Core-Cross-Linking Accelerates Antitumor Activities of Paclitaxel

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Core-crosslinking accelerates anti-tumor activities of paclitaxel-conjugate micelles to prostate multicellular tumor spheroids: a comparison of 2D and 3D models Alice W. Du, Hongxu Lu, and Martina Heide Stenzel Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00282 • Publication Date (Web): 09 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Core-crosslinking accelerates anti-tumor activities of paclitaxelconjugate micelles to prostate multicellular tumor spheroids: a comparison of 2D and 3D models Alice W. Du†, Hongxu Lu‡ and Martina H. Stenzel‡* Centre for Advanced Macromolecular Design (CAMD), †School of Chemical Engineering, ‡School of Chemistry, University of New South Wales, New South Wales, 2052, Australia

Abstract The 2D monolayer cell culture model is often the first step in the prediction of the success or failure of a nanoparticle-based drug delivery system. However, there is often poor translation between the 2D monolayer in vitro results and the nanoparticle-drug performance in vivo. One possible way of bridging this gap is the use of multicellular tumour spheroids (MCTS) as an intermediate in vitro model due to its 3D structure. This paper aims to quantify and compare the results obtained from traditional 2D monolayer cell cultures and 3D MCTS by studying the cytotoxic effects of free paclitaxel and paclitaxel which has been conjugated to a poly(ethylene glycol methyl ether acrylate)b-poly(carboxyethyl acrylate) block copolymer and self-assembled to give a micellar delivery system. The core of the micelle was crosslinked with a diamino non-degradable crosslinker to compare the effects of micelle stability on the results. Although the 2D prostate tumor cell culture results indicated that all micellar variants (IC50: 193 – 271 nM) were significantly less toxic than free paclitaxel (IC50: 15.2 nM), the micelles showed faster and higher cytotoxicity than free PTX in the 3D prostate MCTS. The crosslinking of micelles even showed accelerated anti-tumor activities to the MCTS compared with uncrosslinked micelles. The results indicate that DAO-crosslinked POEGMEA-bPCEA-PTX conjugate micelles will be a useful nano-drug carrier for prostate cancer therapy. MCTS offers a very promising method of incorporating 3D structures into in vitro testing.

Introduction The use of nanoparticles for the delivery of therapeutic compounds is a highly active and innovative field which stands to offer many medical benefits. In particular, the use of polymeric nanoparticles for the delivery of strongly hydrophobic drugs has seen much research in recent years as researchers seek to maximise the benefits of therapeutic agents whilst minimising their harmful side effects in oncology treatment 1-3. Nanoparticles have been widely proposed as a way to enhance the delivery of drugs by altering their biodistribution and pharmacokinetics properties. This includes altering the three phases of drug delivery pertaining to systemic circulation and reticuloendothelial (RES) clearance, extravasation and tumour uptake, and interactions with the target cell. 4

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Polymeric micelles form one important subset of nanoparticles often utilized as a delivery vehicle for cancer treatment. Due to the hydrophobic environment of the micellar core, it is able to simultaneously solvate the hydrophobic drugs and protect them. Micelles formed from amphiphilic block copolymers harness many advantages which make them ideal therapeutic carriers 5-9. These advantages include ease of structure modification, passive targeting ability to tumour sites due to a combination of their small size and the enhanced permeability and retention (EPR) effect 10 and ability to avoid detection by the mononuclear phagocyte system (MPS) 11. In particular, crosslinking of the shell or core increases the stability of the micelles, which is especially important as the micelles are significantly diluted when evaluations are conducted on their activity 12, 13. Micelles have been used to deliver a range of drugs including paclitaxel, which is often the focus of attention because of its wide use. Paclitaxel is a highly potent anti-cancer agent which has demonstrated toxicity against a wide range of different cancers. The cytotoxicity of paclitaxel lies in its ability to inhibit the depolymerisation of microtubules, thereby disrupting the dynamic equilibrium required, and normally exhibited, by tubulin in cell division and interphase processes 14. Paclitaxel is also extremely hydrophobic, with an aqueous solubility of 1 µg/mL 15, thereby limiting the therapeutical uses of the compound. Indeed, paclitaxel is delivered to patients with Cremophor EL and ethanol, a vehicle with its own harmful side effects such as anaphylactoid hypersensivity, hyperlipidaemia and peripheral neuropathy 16. As a result, paclitaxel represents the perfect drug for nanoparticle delivery and research involving micellar delivery of paclitaxel have targeted a range of cancers 17-27, including prostate cancer 28. Although physical encapsulation of paclitaxel remains a popular method of delivery of the drug 18, 20, 22, 29, chemical conjugation of the drug to a polymer chain has also been explored 17, 30-32. The most common in vitro method to evaluate the success of micellar systems as delivery vehicles to cancer sites has traditionally been a 2D monolayer cell culture study as it is a relatively quick and simple method to test the biological response of the target cells. However, interest in multicellular tumour spheroids (MCTS) as a 3D cell culture model has grown rapidly in recent years as it becomes increasingly clear that 2D monolayer models are too unrealistic when compared to their in vivo counterparts 33-37. Compared to 2D monolayer cell culture, MCTS as an in vitro model mimics the complexity found in in vivo tumours to a greater degree and as such, provides a more accurate reflection of the interaction between tumours and the nanoparticle delivery of an anti-cancer drug 38-40 . MCTS models have already been used to evaluate the effectiveness of a range of polymeric nanoparticles in drug delivery, including doxorubicin 41 and paclitaxel 42 delivery using poly(ethylene glycol)-phosphatidyl ethanolamine (PEG-PE), docetaxel delivery using poly(ethylene glycol)-bpolycaprolactone (PEG-b-PCL) 43 and siRNA delivery using branched poly(disulfide amine) (B-PDA) 44. Although the use of MCTS to determine the activity of drug loaded nanoparticles is a growing field and it is still in its infancy.32 The chemical binding of the drugs and the often unknown slow cleavage of the drug from the carrier results frequently in IC50 values in the traditional 2D model which are significantly higher than the parent drug. This may lead to the conclusion that the drug carrier is not suitable. However, tumors require the penetration of nanoparticles into the 3D structure itself and a slow cleavage may, in that scenario, be advantageous. In this paper, we therefore developed a micellar delivery vehicle for paclitaxel in the treatment of prostate cancer. For that purpose, a block copolymer was synthesized via RAFT polymerization, which was subsequently reacted with paclitaxel to generate a drug conjugate (Scheme 1a). The


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amphiphilic block copolymers were then self-assembled and crosslinked with a non-degradable diamino crosslinker as shown in Scheme 1b. In addition to results obtained from traditional 2D monolayer cell culture, the polymer-paclitaxel conjugate micelles were tested on a 3D MCTS model to showcase the difference in results between the two studies and the importance of introducing 3D-based cell culture models to drug delivery studies. Both uncrosslinked and core-crosslinked micelles were tested to determine whether increasing the stability of the micelles 29, 45 will have an appreciable effect on paclitaxel delivery in both systems.

Scheme 1: Conjugation of paclitaxel to block copolymer as prepared by RAFT polymerisation using solid phase peptide synthesis (SPPS) reagent (a); Self-assembly and core crosslinking of polymer-paclitaxel conjugate micelle using nondegradable diamino crosslinker (b).

Materials and Methods Materials All materials were reagent grade and used as received, unless otherwise specified. 1,4-dioxane (>99%, Sigma-Aldrich), 1,8-diaminooctane (98%, Sigma-Aldrich), 2-(5-norborene-2,3-dicarboximido)1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU, >99.9%, Chem-Impex International), acetonitrile (ACN, >99.5%, Ajax Finechem), carboxyethyl acrylate (CEA, Sigma-Aldrich), diisopropylethylamine (DIPEA, 99.9%, Sigma-Aldrich), fluorescein O-methacrylate (97%, SigmaAldrich), methanol (>98%, Ajax Finechem), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, BioXtra, Sigma-Aldrich), N,N-dimethylacetamide (DMAc, >99.9%, Sigma-Aldrich), N-hydroxysuccinimide (NHS, 98%, Sigma-Aldrich), paclitaxel (PTX, >99%, Dato Chemicals), poly(ethylene glycol) methyl ether acrylate (OEGMEA, Mn = 480, Sigma-Aldrich) and toluene (>99.5%, Ajax Finechem) were used without further purification. The RAFT agent, 3benzylsulfanylthiocarbonylsufanylpropionic acid (BSPA), was synthesised previously 46. The initiator, 2,2’-Azobisisobutyronitrile (AIBN) was re-crystallized twice from methanol. MilliQ was produced internally and had a resistivity of 18.2 mΩ/cm.


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Experimental BSPA-POEGMEA MacroRAFT synthesis OEGMEA (4.995 g; 1.04 × 10-2 M) and BSPA (59.1 mg; 2.17 × 10-4 M) were dissolved in toluene (9.4 mL) before AIBN was added (1 mL of 4.17 × 10-2 M stock solution). The reaction solution was purged with nitrogen gas (30 min) before the reaction vessel was sealed. The polymerisation was allowed to proceed at 60°C for 2 h before it was quenched by simultaneously exposing the reaction mixture to atmosphere and cooling in an icebath for 5 min. The contents were purified via dialysis against methanol (changed 3 times) and then MilliQ water (2 d). The product was isolated through lyophilisation. The conversion of PEGMEA, as determined by 1H-NMR, after 2 h was 32%.

Fluorescent POEGMEA-b-PCEA copolymer synthesis The BSPA-POEGMEA macroRAFT agent (70.2 mg, 8.76 × 10-6 mol), CEA (129.3 mg, 8. 97 × 10-4 mol) and fluorescein O-methacrylate (3.8 mg, 9.46 × 10-6 mol) were dissolved in 1,4-dioxane (0.965 mL) before AIBN (0.20 mL of 1.77 × 10-2 M stock solution) was added to the mixture. Oxygen was removed from the polymerisation solution via three freeze-pump-thaw cycles after which the flask was sealed. After 8 h at 60 °C, the polymerisation reaction was quenched via simultaneous exposure to the atmosphere and cooling in an icebath. The polymer was purified via dialysis against MilliQ water for 24 h (4 × water change) and isolated via lypophilisation. All experimental steps were conducted in the dark. The conversion of CEA, as determined by 1H-NMR, after 8 hours was 86%. A non-fluorescent version of the block copolymer was similarly produced where the final block copolymer structure was POEGMEA16-PCEA114 as determined by 1H-NMR.

Paclitaxel-polymer conjugate synthesis The fluorescent POEGMEA-b-PCEA copolymer (15.7 mg, 7.46 × 10-7 mol) was dissolved in DMSO (300 µL) before the addition of DIPEA (31.9 µL, 1.83 × 10-4 mol) and TNTU (23.8 mg, 7.91 × 10-5 mol). Once TNTU was completely dissolved, PTX (7.7 mg, 9.02 × 10-6 mol) was added and the reaction was left to stir at 40 °C. After 48 h, the reaction solution was removed and purified via successive dialysis (MWCO = 6000 - 8000) against acetonitrile (3 × solvent change), acetonitrile/MilliQ (50 : 50, 3 × solvent change) and MilliQ water (5 × solvent change). The purified conjugate (POEGMEA-b-PCEAPTX) was isolated via lypophilisation. All experimental steps were conducted in the dark. The number of PTX moieties attached per polymer chain, as determined by 1H-NMR, was 6.9 units.

Formation of micelles Micelle formation was completed via dialysis and occurred simultaneously with the purification of the paclitaxel-polymer conjugate. Analysis and size determination of micelles containing fluorescence was confirmed via TEM.

Core-crosslinking with 1,8-diaminooctane A micellar solution of the paclitaxel-copolymer conjugate was prepared by dissolving the lypophilised solid (2.0 mg) in MilliQ water (0.6 mL). Stock solutions of 1,8-diaminooctane (0.36 mg/mL; uniform suspension was achieved after 30 min sonication), EDC (1.92 mg/mL) and NHS (1.14


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mg/mL) were prepared in MilliQ water. 100 µL of the EDC and NHS stock solution was added to the micellar solution and allowed to stir for 5 min before the 1,8-diaminooctane stock solution (100 µL) was added. The flask was sealed and the reaction proceeded at room temperature for 24 h. After 24 h, the solution was dialysed against MilliQ water (3 × solvent change) to remove unwanted reagents. The final 1,8-diaminooctane crosslinked micelle (denoted DAO micelles) solution had a concentration of 1.68 mg/mL.

Enzymatic degradation of polymer-paclitaxel ester bond Lipase from Aspergillus niger 47 was dissolved in PBS (pH 7.4) to give a pale brown solution (10 mg/mL). Uncrosslinked micelles (0.30 mg) were dissolved in PBS before 200 µL of lipase solution was added to the micelles to give a final micelle concentration of 0.45 mg/mL and lipase concentration of 3.0 mg/mL. Similarly, DAO micelles (0.48 mg) were dissolved in PBS and then lipase solution (310 µL) added to the solution to give the same micelle and lipase concentration as that for uncrosslinked micelles. Both micelle variants were incubated at 37 °C with gentle shaking for 72 h and samples were removed every 24 h for analysis. The removed samples were lyophilised and dissolved in acetonitrile/water solution (50 : 50) for high performance liquid chromatography (HPLC) analysis. The digestion was repeated at pH 4.5 and 5.5 where the samples were removed after 72 h, lyophilised and dissolved in acetonitrile/water for HPLC analysis.

In vitro studies on the toxicity of paclitaxel-polymer conjugate micelles Cell culture Human prostate carcinoma LNCaP cells were used for the in vitro testing of the paclitaxel-copolymer micelles. All in vitro testing was conducted on LNCaP cells which have been passaged less than 15 times. The cells were cultured in RPMI-1640 medium (Life Technologies, supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM sodium pyruvate) at 37 °C and 5 % CO2. Upon confluence, the cells were detached from tissue culture flask with trypsin/EDTA treatment (3 min) before being seeded into a new tissue culture flask. Intracellular micelle distribution via confocal microscopy LNCaP cells were seeded in a 35 mm Fluoro dish at 1 × 105 cell per dish and cultured for 2 d. The cells were incubated with micelles (250 µg/mL) for 1 hr and then washed three times with phosphate buffered saline (PBS, pH 7.4). The cells were stained with 2.0 µg/mL Hoechst 33342 (5 min) and excess stain removed with PBS washing (3 ×). The cells were then stained with 100 nM LysoTracker (1 min) and rinsed once with PBS before they were mounted in PBS for confocal observation IC50 determination via sulforhodamine B assay (2D cell studies) LNCaP cells were seeded in a 96-well plate at a density of 3 × 103 cells/well in 100 µL of cell culture medium. The cells were incubated for 24 h at 37 °C and 5% CO2 before the medium was discarded from the plate. Twice-concentrated RPMI-1640 medium was then added to each well (100 µL). For wells which acted as the control, the total volume was increased to 200 µL by the addition of sterile MilliQ water (100 µL).


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The sample solution to be tested was sterilised via UV irradiation (20 min) before the solution was serially halved via dilution in sterile MilliQ water. The micellar solutions were then loaded into the plate at 100 µL per well. The plate was incubated at 37 °C and 5% CO2 for 72 h. The samples which were tested include paclitaxel (free), uncrosslinked POEGMEA-b-PCEA-PTX conjugate micelles, and 1,8-dimainooctane crosslinked POEGMEA-b-PCEA-PTX conjugate micelles. The highest concentration of paclitaxel (equiv.) tested was 100 nM for free paclitaxel and 5 µM for the micellar solutions. Free PTX was initially dissolved in DMSO (1 × 10-4 M) and the appropriate solution was made by dilution with MilliQ water. The final working concentration of DMSO is 0.1% in the cell culture medium. After 24 or 72 h, the culture medium was discarded and 10% cold trichloroacetic acid (TCA) was added to each well (100µL). The plate was incubated at 4°C for 30 min before the TCA was discarded and the plate was washed with water (5 ×). Sulforhodamine B (SRB) solution (0.4 wt% in 1% acetic acid) was added to each well (100 µL) and the plates incubated at room temperature for 20 min in dark. SRB solution was discarded and excess dye removed via washing with 1% acetic acid (5 ×). After air-drying the plate, tris(hydroxymethyl)aminomethane (TRIS) buffer solution (200 µL) was added to each well to dissolve bound SRB. The absorbance of the plate was measured at 572 nm on a Multiskan Ascent plate reader and the data were analysed with GraphPad Prism 6.0. 3D prostate MCTS formation The prostate MCTS were prepared by a liquid overlay method 48. LNCaP cells were suspended in RPMI-1640 media at a density of 7.5 × 103 cells/mL. 200 µL of the cell suspension was seeded into each well of an ultra-low attachment 96-well plate (Corning). The plate was centrifuged for 5 min at 500 × g and incubated at 37 °C and 5% CO2 for 4 d. LNCaP cells will form spheroids after 4 d’s culture and the morphology of the spheroids was recorded by an inverted microscope with CCD camera (Leica). Micellar treatment of prostate MCTS After the LNCaP MCTS was formed, 170 µL of culture medium was removed and replenished with 200 µL of fresh medium supplemented with PTX (free), UC or DAO micelles. Firstly, micelle distribution in MCTS was observed with laser scanning confocal microscopy. After incubation with micelles (250 µg/mL) for 2 h, the spheroids were washed with PBS thrice. The spheroids were then mounted in PBS for confocal observation. The MCTS were also incubated with micelles or PTX for up to 14 days to evaluation the anti-tumor efficiency in the 3D MCTS model. The concentration of paclitaxel from the micelles was 15 µM and was the same across all samples. The medium and micelles/free PTX were replenished once at Day 7. At day 4, day 7 and day 14, spheroid morphology was recorded with the microscope and cell viability of MCTS was determined by an acid phosphatase (APH) assay described elsewhere 49. Briefly, the MCTS and entire supernatant with cells were gently transferred into standard flat-bottomed 96-well microplates. The plates were centrifuge for 5 min at room temperature at 500 × g to spin down spheroids, clusters and single cells. The spheroids were washed carefully with PBS twice and finally supernatant was discarded to a final volume of 100 µL. Then 100 µL of APH assay buffer was added to each well and incubated for 90 min at 37 °C and 5% CO2. The reaction was terminated with 10 µL of 1 M NaOH and the absorption at 426 nm was measured with a microplate reader (Ascent). APH


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assay buffer was composed of 2 mg/mL p-nitrophenyl phosphate, 0.1M sodium acetate and 0.1% (v/v) Triton X-100 (pH 4.8). P-nitrophenyl phosphate was added just before use. Statistical analysis All data for In vitro studies were reported as mean ± standard deviation (SD). A one-way analysis of variance was performed for the statistical analysis followed by a Tukey’s post hoc test for pairwise comparison. A P value less than 0.05 was considered statistically significant. All the statistical analysis was done with GraphPad Prism 6.0.

Instrumental Analysis Nuclear magnetic resonance (1H-NMR) All NMR analysis was conducted on a Bruker Avance III HD instrument (1H, 600MHz) using a TCI cryoprobe. Either deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) was used and are assigned within each respective spectra. All spectra were collected using a minimum of 8 scans and all chemical shifts are reported in ppm (δ). DMAc gel permeation chromatography (GPC) The DMAc GPC system is a Shimadzu modular system consisting of a DGU-12A degasser, LC-10AT pump (1 mL/min), SIL-10AD automatic injector (50 µL), CTO-10A column oven (50 °C) and RID-10A refractive index detector (290 nm). A Phenomenex 50 × 7.8 mm guard column and four linearly arranged Phenomenex 300 × 7.8 mm columns (105, 104, 103 and 102 Å pore size, 5 μm particle size) were used for the analyses. DMAc (HPLC grade) was used as the mobile phase and it was supplemented 0.05 w/v% BHT and 0.03 w/v% LiBr. Calibration of the instrument was conducted with commercial polystyrene standards with molecular weights in the range of 200 - 106 g/mol. Samples were dissolved in DMAc (final concentration ca. 4 - 5 mg/mL) and then filtered through a 0.45µm filter to remove particulates prior to injection. Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Dynamic light scattering (DLS) DLS measurements were conducted Bookhaven ZetaPlus Particle Sizer with a Dust-Cutoff value of 40. For size determination, polymer samples were prepared in MilliQ water at a concentration of 0.5 mg/mL and filtered through a 0.45 µm filter prior to measurement to remove any dust particulates. Polymer samples used to confirm crosslinking presence was prepared in DMSO at a concentration of 0.5 mg/mL and measured as is without filtration. Each measurement was repeated in triplicate and averaged to give the listed values. High performance liquid chromatography (HPLC) The HPLC system is a Shimadzu modular system consisting of a SPD-20A UV-Vis detector, DGU-20A3 degasser, LC-20AD pump (1 mL/min), Rheodyne 7725i manual injector (150 µL), and a Cole-Palmer single compact HPLC column heater (30 °C). The column was a Phenomenex Viva C18 (250 × 4.6 mm) with a pore size of 5 µm. The injection volume of each sample was 75 µL. A 50:50 mixture of acetonitrile (HPLC grade) and MilliQ water was used as the mobile phase. A range of paclitaxel


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solutions with different, and known, concentrations was prepared in the same solvent and used to prepare standard curve. The elution time of paclitaxel was 8.89 min. Transmission electron microscopy (TEM) TEM images were obtained with a JEOL1400 or GEI Techani-G2 instrument where the working beam voltage was 80-100 kV on both machines. Samples were prepared by loading a drop of the sample solution onto a FORMVAR-coated copper grid (JEOL 1400) or carbon-coated copper grid (TechnaiG2) and letting the solution sit, under cover, for 30 min. Excess solution was removed using filter paper and the grids allowed to air dry before staining. The grids were stained using uranyl acetate (3 %) for 5 min and air dried before imaging. Laser scanning confocal microscopy Micellar internalisation and penetration in spheroids were observed under a Zeiss LSM 780 laser scanning confocal microscope system. The system equipped with a Diode 405-30 laser, an argon laser and a DPSS 561-10 laser connected to a Zeiss Axio Observer.Z1 inverted microscope. The ZEN2012 imaging software (Zeiss) was used for image acquisition and processing. The 2D cultured cells were observed with a 100 × 1.4 NA oil objective and the spheroid was observed with a 20 × 0.8 NA air objective.

Results and Discussion Polymer Synthesis Fluorescent POEGMEA-b-CCEA Synthesis The RAFT polymerisation of OEGMEA by BSPA resulted in a macroRAFT agent with 16 OEGMEA units which corresponds to 32% conversion of the initial added amount of OEGMEA monomer. Further chain extension with CEA resulted in the addition of 88 units of CEA on to the polymer chain (equal to 86% conversion). It was assumed that the number of fluorescein O-methacrylate units, which copolymerised simultaneously with CEA, occurred so in the same ratio as that added to the reaction solution. That is, as the initial working ratio of macroRAFT to CEA to fluorescein O-methacrylate was 1:102:1.08, it was assumed that one unit of fluorescein O-methacrylate was added per polymer chain. The presence of fluorescein O-methacrylate was visually confirmed after purification of the POEGMEA-b-PCEA copolymer as the solid was the distinctive yellow colour associated with fluorescence. Hence, the final fluorescent POEGMEA-b-PCEA copolymer used for further experiments was characterised as POEGMEA16-b-PCEA88.

Conjugation of PTX to POEGMEA-b-PCEA Attachment of PTX to the block copolymer was conducted with solid phase peptide synthesis (SPPS) reagents due to the robust nature of SPPS reagents. Although chemical conjugation of PTX have been previously conducted with traditional carbodiimide regents 23, 26, the use SPPS reagents allows the reaction to proceed at room temperature and bypasses the requirement for anhydrous and/or inert conditions. The successful conjugation of PTX highlights the versatility of SPPS reagents as an


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esterification reagent in addition to its main purpose as a facilitator of amide bonds. The polymer was incubated with paclitaxel at 40 °C for 48 h where the ratio between the carboxylic acid on the polymer backbone and paclitaxel units was 8:1. Under these conditions, it was determined via 1HNMR that each polymer chain contained 7 units of PTX, which equates to approximately 22 wt% of the final conjugate. Thus, the actual the conjugation efficiency using SPPS reagents was 62%. In all likelihood, conjugation past this point is limited due to the steric hindrance caused by the paclitaxel units already attached to the polymer backbone. Use of carbodiimide reagents for the conjugation of paclitaxel to polymer chains have resulted in varying conjugation efficiencies of 60.8 – 87.5% 17, 23, 30, 31, 50 . However, carbodiimide reactions often require anhydrous and/or inert conditions and an excess of coupling reagents and/or paclitaxel itself. Thus, SPPS reagents deliver comparable conjugation efficiencies without the need for such stringent reaction conditions even where the coupling reagent and paclitaxel were only added at 10-15 mol% of the available carboxylic acid groups. Table 1 summarises the characterisation of the block copolymer and POEGMEA-b-PCEA-PTX. In particular, the similar Ð values between the copolymer itself and the final conjugate (1.26 and 1.20, respectively) shows that the number of PTX moieties per polymer chain can be assumed to be similar between all polymer chains. More importantly, no crosslinking occurred during the conjugation reaction. The GPC traces of the POEGMEA macroRAFT, the block copolymer and the polymer-paclitaxel conjugate are shown in Figure 2. As the GPC system was calibrated on polystyrene standards the analysis was only used to determine the polydispersity of each polymer sample and 1H-NMR was used to determine the conversion of each block as well as the conjugation efficiency of paclitaxel to the block copolymer. 1

H-NMR, GPC and HPLC analysis was used to confirm the successful conjugation of paclitaxel to the copolymer. As shown in Figure 3, the characteristic peaks from paclitaxel, especially in the aromatic region (δ 7.35 – 8.1 ppm) can be clearly seen in the final conjugate after purification. The lack of free paclitaxel in the final conjugate was determined by both GPC and HPLC. In the GPC trace, the characteristic sharp paclitaxel peak (retention time > 29 min) was not seen in the polymer-conjugate trace. In addition, the shift in retention time of conjugate trace when compared to the block copolymer trace indicates a clear change molecular weight as well as copolymer amphiphilicity due to the conjugation of paclitaxel. Similarly, when the polymer-conjugate was analysed via HPLC, the characteristic free paclitaxel peak at 8.89 min was not seen. Instead, the conjugate eluted at 2.8 min, where the copolymer is typically seen, again indicating successful conjugation of the drug (data not shown).

The conjugation efficiency of paclitaxel was determined by comparing the aromatic protons on the paclitaxel (Figure 3, h, i, j, δ 7.35 – 8.1 ppm) to the –CH2– groups on the polymer backbone (Figure 3, a, b, δ 3.9 – 4.4 ppm). It was calculated that for every polymer chain, 7 units of paclitaxel was coupled to the carboxylic acid on the PCEA block. That is, the final polymer-paclitaxel conjugate had the structure POEGMEA16-b-PCEA88-PTX7. Table 1: Conversion, calculated molecular weight and PDI of BSPA-POEGMEA

Sample POEGMEA16 MacroRAFT Fluorescent POEGMEA16-b-PCEA88

Conversion (%) 32 a 86 a


Mn (g/mol)b 7950 21000

Ðc 1.12 1.26

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copolymer POEGMEA16-b-PCEA88-PTX7 conjugate

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1.20

a

monomer conversion b Determined via NMR c Determined via GPC

Figure 2: GPC traces of POEGMEA16 macroRAFT, POEGMEA16-b-PCEA88 block copolymer and POEGMEA16-b-PCEA88-PTX conjugate

1

Figure 3: H-NMR spectra and assignment of POEGMEA-b-PCEA-PTX conjugate.

Crosslinking of POEGMEA-b-PCEA-PTX conjugate micelles with diaminooctane Although the PTX conjugate naturally formed stable uncrosslinked micelles (UC micelles) during the purification process, crosslinking has been shown to introduce additional stability which can


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enhance the cellular uptake, 13 but also accelerate exocytosis of micelle from the cell.51 1,8diaminooctane, a permanent crosslinker, was chosen for this study to showcase the effects of different micelle structures on cytotoxicity. The crosslinker was introduced into the micelles via amide bonds between the amine groups on the crosslinker and the free carboxylic acid groups of the PCEA block on the copolymer chain (as shown in Scheme 1). The size of the micelle variants were determined by DLS (Figure 4) and the results are given in Table 2. The success of the corecrosslinking was also confirmed via DLS (Figure 4) where the 1,8-diaminooctane crosslinked micelles (DAO micelles) were dissolved in DMSO (a good solvent for both blocks of the copolymer). The intensity-average diameter of the micelle was 236.6 nm, signifying that crosslinking was a success as the micelles retained their morphology in DMSO.

Figure 4: Intensity-average particle size distribution as determined by DLS of POEGMEA-b-PCEA-PTX conjugate micelles (with and without core-crosslinking) in water and DMSO. Table 2: Sizes of UC micelles, CYS micelles and DAO micelles as determined via DLS and TEM (n > 40)

Micelle Sample UC DAO

Size of micelles via DLS Intensity-average diameter (nm) 178.4 181.7

PDI 0.173 0.188

Size of micelles via TEM Diameter (nm) 70.1 42.4

The TEM images of both micelle variants are shown Figure 5. Size measurements of the micelles using the TEM images were significantly smaller when compared to their hydrated counterparts, which is to be expected as micelle size contracts upon drying (as is the case when loaded on to a TEM grid). The sizes of each family of micelles were measured over the population shown on two separate TEM images (numbering at least 40 measurements) and averaged to 70.1 nm and 42.4 nm for UC and DAO micelles respectively, highlighting the effect of the environment on micelle size. When dry, crosslinked micelles were shown to be smaller than that of the uncrosslinked micelles, indicating the contracted nature of the core due to the crosslinking agent.


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Figure 5: Transmission electron microscopy images of POEGMEA-b-PCEA-PTX conjugate micelles (with and without core crosslinking). Scale bar = 100 nm.

Cellular uptake of uncrosslinked micelles via confocal microscopy The internalisation of POEGMEA-b-PCEA-PTX conjugate micelles was visualised using laser scanning confocal microscopy. Figure 6 depicts the uptake and distribution of uncrosslinked micelles in LNCaP cells after incubation with the micelles for 1 h. Fluorescent micelles (green) were observed in the cytoplasm of the cells and external to the Hoechst 33342-stained cell nuclei (blue). The lysosomes were stained with LysoTracker (red) to help identify the location of the micelles. The merged image clearly shows the overlap of the micelles and lysosomes, confirming the lysosomal distribution of the micelles after uptake by the cells.

Figure 6: The uptake of POEGMEA-b-PCEA-PTX conjugate micelles (uncrosslinked) by LNCaP cells as determined by confocal microscopy. Scale bars = 10 µm.

Cytotoxicity in 2D monolayer cell culture The first step in the testing the effectiveness of the nanoparticle delivery system of interest often lies in determining cytotoxicity of the compound. A SRB-based assay was used to determine the IC50 value of the compounds on 2D cultured cells. However, it is necessary to first establish the lack of


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cytotoxicity of the delivery vehicle. The cytotoxicity of POEGMEA-b-PCEA was tested with a sample of the non-fluorescent block copolymer and it was shown that the copolymer was nontoxic up to concentrations of 1 mg/mL (Figure 7), which is far in excess of the concentration of copolymer used throughout this body of work. Hence, the copolymer used for all the in vitro studies can be taken to be biocompatible and to have no influence on the overall effectiveness of the micelles.

Figure 7: Viability of LNCaP cells treated with blank POEGMEA-b-PCEA micelles.

The cytotoxicity of free PTX and the different micelles were evaluated in vitro after 24 and 72 h. After 24 h, the IC50 of paclitaxel, UC micelles and DAO micelles were 70.8 nM, 1.81 µM and 857 nM, respectively. However, after 72 h, the IC50 of all compounds were significantly lower (shown in Figure 8 and summarised in Table 3), demonstrating the time-dependent therapeutic action of macromolecular drug formulations. After 24 h, it was obvious that free paclitaxel exhibited the most toxicity towards LNCaP when compared to the micelle variants and this trend is also seen after 72 h incubation. After 72 h incubation, the IC50 of paclitaxel was determined to be 15.2 nM, again emphasising the extreme cytotoxicity associated with the compound. All the micelles formulations, however, had significantly higher IC50 values, with the UC and DAO micelles exhibiting IC50 values at 270.9 and 190.3 nM, respectively. The lower cytotoxicity of UC micelles might be attributed to the inherent instability of uncrosslinked micelles, leading to the possible dissociation of the micelle before it could be taken into the cell.13, 52 DAO micelles had an IC50 value of 193.2 nM, corroborating the idea that more stable micelles were better able to enter the cell 53. The IC50 ratio of UC to DAO at 24 h (2.11) decreased to 1.42, which also indicates that core-crosslinked DAO micelles also accelerated delivery of PTX into 2D culture prostate cells. The phenomenon where the cytotoxicity of free paclitaxel remains lower than the cytotoxicity of the drug-loaded micelles in 2D monolayer experiments has been previously observed 30, 31, 50, 54. In all these cases, the IC50 value of free paclitaxel on the cancer cell line of interest was already extremely low with IC50 values often being less than 10 nM. Similarly, micellar delivery of doxorubicin, another extremely cytotoxic anti-cancer drug, has displayed a similar trend to that seen in Table 3 where the IC50 of the free drug continues to be lower than that of the nanoparticle 12, 45, 55. As paclitaxel has inherent low IC50 values against LNCaP cells (with other literature values ranging from 1.1 nM to 40 nM depending on culture conditions 56-58), it is conceivable that micellar delivery of paclitaxel follows


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the trend seen with other polymer-PTX and polymer-doxorubicin conjugates where no apparent improvement in the cytotoxicity of the drug can be achieved via assisted delivery. However, due to the extreme hydrophobicity of PTX, assisted delivery is a requirement and micellar PTX still demonstrated significant cytotoxicity against LNCaP cells. Although the cytotoxicity of the conjugate is lower when compared to free paclitaxel, the protection offered by the polymeric vehicle ensures that not only is more PTX solubilised, delivery of the drug can also be more specifically targeted due to inherent advantages offered by nanoparticle delivery systems (such as the EPR effect and conjugation of targeting ligands to the vehicle itself). In addition, multidrug resistance (MDR) involving the transporter protein P-glycoprotein has been implicated in the resistance of cancer cells to paclitaxel and such MDR can be overcome via nanoparticle delivery 59.

Figure 8: Cytotoxicity curves of free paclitaxel and POEGMEA-b-PCEA-PTX conjugate micelles (with and without core crosslinking) on LNCaP cells

Table 3: IC50 values of free paclitaxel and POEGMEA-b-PCEA-PTX conjugate micelles after 24h and 72 h incubation.

Compound

IC50 (nM) 24h

IC50 (nM) 72h

Free Paclitaxel UC micelles DAO micelles

70.8 1810 857

15.2 ± 4.7 270.9 ± 30.0 193.2 ± 16.6

Simulation of drug release from its polymeric delivery system via enzymatic degradation has previously been successfully completed using commercially available enzymes such as lipases 47. A similar degradation procedure was attempted with both micelle variants in an effort to quantify the amount of paclitaxel predicted to be released in an in vitro environment. However, no appreciable amount of free paclitaxel was detected via HPLC analysis across all treatment times (24, 48, 72 h) and all pH values (4.5, 5.5 and 7.4). This might have resulted from an incompatibility between the lipase and the micellar system. If, however, the ester linkage between paclitaxel and the polymer backbone cannot be hydrolysed by intracellular enzymes, the significant IC50 demonstrated by all three variants suggest that activity is still retained, even if the paclitaxel is in a macromolecular form.


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Evidently, further elucidation of micelle behaviour after it has entered the lysosome is required as it is an area that is not well understood. MCTS treated with micelles and free PTX Micelle penetration into MCTS via confocal microscopy Although the 2D cell culture model provided useful information and allowed comparison of the toxicity of micellar-paclitaxel and free paclitaxel, the effects of the nanoparticle delivery system on a 3D cell culture model was also of great interest due to its greater similarity to in vivo tumors. Hence, PTX-conjugate micelles were also used to treat prostate MCTS to evaluate the micellar cytotoxicity and drug delivery in 3D tumor models. Laser scanning confocal microscopy was utilised to envisage the uptake of micelles into MCTS. The micelle variants were tested on LNCaP MCTS and Figure 9 shows the penetration of the micelles scanned at a depth of 90 µm in the spheroids. From the Figure 9, it can be seen that although crosslinked micelles penetrate slightly deeper into the spheroid, both micelles exhibited significant uptake by the spheroids. A recent study by Lu et al 60 investigated the effects of size and crosslinking of micelles on spheroid penetration and their results corroborate those seen in this work. They found that both size and crosslinking play an integral part in the uptake of micelles and as the micelles used in this work were extremely similar in size, it can be argued that the deeper penetration of the DAO micelles was due to its more stable structure.

Figure 9: Penetration of POEGMEA-b-PCEA-PTX conjugate micelles (uncrosslinked, UC and crosslinked, DAO) by LNCaP spheroids as determined by confocal microscopy at a depth of 90µm (scale bars = 100µm).

Inhibition of MCTS growth after 4, 7 and 14 days’ treatment After incubation of LNCaP MCTS with the micelle variants and free PTX, the morphological changes of MCTS were shown in Figure 10. None of the three treated spheroid samples increased in size as much as that of the control. There was no change to both the size and morphology of free PTX treated spheroids. Although the structures of spheroids treated with UC and DAO micelles were not completely destroyed, there was a slight decrease in the spheroids’ size within these two groups. It is very clear that MCTS treated with both micelle variants and PTX all underwent significant growth inhibition. Thus, the POEGMEA-b-PCEA-PTX conjugate micelles retained similar activity to free PTX, despite the implications of the results from the 2D monolayer cell study.


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Figure 10: Effect of POEGMEA-b-PCEA-PTX conjugate micelles (UC and DAO) on the growth of LNCaP spheroids when compared to free paclitaxel (scale bar = 300 µm).

Although size changes in spheroids provide an excellent visual indication of the effects of the micelle variants when compared to free PTX and the control, it is not a definite quantitative method. A more robust method to determine the cytotoxic effects of the micelles and free PTX in MCTS models is to determine the activity of acid phosphatase, which indicates the cell viability changes in 3D spheroids during the treatment 49. Figure 11 shows the APH activities of MCTS after 4, 7 and 14 days’ treatment with UC micelles, DAO micelles and free PTX. As expected, the cell viability per spheroid in the control population is obviously higher than that of all other spheroids at every time point. The control spheroids continued to proliferate throughout the entire culture period and this was observed with the increasing reading from Day 4 to Day 14. However, cell viabilities of the spheroids in UC, DAO and free PTX groups show decreasing trends during the treatment. At day 4, DAO micelle treated MCTS exhibited significantly lower cell viability than both UC micelle and free PTX treatment groups. In addition, UC micelle treated spheroids had statistically lower cell viability than PTX treated micelles. At day 7, there was no statistical difference among UC, DAO and free PTX groups even though the viability reading kept Day 4’s sequence where spheroid viability decreased in the order of free PTX ˃ UC ˃ DAO. At day 14, the spheroids treated with UC, DAO and free PTX had almost the same viabilities. The result that the cell viabilities in micellar treatments groups were lower than the free PTX treatment group is consistent to the size growth of MCTS. In contrast to the results obtained from 2D cell culture model, free PTX showed no superiority in the cytotoxicity against prostate spheroids: it even induced less cell death at Day 4 and Day 7. Compared with free PTX, PTX-conjugate micelles exhibited accelerated anti-tumor efficiency in the 3D MCTS model. Particularly, the crosslinking of micelles with DAO promoted the accurate cytotoxicity for MCTS compared with the uncrosslinked micelles. The reason may be related to the deeper penetration of micelles which have relatively high


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stability 60. At Day 14, all the three treatments showed similar cytotoxicity to MCTS because the long-time incubation enables the abundant penetration of both free drug and micelles.

Figure 11: Cell viability of LNCaP spheroids after 4, 7 and 14 days treatment with POEGMEA-b-PCEA-PTX conjugate micelles (with and without crosslinking) and free paclitaxel. Data represent means ± SD, n = 8. *, significant difference, P ˂ 0.05; **, significant difference, P ˂ 0.01; ***, significant difference, P ˂ 0.001.

The discrepancy between 2D monolayer cell studies and 3D MCTS models is highlighted. Both UC and DAO micelles demonstrated a significant increase in IC50 values when compared to free PTX in the 2D monolayer model, but the results are significantly different in the MCTS model. Whereas the 2D monolayer study would indicate that none of the micelle variants could compare to the activity of free PTX, the MCTS model emphasises the effectiveness of the nanoparticle delivery method, especially for core-crosslinked stable micelles. Crosslinking is required to negate the rapid breakup and clearance of micelles from the body 61. In addition to increasing stability, crosslinking accelerated the cytotoxic effects of the polymer-paclitaxel conjugate in 3D MCTS models and hence, represents a beneficial nanoparticle modification for drug delivery. The distinctly different results obtained from 2D and 3D studies provides a compelling reason to incorporate MCTS models as a method for bridging the gap between in vitro and in vivo studies.

Conclusion Through the incorporation of a 3D cell culture model in this study, it was determined that POEGMEA-b-PCEA-PTX conjugate micelles retained the same effectiveness as free paclitaxel when treating prostate cancer spheroids over a period of 14 days. Accelerated anti-tumor activities were also demonstrated by core-crosslinked micelles, especially when compared to uncrosslinked micelles and free paclitaxel, during the first 7 days of treatment. The enhancement in penetration characteristics of the crosslinked micelles were thought to be facilitated by their increase in stability as it allowed the micelles to penetrate faster and deeper within the LNCaP MCTS. Although the 2D cell culture model indicated that the micelle variants were not as effective as free paclitaxel, the MCTS model demonstrated otherwise. Hence, the incorporation of 3D cell models will be of great benefit when elucidating the effectiveness of nanoparticle drug delivery.


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Acknowledgement The authors would like to thank the staff at the Electron Microscope Unit and Nuclear Magnetic Resonance Facility at UNSW for the support provided.

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Core-crosslinking accelerates anti-tumor activities of paclitaxel-conjugate micelles to prostate multicellular tumor spheroids: a comparison of 2D and 3D models Alice W. Du, Hongxu Lu and Martina H. Stenzel


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Core-Cross-Linking Accelerates Antitumor Activities of Paclitaxel

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