Introduction
Olaparib is a poly (ADP-ribose)polymerase (PARP) inhibitor with high selectivity to inhibit DNA damage repair and induce apoptosis.1 Clinically, this has been approved for the treatment of advanced ovarian cancer.2 However, due to poor water solubility, the dosage forms of olaparib on the market are mainly tablets and capsules, which have not yet achieved the expected therapeutic effect.3,4 Furthermore, due to its intrinsic pharmacokinetics, the clinical dosage of olaparib required for achieving a therapeutic effect remains quite large, e.g., 600 mg (tablets, twice daily) per day for patients with newly diagnosed advanced ovarian cancer,5 leading to high costs, and hindering its clinical application to some extent. On the other hand, the widespread distribution of olaparib in normal tissues resulting from non-specificity can lead to common adverse reactions, such as vomiting, nausea, diarrhea, and anemia.3 Therefore, the development of a new olaparib dosage form is required to improve its solubility, and intrinsic pharmacokinetic profiles.
In recent years, nano-delivery systems have attracted attention in anti-cancer drug research due to their ability to improve drug solubility, pharmacokinetics, efficacy, and pathological tissue targeting.6–8 Among them, polymeric micelles have been one of the most commonly used nanocarriers due to their advantages in drug delivery.9 First, polymeric micelles have a “shell-core” structure, which is formed by the spontaneous assembly of amphiphilic polymers dispersed in an aqueous solution. Poorly soluble drugs are loaded in the hydrophobic core of micelles, thereby improving their solubility in water. Second, the hydrophilic shell, which is most commonly based on poly (ethylene glycol) (PEG), provides a long circulation time of micelles by reducing the uptake of micellar particles through the reticuloendothelial system (RES), thereby allowing the micelles to eventually accumulate in the tumor microenvironment.10,11 Third, micelles can be obtained in customizable sizes, typically <200 nm, making them favorable for the passive targeted delivery of cargos to tumor sites via the enhanced permeability retention (EPR) effect.12,13
We previously reported that polymeric micelles constituted of poly (ε-caprolactone)-co-poly (benzyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate, i.e., benzyl-functionalized trimethylene carbonate-b-poly (ethylene glycol) (P(CL-co-TMC-Bz)-PEG), was not only an excellent and safe solubilizer of a hydrophobic photosensitizer but also displayed prolonged circulation times in mice, as a result of the π-π stacking interactions of the micellar core.14 In the present study, π-π stacked P(CL-co-TMC-Bz)-PEG micelles were used to investigate its capability as a solubilizer of olaparib to improve the solubility, while ensuring its safety to different organs. Meanwhile, the micelles were evaluated as a drug delivery system for the prolonged circulation time of olaparib responsible for the improvement of its tumor site accumulation through the EPR effect. To this end, the P(CL-co-TMC-Bz)-PEG block copolymer was synthesized and characterized by gel permeation chromatography (GPC) and 1H-NMR spectroscopy. A series of olaparib-loaded micelles with different polymer concentrations and wt% loadings were prepared using different methods, in order to investigate the effect of formulation variables, including the preparation methods and type of solvent of the polymer, on the size of polymeric micelles and drug loadings. In addition, the in vitro release of olaparib from the micelles, and the cytotoxicity of the micellar olaparib formulations on the SKOV3 tumor cell line were evaluated by UV spectrophotometry and CCK-8 assay, respectively. Finally, the blood circulation kinetics and side effects of the incorporated olaparib in micelles and free olaparib were investigated in Sprague-Dawley (SD) rats using ultra-high performance liquid chromatography analysis and H&E staining, respectively.
Materials and methods
Materials
The 2,2-Bis (hydroxymethyl)propionic acid (bis-MPA) (98%), triphosgene (99%), methanesulfonic acid (≥99.0%), benzyl bromide (98%), and pyridine (99.8%) were obtained from MACKLIN (Shanghai, China). The ε-caprolactone (CL) and methoxy-poly (ethylene glycol) (mPEG-OH, 5,000 g/mol) were obtained from Sigma-Aldrich (Shanghai, China). The human ovarian carcinoma SKOV3 cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The potassium hydroxide (KOH), N,N-dimethylformamide (DMF), ethyl acetate, n-hexane, ammonium chloride, sodium bicarbonate, 4Å molecular sieves, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), and dichloromethane (DCM) were obtained from Sinopharm Chemical Preparation Co., Ltd. (China). The hydrochloric acid, toluene, and ether were purchased from Yantai Far East Fine Chemical Co., Ltd. (China). All the above reagents were used as received, except for the DCM, toluene and DMF dried on 4Å molecular sieves before use.
The 1H-NMR spectra were recorded using a Bruker NMR spectrometer (400 MHz; Billerica, MA, USA). The reference line at 7.26 ppm was used for the residual solvent of the chloroform.
Synthesis of the monomer and polymer
Synthesis of the monomer
Synthesis of benzyl 2,2-bis (hydroxymethyl)propionate
The benzyl 2,2-bis (hydroxymethyl)propionate was synthesized according to a previous report.15 Briefly, the mixture of bis-MPA (50 g, 0.37 mol) and KOH (21.2 g, 0.38 mol) in DMF (280 mL) was heated to 100°C for one hour, followed by the addition of benzyl bromide (55 mL, 0.45 mol). After 16 hours, the reaction solution was concentrated by reduced pressure distillation, and extracted by ethyl acetate (350 mL), n-hexane (250 mL), and water (250 mL). Then, the collected organic layer was concentrated to obtain the crude product (73.5 g) as white powder. The pure product was obtained as a white needle-like crystal after the recrystallization of the crude product from toluene (53.9 g, yield: 64%).
Synthesis of benzyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate
The synthesis of benzyl 5-methyl-2-oxo-1,3-dioxane-5-carboxlate (i.e. the trimethylene carbonate functionalized by the benzyl group, abbreviated as TMC-Bz) was carried out based on a previous report.15 Briefly, pyridine (55 mL, 0.67 mol) was added to 2,2-bis (hydroxymethyl)benzyl propionate (25 g, 0.11 mol) in DCM (330 mL). Then, the mixture was cooled down to −78°C under a N2 atmosphere, followed by the slow addition of the triphosgene (16.5 g, 0.06 mol) solution in DCM (40 mL). Next, the mixed solution was allowed to react at −78°C for 30 minutes, and subsequently at room temperature for 1.5 hours. Afterwards, the reaction solution was terminated by the addition of the saturated NH4Cl solution (270 mL). After being washed with 1 M of hydrochloric acid and the saturated NaHCO3 aqueous solution, and dried by anhydrous Na2SO4, the organic layer was collected and concentrated to yield the product as a white solid (25.2 g, yield: 91%). Then, the product was further purified by recrystallization from the ethyl acetate before polymerization. 1H-NMR (400 MHz, CDCl3): δ 7.39 (m, OCOCH2C6H5), 5.21 (s, OCOCH2C6H5), 4.69 (m, COOCH2CCH2OCO), 4.22 (m, COOCH2CCH2OCO), and 1.34 (s, OCH2CCH3).
Synthesis of the P(CL-co-TMC-Bz)-PEG copolymer
The poly (ε-caprolactone)-co-poly (benzyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate)-b-poly (ethylene glycol) (P(CL-co-TMC-Bz)-PEG) was synthesized, as previously described.14 Briefly, CL (502.2 mg, 4.4 mmol), TMC-Bz (600.6 mg, 2.4 mmol), and mPEG-OH (2,000.0 mg, 0.4 mmol) were dissolved in dry DCM (4.8 mL), followed by the addition of methanesulfonic acid (50.0 mg, 0.5 mmol) with stirring. The reaction proceeded under a N2 atmosphere at 37°C for 24 hours. Then, the cooled reaction solution was added dropwise to an excess of cold ether (−20°C), and the collected precipitate was dried under vacuum to obtain the final product. 1H-NMR (400 MHz, CDCl3): δ 7.33 (m, OCOCH2C6H5), 5.12 (s, OCOCH2C6H5), 4.29 (m, COOCH2CCH2OCO), 4.10 (m, OHCH2CH2CH2), 3.37 (s, CH3O), 2.27 (m, CH2CH2CH2OCO), 1.63 (m, CH2CH2CH2CH2CH2), 1.37 (m, CH2CH2CH2CH2CH2), and 1.26 (s, OCH2CCH3).
Characterization of the polymer
The average degree of polymerization (DP) of CL or TMC-Bz was determined by 1H-NMR, as previously described.14 Briefly, DP was calculated using the ratio of the integral of the CH2 protons of CL at 1.38 ppm, or the CH2 protons of benzyl groups in the poly (TMC-Bz) at 5.16 ppm, to the CH3 protons of mPEG-OH at 3.36 ppm. The average molecular weight (Mn) of the polymer was calculated based on the molecular weight of the DP of each unit.
The Mn, weight average molecular weight (Mw), and polydispersity (Mw/Mn) of the synthesized polymer were detected by gel permeation chromatography (GPC). The used column was the Shodex™ KD803 column (300 × 8 mm, including a guard column, 10.0 × 4.6 mm), and the detector was a differential refractive index (RI) detector. The poly (ethylene glycol)s of narrow molecular weights (840–30,000 g/mol) were used as the calibration standard. The eluate was DMF with 10 mM of LiCl, the elution rate was 1.0 mL/min, and the column temperature was set to 65°C.
Preparation and characterization of empty micelles
The following methods were used to prepare the empty micelles: the thin-film hydration and nanoprecipitation of polymer dissolved in THF or DMSO.14,16,17 For example, in order to prepare the micelles with 10 mg/mL of polymer by thin-film hydration, the block copolymer (10 mg) was dissolved in DCM (1 mL). Then, the solvent was removed using rotovap to form a thin film. Afterwards, the block copolymer film was hydrated in deionized water (1 mL), followed by being heated at 65°C for five minutes, in order to form a uniform micellar dispersion.
For the first nanoprecipitation method, the block copolymer (10 mg) was dissolved in DMSO (100 µL), followed by being heated up to 70°C for five minutes. Then, the cooled solution was added dropwise to deionized water (900 µL) at a 1:9 volume ratio with gentle shaking, in order to form a homogenous micellar dispersion. Afterwards, the micellar dispersion was dialyzed using dialysis tubing (MWCO = 1 kDa) against deionized water at room temperature for 12 hours to remove the DMSO.
For the second nanoprecipitation method, the copolymer dissolved in THF (10 mg/mL) was added dropwise to deionized water at a 1:1 volume ratio with gentle agitation. Then, the mixed solution was kept at room temperature for 36 hours to remove the THF.
The micellar dispersions obtained using the above methods were filtered through a 0.2 µm syringe filter. Dynamic light scattering (DLS) was used to determine the Z-average hydrodynamic diameter (Zave) and polydispersity index (PDI) of the resulting micelles. The scattering angle was fixed at 173°, and the measured temperature was at 25°C using a Malvern nano ZS (Malvern, Surrey, UK). The morphologic characteristics of the micelles were determined by transmission electron microscopy (TEM; JEOL JEM-2100Plus, Japan).
Preparation and characterization of olaparib-loaded micelles
The olaparib-loaded micelles (at different loading percentages) were prepared by thin-film hydration, as described above, with slight modifications. Briefly, a certain volume of olaparib solution in DCM (the additional volume was based on the predetermined wt% loading) was added to the polymer, followed by the addition of a certain volume of DCM, in order to obtain the predetermined polymer concentration. Then, the solvent in the mixture was evaporated to form a homogeneous film of polymer and olaparib, and this was hydrated and heated to form the olaparib-loaded micelles.
In addition, as described in above, with slight modifications, the olaparib-loaded micelles (at different loading percentages) were prepared through the nanoprecipitation of the polymer dissolved in DMSO using water as the non-solvent. Briefly, the olaparib solution in DMSO (the additional volume was based on the predetermined wt% loading) was added to the weighted polymer, followed by the addition of DMSO, to obtain the final polymer concentration (10 mg/mL). Then, the mixture of olaparib and polymer in DMSO was heated, and the cooled solution was added to deionized water (1:9 volume ratio) with gentle shaking, followed by dialysis against deionized water.
The resulting olaparib-loaded micelles were filtered through a 0.2 µm syringe filter to remove the non-encapsulated and thus precipitated Olaparib. The absorbance of micellar dispersions diluted with acetonitrile at 276 nm was measured using the UV-2006i Shimadzu Spectrophotometer (Kyoto, Japan). A series of olaparib solutions in acetonitrile was prepared as standard solutions to determine the loading of olaparib. The loading efficiency (LE) and loading capacity (LC) of olaparib were calculated according to equations (1) and (2), respectively:
LE%=Olaparib loaded (mg)Olaparib in the feed (mg)×100%
LC%=Olaparib loaded (mg)polymer used (mg) + Olaparib loaded (mg)×100%
In vitro release of olaparib-loaded micelles
The in vitro release of olaparib from micelles, with 10 wt% loading at 10 mg/mL of polymer and 3.5 wt% loading at 30 mg/mL of polymer (equivalent to approximately 1 mg/mL of olaparib), was explored using a dialysis tube (MWCO: 50 kDa) at 37°C in PBS (pH = 7.4) containing Tween-80 (0.5%, w/w), with shaking (900 rpm). Typically, 2 mL of olaparib-loaded micellar dispersion was dialyzed against 60 mL of release media. At predetermined intervals, 2 mL of release medium was withdrawn and renewed with an equal volume of fresh medium. As a control, the release of free olaparib was conducted following a similar procedure, with the replacement of olaparib-loaded micelles with the olaparib solution in DMSO. The concentration of olaparib in the medium was measured using the UV-2006i Shimadzu spectrophotometer (Kyoto, Japan) at 276 nm.
Cytotoxicity of empty and olaparib-loaded micelles
Human ovarian cancer SKOV3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose, which was supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotics (1,000 U/mL of penicillin and 1,000 µg/mL of streptomycin). Then, the cells were cultured at 37°C in a humidified 5% CO2 atmosphere.
The empty and olaparib-loaded micelles were formed in water at a polymer concentration of 10 mg/mL using the thin-film hydration method, as described in above. The stock dispersions were diluted in DMEM medium to the predetermined polymer concentration before being exposed to cells.
SKOV3 cells were seeded into 96-well plates at a density of 6,000 cells/well, and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. Then, the culture medium (100 µL) in the wells was replaced by the aforementioned empty or olaparib-loaded micelles (100 µL). Cell viability was determined after 24 hours of incubation by CCK-8 assay, according to manufacturer’s instructions. Briefly, 100 µL of culture medium containing 10% CCK-8 solution (SparkJade, Shandong, China) was added to each well, and the absorbance was determined at 450 nm after one hour of incubation using the Tristar2 S LB 942 multifunction microplate reader (Berthold, Germany). Untreated cells were used as the negative control group, and the assay was repeated at least three times.
Circulation kinetics and side effects of olaparib-loaded micelles in SD rats
The olaparib-loaded P(CL-co-TMC-Bz)-PEG micelles, with 20 and 7 wt% drug feeds at 10 and 30 mg/mL polymer concentrations, respectively (the final olaparib concentration was approximately 2 mg/mL), were prepared, as described in above. The free olaparib solution was prepared through the dilution of the olaparib stock solution (80 mg/mL) in DMSO using DMSO/polyethylene glycol 400/PBS (2:10:88 v/v/v), and a final olaparib concentration of 2 mg/mL was obtained. This was equivalent to the injected dose of olaparib in the micellar formulation.
The animal experiments were carried out according to the Guidelines for the Feeding, Management and Use of Laboratory Animals, and approved by the Ethics Committee of the Institute of Materia Medica, Shandong Academy of Medical Sciences (Jinan, China; Approval no.: 2021011). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. The female SD rats (weighing 195–206 g) were obtained from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Jinan, China). There rats were housed in natural light conditions and ventilated cages at 25°C with 55% humidity. Food and water were given ad libitum.
Three groups of SD rats (each group = 4) were intravenously (i.v.) administrated with free olaparib or olaparib-loaded micelles via the tail vein at an injection dose of 10 mg of olaparib/kg, corresponding to approximately 2 mg of olaparib in approximately 500 µL per rat. Then, blood samples were collected at one minute (used as the control for 100% injection), and at 1, 2, 6, 8 and 12 hours after injection. After 24 hours from the i.v. administration of the formulations, these rats were sacrificed, and the heart, liver, spleen, lungs and kidneys were excised for H&E staining, to evaluate the drug toxicity. Rats treated with PBS were used as the control group.
The collected blood samples were centrifuged at 1,000 × g for 15 minutes at 4°C to obtain the plasma supernatant. To determine the amount of olaparib, 40 µL of the collected plasma was vortex-mixed with 160 µL of acetonitrile for one minute. Then, the mixture was centrifuged at 15,000 × g for 10 minutes, and the clear supernatant was collected and analyzed by ultra-high performance liquid phase chromatography (UPLC). The UPLC system consisted of a ACQUITY UPLC BEH C18 1.7 µm 2.1 × 50.0 mm column, with acetonitrile/water (60/40, v/v) as the mobile phase, using a flow rate of 0.2 mL/min. The injection volume was 7 µL, and olaparib was detected using a UV detector set at 276 nm. The measuring range was 10–100 µg/mL, and the detection limit was approximately 0.1 µg/mL. A calibration curve was obtained from a series of standard solutions of olaparib in DMSO, in which 120 µL of SD rat plasma was added, followed by olaparib extraction using acetonitrile and UPLC analysis, as described above.
Results and discussion
Synthesis and characterization of the monomer and polymer
The monomer trimethylene carbonate containing benzyl group (TMC-Bz) was synthesized using benzylation of 2,2-bis (hydroxymethyl)propionate with benzyl bromide, followed by the reaction with triphosgene, as previously described (Fig. 1a).15 The structure of TMC-Bz was confirmed by 1H-NMR spectroscopy.
The P(CL11-co-TMC-Bz5)-PEG block copolymer was synthesized using the ring-opening polymerization of CL and TMC-Bz, with mPEG-OH (5,000 Da) as the initiator and methanesulfonic acid as the catalyst, at a CL/TMC-Bz/initiator/catalyst molar ratio of 11.0/6.0/1.0/1.3 (Fig. 1b). The copolymer was obtained as a white solid, with a yield of approximately 69%. The characteristics of the obtained copolymer are presented in Table 1. The composition of the resulting copolymer was calculated by 1H-NMR, and the results were in good agreement with that expected from the ratios of monomers in the feed. The GPC analysis revealed that the Mn of the synthesized polymer (6,844 Da) was close to that calculated in the 1H-NMR analysis (7,507 Da), and the marrow molecular distribution (Mw/Mn = 1.08) suggested the absence of significant side reactions.
Table 1Characteristics of the synthesized P(CL-co-TMC-Bz)-PEG copolymer
| Composition of the obtained copolymera | Aimed molecular weight (Da) | Mn (Da, 1H-NMR) | GPC
|
|---|
| Mw (Da) | Mn (Da) | Mw/Mn |
|---|
| P(CL11-co-TMC-Bz5)-PEG | 7,757 | 7,507 | 7,402 | 6,844 | 1.08 |
Preparation and characterization of empty micelles
In order to investigate the effect of the preparation method and type of organic solvent of the polymer on micellar sizes, empty polymeric micelles based on P(CL11-co-TMC-Bz5)-PEG were prepared at a fixed polymer concentration of 10 mg/mL using the thin-film hydration and nanoprecipitation methods, respectively. The TEM revealed that the empty P(CL11-co-TMC-Bz5)-PEG micelles prepared using the thin-film hydration method were close to a spherical morphology, and had an average size of approximately 40 nm (Fig. 2a). This was in line with that determined by DLS (Table 2). In addition, the size of the micelles prepared using the thin-film hydration method was similar to that prepared using the nanoprecipitation method, with DMSO as the solvent of the polymer (Table 2). In contrast, for the polymeric micelles prepared using the nanoprecipitation method, with THF as the solvent of the polymer, although the PDI was narrow, the particle size was significantly larger, suggesting that the particle size is dependent on both the method and solvent used for the polymer. Opposite to the present observation, Mahsa et al. reported that polymeric micelles based on the mPEG-b-poly (N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) prepared using the nanoprecipitation method, with THF as the solvent of the polymer, had smaller sizes, when compared to particles prepared with DMSO as the solvent of the polymer, suggesting that the property of the polymer plays a role in the size of the particles.17,18
Table 2Zave and PDI of empty polymeric micelles formed using different methods
| Method | Solvent of the polymer | Zave (d.nm) | PDI |
|---|
| Thin-film hydration | DCM | 44.00 ± 0.18 | 0.34 ± 0.00 |
| Nanoprecipitation | DMSO | 47.00 ± 1.04 | 0.37 ± 0.01 |
| THF | 255.00 ± 2.26 | 0.10 ± 0.03 |
Preparation and characterization of olaparib-loaded micelles
The P(CL11-co-TMC-Bz5)-PEG micelles prepared by thin-film hydration and nanoprecipitation, with DMSO as the solvent of the polymer, had comparable small sizes (Table 2). Therefore, both micelle preparation methods were used to prepare a series of olaparib-loaded polymeric micelles, to determine the effect of these preparation methods on the drug loading. As shown in Figures 2b and c, regardless of the preparation method and drug feeding, olaparib-loaded micelles had comparable particle sizes as the empty micelles (micelles without drug loading), indicating that drug encapsulation had a negligible effect on the particle size.
The UV-Vis analysis revealed that when olaparib was loaded in the micelles using the thin-film hydration method, the loading efficiency of olaparib at 10 mg/mL of polymer decreased from 100% to 50%, with the increase in olaparib feed ratio from 0.5% to 30%, and this reached a plateau (Fig. 3a). As a result, the loading capacity linearly increased until a maximum of 15% was reached at a feed ratio of 35% (Fig. 3a). Similarly, when the polymer concentration was increased to 30 mg/mL, the loading capacity continued to increase with the increase in olaparib feed ratio, reaching a plateau at 12% at a feed ratio of 20% (Fig. 3b). It is noteworthy that at the present tested condition, the solubility of olaparib in the micelles formed in the aqueous solution reached 4 mg/mL. Compared to that of olaparib in the micelles prepared using the thin-film hydration method, the loading efficiencies of P(CL11-co-TMC-Bz5)-PEG micellar dispersions at olaparib feed ratios of 5%, 10%, and 15% (formed using nanoprecipitation, with DMSO as the solvent of the polymer) were remarkably lower (≤2.1%), and the corresponding loading capacities were less than 0.35% (Fig. 3c). Therefore, the thin-film hydration method was superior to the nanoprecipitation method in improving the solubility of olaparib in the P(CL11-co-TMC-Bz5)-PEG micelles. Overall, these results suggest that the solubility of olaparib in an aqueous solution can be improved by using P(CL11-co-TMC-Bz5)-PEG polymeric micelles as the solubilizing excipient and that the solubilization capacity can be tailored by adjusting the polymer concentration, as needed.
In vitro release of olaparib-loaded micelles
The in vitro release of olaparib from micelles, with 10 wt% loading at 10 mg/mL of polymer and 3.5 wt% loading at 30 mg/mL of polymer (equivalent to approximately 1 mg/mL of olaparib), which was formed using thin-film hydration, was investigated in PBS (pH 7.4) supplied with 0.5% Tween-80 at 37°C. Then, this was compared with free olaparib under the same condition. As shown in Figure 4, the free olaparib demonstrated a rapid release in the first hour, which was indicated by approximately 70% olaparib in the medium, and a total of >80% of olaparib released into the medium within six hours. However, when olaparib was loaded in P(CL11-co-TMC-Bz5)-PEG micelles, the release of olaparib slowed down, and this release became significantly slower as the polymer concentration increased. For instance, the amount of released olaparib from the micelles was merely approximately 40% at 10 mg/mL of polymer, and 25% at 30 mg/mL of polymer in the first hour. Then, this gradually increased to approximately 60% and 40%, respectively, within 12 hours, and remained stable. The higher release of olaparib in micelles at low polymer concentration is probably due to the overloading of the core of the micelles with olaparib, and this can also be observed in photosensitizer-loaded micelles.16 Overall, these results suggest that the retention of olaparib was improved by the encapsulation in micelles, while the extent of improvement relied on the loading percentage of olaparib-loaded micelles, i.e., polymer concentration.
Cytotoxicity of empty and olaparib-loaded micelles
Cell viability assays were conducted on the SKOV3 tumor cell line to determine the cytotoxicity of empty and olaparib-loaded micelles. As shown in Figure 5a, no toxic effect was observed when SKOV3 cells were exposed to empty micelles at a polymer concentration of up to 3 mg/mL. This demonstrated the excellent cytocompatibility that was also observed on HeLa and A431 cells.14 After 24 hours of incubation, regardless of the formulation, the viability of SKOV3 cells treated with olaparib exhibited a dose-dependent decrease (Fig. 5b). It is noteworthy that the IC50 values for the micellar preparations of olaparib on SKOV3 cells were slightly higher than those for free olaparib (Table 3). Furthermore, a higher IC50 value was previously observed in photosensitizer-loaded P(CL-co-TMC-Bz)-PEG micelles, when compared to its free formulation.14 This might be attributed to the less efficient cellular uptake of PEGylated micelles, and/or the relatively time-consuming degradation of polymers to release olaparib.16,19,20
Table 3IC50 for free olaparib and olaparib-loaded micelles on SKOV3 cells (obtained from the curves in Figure 5b)
| Compound | Free olaparib | olaparib-loaded micelles |
|---|
| IC50 (mg/mL) | 0.11 | 0.68 |
Circulation kinetics and side effects of olaparib-loaded micelles in SD rats
The concentrations of olaparib in plasma were analyzed after the injection of free olaparib or olaparib-loaded P(CL11-co-TMC-Bz5)-PEG micelles containing ∼2 mg/mL Olaparib at polymer concentrations of 10 mg/mL (20 wt% drug feed) and 30 mg/mL (7 wt% drug feed), respectively (Fig. 6). It was clearly observed that the free olaparib was quickly eliminated from the blood circulation, and no olaparib was detected in blood after 0.5 hours (Fig. 6, green line). In contrast, when loaded in micelles, olaparib exhibited a significantly slower elimination in circulation. The elimination more significantly slowed down with the increase in polymer concentration from 10 to 30 mg/mL (Fig. 6, blue vs. red line). As a result, the half-life times (t1/2 values) of olaparib improved from approximately 0.6 to 2.0 hours, with the injected polymer concentration increasing from 10 to 30 mg/mL. This is in line with the in vitro release of olaparib from micelles (Fig. 4, blue vs. purple line). Furthermore, the more rapid elimination of olaparib at low polymer concentration is probably related to the overloading of the core of the micelles with olaparib (20 wt% drug loading). It is noteworthy that although the circulation time of olaparib was prolonged by encapsulation in the P(CL11-co-TMC-Bz5)-PEG polymeric micelles, this remained rather short for achieving the target drug delivery through the “EPR” effect. Thus, this needs to be further improved by e.g., chemical crosslinking, drug-polymer conjugation, or increasing the amount of aromatic rings.21–23
The in vivo toxic effect of the different olaparib formulations in the organs of rats was also determined. To this end, rats treated with PBS, free olaparib, or olaparib-loaded micelles (30 mg/mL of initial polymer) for 24 hours were sacrificed. Then, the heart, liver, spleen, lungs, kidneys, and brain were dissected and prepared for H&E staining. As shown in Figure 7, no obvious pathologic changes were observed in the organs of rats, regardless of the treatment groups, suggesting the safety of the micellar olaparib formulation.