Carrier Free Delivery System of Monoacylglycerol Lipase Hydrophobic Inhibitor for Cancer Therapy

Background: Monoacylglycerol lipase (MAGL) is an emerging therapeutic target for cancer. It is involved in lipid metabolism and its inhibition impairs many hallmarks of cancer including cell proliferation, migration/invasion and tumor growth. For these reasons, our group has recently developed a potent reversible MAGL inhibitor (MAGL23), which showed promising anticancer activities. Here in, to improve its pharmacological properties, a nanoformulation based on nanocrystals coated with albumin was prepared for therapeutic applications. MAGL23 was solubilized by a nanocrystallization method with Pluronic F-127 and Cetyltrimethylammonium bromide (CTAB) as surfactants into an organic solvent and was recovered as nanocrystals in water after solvent evaporation. Finally, the solubilized nanocrystals were stabilized by human serum albumin to create a smart delivery carrier. Results: An in-silico prediction (lipophilicity, structure at different pH and solubility in water), as well as experimental study (solubility), have been performed to check the chemical properties of the inhibitor and nanocrystals. The solubility in water increases from less than 0.01 mg/mL (0.0008 mg/mL, predicted) up to 0.82 mg/mL in water. The formulated inhibitor maintained its potency in ovarian and colon cancer cell lines as the free drug. Furthermore, the system was thoroughly observed at each step of the solubilization process till the nal formulation stage by different spectroscopic techniques and a comparative study was performed to check the effects of Pluronic F-127 and CTAB as surfactants. The formulated system is favorable to release the drug at physiological pH conditions (at pH 7.4, after 24h, less than 20% of compound is released). Conclusions: As per our knowledge, we are reporting the rst ever nanoformulation of a which

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Read Full License Background Drug design and combinatorial chemistry have been exploited to develop new drugs, which are generally characterized by high molecular weight, lipophilicity that assist drug to transfer across membranes [1] and therefore low solubility in water [2].Indeed 40-70% of new molecules developed in the context of drug discovery programs are not enough soluble in aqueous media, thus compromising the bioavailability of the drug [3].Therefore, in order to became commercially available (in general almost 10-17 years after its rst synthesis [4]) a poorly soluble drug needs to be formulated through drug delivery technologies.
Many drug discovery programs are dedicated to cancer treatment, which is the second major disease that kills thousands of people around the globe every year.Indeed, despite several therapeutic strategies have been employed to cure cancer, there is still no accurate way to eradicate it by roots [5,6] and chemotherapy which is considered one of the major treatment tools, suffers from several issues and even failure [7,8].To overcome traditional chemotherapy issues, current approaches are focused on the discovery of drugs that target speci c receptors or proteins mostly upregulated in tumor cells.This strategy takes part of a wider concept termed personalized therapy in which each patient is treated with speci c targeted drugs based on the genotype and phenotype.Several research groups are exploring many genes, receptors, and enzymes that are commonly involved in many cancer types and could be a therapeutic target [6,9,10].Among the enzymes, monoacylglycerol lipase (MAGL) is a member of the serine hydrolase superfamily, which primarily degrades the endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid and glycerol in the brain, thus interrupting its signaling on cannabinoid receptors.
MAGL is also involved in the hydrolysis of monoacylglycerols in peripheral tissues [11].For this reason, in cancer cells MAGL was reported to activate oncogenic signaling related to the intracellular pool of free fatty acids [12] and it proved to be involved in sustaining the growth of different cancer cells [13][14][15].Recently, our group developed a reversible and selective MAGL inhibitor (MAGL23, Figure 1), which inhibits the target enzyme with a potency in the nanomolar range (K i = 39 nM) [16].MAGL23 is a compound with suitable properties to be further developed as a drug because it is obtained by a few steps' synthetic preparation.Moreover, its reversible binding mechanism makes it devoid of the typical side effects of irreversible MAGL inhibitors observed in in vivo studies [17].Finally, it also showed selectivity for MAGL inhibition over the other main components of the endocannabinoid system, i.e., cannabinoid receptor 1 and 2, fatty acid amide hydrolase, α/β hydrolase-6 and α/β hydrolase-12.
Interestingly, the ability of MAGL23 to inhibit the viability of ovarian, colon, and breast cancer cell lines was demonstrated [16].
Although the results in terms of antiproliferative activity in cancer cells were quite good (IC 50 values in the low micromolar range), the inhibitor suffered of problems related to poor solubility.Recently, nanotechnology is playing a vital role to improve important drug parameters such as solubility, biodistribution, pharmacokinetics and pharmacodynamics [18][19][20][21][22].Among the possible approaches to increase the solubility, nanocrystallization allows to avoid a matrix such as polymeric or lipid nanoparticles.Indeed, drug nanocrystal particles have an almost 100% hydrophilic surface, avoiding the toxic effects of the matrix and with a high yield of solubilization [23,24].In nanotechnology, two different approaches can be exploited: the "Top-down" and "Bottom-up" methods.When applied to crystals, the main concept is to reduce their size from several microns to a few nanometers that are easily soluble in water.The "top-down" approach foresees the use of high shear stress for the breakage of large crystals while in the "bottom-up" approach, the nanocrystals are generated from the crystallization of drug molecules.Furthermore, the two processes can be used one after the other in a combination process, where usually after a "bottom-up" process of crystallization, size is decreased with a "top-down" method such as ultrasonication [25].However, these methods still have limits due to the variation in the size of nanocrystals during batch preparation and the chemical nature of the compounds [26-28].
Recently, Park et al. introduced a novel nanocrystallization strategy for the solubilization of poorly soluble drugs in water by a combined method.The authors used the Pluronic F-127 and cetyltrimethylammonium bromide (CTAB) as surfactants to obtain a controlled generation of the nanocrystals in water and then covered them with human serum albumin [27].The presence of surfactant during the crystallization allows to control the size of the obtained nanocrystals.More importantly, the bare nanocrystals of therapeutic compounds can lead to the adsorption of non-speci c proteins resulting in the uptake by mononuclear phagocyte system (MPS) [29].Despite the fact that others hydrophilic stealth surfaces could reduce the MPS uptake of nanocrystals, there would always be a possibility that after the distribution of nanocrystals in tumors, they could interfere with the retention and intended cellular interactions [30].On the contrary, albumin is a natural carrier of native ligands and other hydrophobic molecules, it is very stable, digestible and decomposable by cells, provides amino acids for cell metabolism and helps to internalize the nanocrystals in tumoral sites through the interaction with speci c receptors [31][32][33][34].
In the proposed study, it was utilized a combined method to obtain nanocrystals of the MAGL23 inhibitor that was highly hydrophobic.The inhibitor was solubilized with Pluronic F-127 or CTAB surfactants in the organic solvent (chloroform) and the crystals were obtained by sonication in water.We compared the effects of surfactants and the interaction of albumin with the MAGL23 inhibitor.The system was observed at each step of the formulation by using different spectroscopic techniques to ascertain the stability of the formulation.We believe that the proposed system provides an opportunity to increase the solubilization of poorly soluble hydrophobic drugs, such as MAGL23 inhibitor, and create a drug delivery system for in vivo applications.

Results And Discussion
Nano crystallization process increases the solubility of MAGL23 MAGL23 is an organic molecule of medium molecular weight (369.436g/mol) which, basing on the Chemicalize.comprediction (predicted physicochemical properties are reported in Table 1), is highly hydrophobic and insoluble at physiological pH levels (Figure 2a) at which it is mainly in its neutral form (94% at pH=7.4) (Figure 2b).In order to con rm the poor solubility of MAGL23 at neutral pH, a solubility test was performed on drug powder, following the method reported in section 2.2.The experimentally determined solubility is lower than 0.01 mg/ml (the lowest measurable value) in accordance with the prediction.Despite the fact that neutral compounds (having zero charges) are more favorable for biological membrane penetration than energetically charged compounds [4,35], poor solubility is often an impediment for the development and the clinical use of a drug.The drug formulation for enhancing aqueous dispersion often require the use of surfactants or nanoparticles, which may result in an increased systemic toxicity of the drug [36].Nanocrystals having an high ratio between the carried drug and the excipients avoid toxicity and deliver a substantial amount of drug to the cells [24].In fact, to overcome MAGL23 solubility issue, the drug was formulated as nanocrystals covered by albumin, which strongly binds to lipophilic drugs [24].Wrapping MAGL23 nanocrystals with albumin was important for the safe delivery without a toxic nanocarrier and helped to stabilize and to internalize the nanocrystals into the cells [27].The formulation proposed with both surfactants led to a signi cant increase in drug solubility as experimentally con rmed, reaching 0.82±0.06mg/mL with at least eighty-fold increase of solubility in comparison with the stand-alone drug.

Yield and coating
The absorbance in the UV region was utilized to measure the loading of the MAGL23.Six separate synthesis were performed; Table 2 shows the average of the percentage of yield for each analyzed compound and the average of the percentage of HSA bonded to MAGL-AF and MAGL-AC nanocrystal samples.The results showed that the nanocrystallization protocol solubilizes MAGL23 up to 0.82 mg/ml and the percentage of HSA bound to the nanocrystals were 22% and 34% by weight in MAGL23-AF and MAGL23-AC, respectively.
Table 2. Percentage yield of nanocrystal samples and percentage of interacting HSA (data are reported as mean ± standard deviation).

Morphological Analysis
The morphological analysis was performed by transmission electron microscopy (TEM) on nanocrystal samples and drug powder (MAGL23).The formulation with both surfactants produced a reduction in crystal size with similar size frequency distributions (Figure 3 a,b,c) with average sizes of 389 nm (95% CI: 336-442) and 391 nm (95% CI: 344-439) for MAGL23-AC and MAGL23-AF, respectively.The images of nanocrystal samples at intermediate state of formulation (MAGL23 and MAGL23-F) are reported in supporting information Figure S1.Clearly, the size decreased and monodispersibility increased compared to MAGL23 while employing pluronic acid as surfactant.
Dispersion and homogeneity of the crystals were evaluated for MAGL23-AF and MAGL23-AC (Figure 3 d,e,f).TEM data support the fact that surfactants allowed to increase the solubility of the drug by creating smaller and more monodisperse nanocrystals with a hydrophilic coat.The size of MAGL23-AF suspended particles measured using DLS technique was around 395.4 nm (PdI 0.1), a value close to that identi ed by analysis on the TEM pictures, highlighting good dispersion of the crystals.Conversely, the hydrodynamic diameter of MAGL23-AC measured by DLS was bigger with an average crystal size of 477.2 nm (PdI 0.2) and it could be due to self-agglomeration of the particles in solution as it was possible to ascertain from in vitro observations.This unfavorable behavior and the higher toxicity of CTAB (see IC 50 results, Table 3) led us to proceed further with pluronic F127 formulation only.

Characterization of nanocrystals, MAGL23 and coating components
The nano-crystallization process should not change the chemical and structural properties of the drug to maintain its e cacy, while increasing its pharmacokinetic properties by size reduction and coating.
To investigate the structure of the nanocrystals and study them during the crystallization process, X-ray diffraction (XRD) measurements were performed.XRD diffractograms of nanocrystal samples MAGL-F, MAGL-AF and MAGL23 are reported in Figure 4a for values of 2θ ranging from 10 to 40° since for higher value of 2θ there were no peaks.Nanocrystals samples and MAGL23 diffractograms show intense and sharp diffraction peaks, which evidence their crystalline structure.All the major peaks of MAGL inhibitor diffractogram are present in nanocrystal samples, at 2θ values of 13.5, 15.75, 17.95, 18.95, 19.75, 23.5°, despite the dilution effect in uences the nanocrystals samples.These results indicate that the crystallization process did not interfere with the inner crystalline structure of the drug.
To assess the possible interactions between the drug and the surfactants present on crystal surface in the nanocrystal formulation, FTIR spectroscopy analysis were performed for nanocrystal samples and MAGL23 (Figure 4b).Nanocrystal samples (MAGL-AF and MAGL-F) show mostly all MAGL23 bands (around 3430 cm -1 OH, 3167 cm -1 aromatic CH, 2865,2929,2961 cm -1 aliphatic CH, 1683 cm -1 chetonic CO, 1621 cm -1 ammidic CO, 1320 cm -1 CF [37]) without any signi cative shift.The only difference is the strong band at 3438 cm -1 steeper in MAGL-F and MAGL-AF, evidencing the presence of water in the samples probably due to the incomplete lyophilization [38,39].The Pluronic F-127 weak band at 1100 cm - 1 in MAGL-F is the only coating components band visible in the nanocrystal samples.Therefore, FTIR spectra evidence that the chemical structure of the drug remains unchanged during the crystallization process and that the drug delivery system is mainly formed by the active compound with little amount of coating components not strongly interacting with it.The chemical stability of MAGL23-AC and MAGL-C were also tested by FTIR spectra (supporting information Figure S2).No change was observed in the structure even by employing CTAB as surfactant.
Eventually, the FTIR and XRD spectra evidenced that during the crystallization process, the drug remained chemically and structurally unchanged and the drug delivery system was mainly formed by the active compound.
MAGL23-AF has an optimal release pro le MAGL23-AF in vitro release test results are reported in Figure 5 as percentage of the released drug over the initial drug amount.It is evident that the dissolution rate is low.Indeed, after one day only the 19 ± 7% of the drug was released and this value reached the 42 ± 6% after three days.The low release rate is in agreement with the stability of the complex [40].This means that the nanocrystal formulation allows to increase the solubility, avoiding burst release of the drug and toxic effects during the blood circulation site.Taking advantages from the Enhanced Permeability Retention (EPR) effect and the targeting ability of the albumin, the drug should be released mainly at the targeted site reducing the side effects and promoting the uptake in cancer cells over more cycles of cell division [41,42].

Cell viability assay
MAGL enzyme is overexpressed in a large variety of cells deriving from ovarian and colorectal cancers, and also has a key role in tumor progression [43,44].Therefore, the new formulations of MAGL23 were tested on tumor cell lines inherent to ovarian [45] (A2780, Skov3 and Ovcar3) and colorectal [46] cancers (Colo205 and HCT116).
Before testing MAGL23 nanocrystals, pluronic acid F127 and CTAB were evaluated.The IC 50 values are reported in the Table 3.The IC 50 of the CTAB are signi cantly lower than those of F-127, which suggests a marked self-cytotoxic activity.Conversely, pluronic acid F-127 did not display a signi cant cytotoxic activity and should be considered the best biocompatible formulant [47,48] among those tested.We therefore focused only on compounds treated with F-127, as a biocompatible surfactant with low intrinsic cytotoxicity.In Table 3  MAG23-AF and MAGL23 have comparable e cacy, in agreement with the results obtained from the nanocrystal characterization, which evidenced the maintenance of structural and chemical characteristics of the drug during the nanocrystallization procedure.Therefore, the drug delivery system allows to increase the water solubility without perturbing the molecule and consequently maintaining unaltered its activity on cells.Furthermore, the albumin coating should improve the biodistribution in vivo because albumin works rst of all as a carrier to transport molecules in the blood [49,50], and secondly because albumin has a high a nity with sialoglycoprotein gp60, a protein present in the vascular endothelium and closely related to the formation of caveoles and transcytotic processes and SPARC (Secreted Protein, Acidic and Rich in Cysteine), which are overexpressed in the tumors [42,51,52].These characteristics allow MAGL23-AF to extravasate more easily in vivo than MAGL23, signi cantly increasing the possibility of concentrating the drug around the tumor mass and therefore improving its effectiveness.

MAGL23-AF internalized into the cells through lysosomes
To determine the intracellular localization of MAGL23-AF, A2780 cells were probed with Hoechst 33342 (blue nucleus) and Lysotracker (green Lysosomes).MAGL23-AF was probed with rhodamine as reported previously [27].A time course analysis at 1 hour and 24 hours showed that MAGL23-AF accumulates in the lysosomes with a Pearson's correlation coe cient (R) of 0.73 and 0.67, respectively (yellow signals, Figure 6).These data suggest an active mechanism of cellular import with a partial tra cking to lysosomes (Figure 6).

Conclusions
The rst nanoformulation of a potent MAGL inhibitor, MAGL23, which is a promising compound as anticancer agent, was developed.MAGL is an enzyme that is highly expressed in tumors, where it plays a fundamental role in the oncogenic lipid signaling that promotes invasion, migration, survival, and in vivo tumor growth [12].Many MAGL inhibitors have been reported in the literature and they showed positive effects when tested in in vivo studies.However, most MAGL inhibitors act by an irreversible mechanism of action, which provokes many negative effects in animal models, thus hampering their further development as drugs [17].On the other hand, potent MAGL reversible inhibitors are usually characterized by a high lipophilicity.MAGL23 is a recently published reversible MAGL inhibitor, whose development is facilitate by its high selectivity towards other members of the endocannabinoid system as well as by its straightforward synthetic preparation [16].Unfortunately, not a single delivery system is available (for reversible and irreversible inhibitors).A challenge in drug delivery systems is to have one single system able to protect and transport the drug to the target site without compromising the potency.Most of the systems are not stable in in vivo, suffer of a low loading e ciency and require drug pre-solubilization (because of their intrinsically hydrophobic nature).Hence, a nanocrystallization method was employed to decrease the size of the crystals of MAGL23.They were dissolved in an organic solvent together with different surfactants, which helps to decrease the size of nanocrystals and provides their hydrophilicity, in turn increasing the solubility at about 1 mg/ml.Usually, the pharmacokinetics pro le of the therapeutic compounds is altered by decreasing the size (while employing different physical or chemical methods to achieve the solubility) in turn in uencing the potency of the compound.But in our case, the nanocrystal formation was thoroughly observed by different spectroscopic techniques and results demonstrated that there was no change in the structural properties and the IC 50 data on cells are also in line with the results obtained with the compound stand-alone.The nanocrystals were formulated with albumin for safe delivery applications.Indeed, albumin can direct the therapeutic compound at tumoral sites via the GP60 receptor and SPARC, and also it stabilizes the nanocrystals avoiding unwanted protein adsorption during circulation in the body.Furthermore, our future research is directed to investigate the in vivo properties of the formulated nanocrystals in animal models.In conclusion, in this article, we developed the rst soluble and easily absorbable nanoformulation system for a potent reversible MAGL inhibitor for a future use in clinical applications.

Nanocrystallization and Albumin Formulation
The nanocrystallization of MAGL23 was performed by the method reported in the literature [27].Brie y, 6 mg of the drug and 24 mg of Pluronic F-127 (F-127) were mixed in 3 mL of chloroform inside a roundbottom ask.The mixture was carefully mixed to ensure the drug dissolution, then chloroform was evaporated in a rotary evaporator at 30°C until a thin amorphous layer of the mixture was obtained.The sample was recovered in 6 mL of milli-Q water achieving a nal drug concentration of 0.82 mg/ml.Bath sonication (1 min) and probe sonication (10 min, 40% frequency, in a mixture of ice and water to avoid heating the sample) were used to form nanocrystals from the thin lm and to homogenize the sample.This method was replicated also replacing 24mg of F-127 (MAGL23-F) with 2.4 mg of CTAB (MAGL23-C).
In the second step, 4 mg of HSA were added for each mL of the solution containing water-soluble nanocrystals of drug.Each mL of the mixture was left in rotation at 30 rpm, room temperature, for 24h to ensure the maximum coverage and interaction between HSA and nanocrystal surface.The nanocrystal samples were washed two times by centrifugation at 38000 rpm, 4°C for 1h to remove the unbounded albumin and extra surfactants.The pellet obtained after the second centrifuge was lyophilized and resuspended in milli-Q water while maintaining the same starting volume.MAGL23-C plus HSA were later referred as MAGL23-AC while the ones obtained from MAGL23-F as MAGL23-AF.

Solubility Test
The solubility of the MAGL23 inhibitor was tested in silico as well as experimentally.The in silico prediction was performed by the "Chemicalize" software (Chemicalize.com;ChemAxon Ltd., Budapest, Hungary) while experimental results were obtained by mixing 1 mg/mL of drug powder in milli-Q water for 1 hour.The solution was centrifuged at 10000 rpm for 10 minutes to eliminate the insolubilized fraction and quanti ed through UV-Visible spectroscopy method.

Yield and coating
The nanocrystals were analyzed spectrophotometrically in the UV region at 252 nm using the Agilent 8453 spectrophotometer through a calibration curve method.Samples solutions of MAGL-C and MAGL-F were diluted in methanol to be quanti ed.The concentrations of MAGL23-AC and MAGL23-AF were measured by diluting the solution in methanol, following by centrifugation (12000 rpm, 2 min) to precipitate HSA and then quantifying the drug in the supernatant.The yield % of the nanocrystal formulation was determined as the concentration of the nano crystallized material, over the quantity of the drug used for the nanocrystal production, the ratio was multiplied by 100.The quantity of HSA in MAGL23-AF and MAGL23-AC was determined by Bradford protein assay (Bio-Rad reagent) using the Agilent 8453 spectrophotometer (λ=595nm) by comparison with a HSA calibration curve.The percentage of HSA bound to MAGL23-AF and MAGL23-AC, was calculated by dividing the albumin concentration in the sample, by the concentration of the drug encapsulated in the nanocrystals and multiplying by 100.

XRD analysis
After spectrophotometric quanti cation, compounds were re-centrifuged at 38000 rpm for 1h, then the supernatant was discarded, and the pellet was lyophilized.The obtained powders, and the original drugs were analysed by Philips X-ray diffractometer with PW1050/70 goniometer.CuKα radiation (λ = 1.54178Å) was used, the diffractograms were recorded by setting a diffracted intensity detection step equal to 0.05°.The unit of measurement of the detected intensity is the number of pulses / second.In order to compare the spectra they have been normalized, the signal to noise ratio is in uenced by the quantity of the samples used for the measurement.

FTIR analysis
Solid nanocrystal samples were obtained as already described in X-ray diffraction technique section.The lyophilized samples of nanocrystals were used to prepare the pellet with KBr.Fourier-transform infrared (FTIR) spectrum were measured with FTIR spectrophotometer (Perkin Elmer-Spectrum One).

TEM analysis
A drop of solution (about 25 µl) was deposited on a 400-mesh holey lm grid; after staining with 1% uranyl acetate (2 min), the sample was observed with a FEI Tecnai G2 transmission electron microscope operating at 100 kV (Hillsboro, Oregon, US).The images were taken with a Veleta digital camera (Olympus Soft Imaging System).Image processing to outline crystals and coating proteins and to measure Feret diameter was carried out by ImageJ (1.52a) software (the point-by-point set of operations for image processing is described in a dedicated section of the supplementary material).

Dynamic light scattering analysis
Dynamic light (DLS) analysis were performed by Zetasizer Nano particle analyzer (Malvern Panalytical, Malvern, UK) on nanocrystals samples diluted with Phosphate-buffered saline (PBS).

Cell viability
Ovarian cancer (A2780, KURAMOCHI, SKOV3, OVCAR3) and colorectal cancer (Colo201, Colo205, HCT116) cell lines and lung broblasts (MRC-5) were grown at 37 °C in a controlled atmosphere containing 5% CO 2 according to the supplier instructions.A thousand of cancer cells were plated in 96multiwell culture plates or 5000 cells for MRC-5.The day after seeding, drugs were added with a serial dilution 1:10 to have a nal concentration ranging from (100 µg/mL) to (0.001µg/mL).Cell viability was measured with Tecan In nite M1000 PRO (Tecan, Mannedorf, Switzerland) after 96h with CellTiter-Glo® assay according to the supplier (Promega, Madison, WI, US).IC 50 values were calculated from nonlinear regression dose-response curves by GraphPad Prism 8 Software.Averages were obtained from triplicates and the errors are standard deviations.

Drug Release
In order to reproduce in vivo conditions, the release test was performed dialyzing 1 mL of MAGL23-AF against PBS (pH 7.4, isotonic solution) placing the nanocrystal sample in a Slide-A-Lyzer MINI Dialysis Device 20k MWCO (Thermo Scienti c) kept at 37°C.Aliquots of the solution were sampled at different time points and then measured with Agilent 1260 In nity II HPLC with Variable Wavelength Detector, equipped with an Accucore-150-C18 column (5 cm x 2.1 mm, particle size 2.6 μm) (Thermo Fisher Scienti c, Waltham, MA, US).A mixture of acetonitrile and MQ water 50:50 was used as mobile phase with a ow rate of 0.5 mL/min.The quanti cation of the inhibitor has been obtained measuring the absorbance at 252 nm through a calibration plot.Aliquots were processed before HPLC analysis to remove HSA.The nanocrystal samples were diluted into acetonitrile and centrifuged at 13000 g for 15 minutes, then the supernatant has been diluted 1:1 with MQ water.

Lyso tracker analysis
The internalization and localization of the drug nanocrystals into the cells were analyzed.Typically, 150,000 cells were plated on top of a sterile coverslip into transparent microplates wells.The uorescent dye rhodamine B was mixed at 30 µg/mL with nanocrystals under continuous rotation at room temperature for one hour.Then, 50 µg/mL of the rhodamine B-nanocrystals were added into each well for different time points (1h, 6h, and 24h).After incubation, the media was removed from the cells and new media was added with 200 nM of Lysotracker TM Green DND-26 (Thermo Fisher Scienti c, Waltham, MA, US) to label lysosomes and 0.02 ng/mL Hoechst 33342 for three hours to label the cell nucleus.After that, the media was removed, and cells were washed three times by PBS and xed with paraformaldehyde 4% for 20 minutes.The cells were analyzed with an inverted uorescence microscope (Nikon, Tokyo, Japan).Release pro le of MAGL23-AF reported as percentage of drug released percentage (%) over time (h).
Funding and AcknowledgementsThis work was nancially supported by SPIN-Supporting Principal Investigators, Ca'Foscari University of Venice, Italy; by MIUR (PRIN 2017, project 2017SA5837) and by the Italian Ministry of Health -Ricerca Finalizzata 2016 -NET-2016-02363765.Consent for publicationAll the authors read and approved the original draft of the manuscript and declare no competing/con ict of interests.

Figures
Figures

Figure 2 a
Figure 2 (a,b,c) Relative frequency of crystals size of MAGL23, MAGL23-AF and MAGL23-AC from left to right; (d,e,f) TEM images of MAGL23, MAGL23-AF and MAGL23-AC.

Figure 4 a
Figure 4

Table 1 .
Physicochemical properties of the drug predicted (by Chemicalize.com)

Table 3 .
Calculated IC 50 results for drug solution, nanocrystals samples and surfactants (data are reported as mean ± standard deviation).*Datafrom reference[16] are reported the calculated IC 50 values of MAGL23 initially dissolved in DMSO and MAGL23-AF dissolved in aqueous solution.In general, IC 50 values were in the same range for each tumor cell line treated with MAGL23-AF compared to MAGL23 stand-alone.