoparticles to Sprague-Dawley rats.The dose was 14.5 mg/kg.Results are expressed as mean ± S.E.M. (n = 5)