Influence of Size and Shape on the Biodistribution of Nanoparticles

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Influence of Size and Shape on the Biodistribution of Nanoparticles Prepared by Polymerization-Induced Self-Assembly (PISA) Sadik Kaga, Nghia P. Truong, Lars Esser, Danielle Senyschyn, Amitav Sanyal, Rana Sanyal, John F. Quinn, Thomas P. Davis, Lisa M. Kaminskas, and Michael R. Whittaker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00995 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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Influence of Size and Shape on the Biodistribution of Nanoparticles Prepared by PolymerizationInduced Self-Assembly (PISA) Sadik Kaga,a,b,c Nghia P Truong,b Lars Esser,b Danielle Senyschyn,a,b Amitav Sanyal,c Rana Sanyal,c John F. Quinn,b Thomas P Davis,b,d Lisa M Kaminskas,a,e* Michael R Whittakerb*

a

Drug Delivery Disposition Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia

b

ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia c

d

e

Department of Chemistry, Bogazici University, Istanbul, 34342, Turkey

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom

School of Biomedical Sciences, University of Queensland, St Lucia, QLD 7052, Australia

Keywords: Polymerization-induced self-assembly, Nanoparticle, Biodistribution, Tumour xenograft.

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ABSTRACT: Polymerization-induced self-assembly (PISA) is a facile one-pot synthetic technique for preparing polymeric nanoparticles with different sizes and shapes for application in a variety of fields including nanomedicine. However, the in vivo biodistribution of nanoparticles obtained by PISA still remains unclear. To address this knowledge gap, we report the synthesis, cytotoxicity and biodistribution in an in vivo tumour bearing mouse model of polystyrene micelles with various sizes and polystyrene filomicelles with different lengths prepared by PISA. First, a library of nanoparticles was prepared comprised of poly(glycidyl methacrylate)-bpoly(oligo(ethylene glycol) methyl ether methacrylate)-b-polystyrene polymers and their size and morphology was tuned by varying the polystyrene block length without affecting the surface chemistry. The non-cytotoxicity of these nanoparticles after hydrolysis of the epoxide groups was confirmed by MTT assays against HT1080 cancer cells and AlamarBlue assay against human umbilical vein endothelial cells (HUVECs). These nanoparticles were then radio-labelled with tritiated (3H) ethanolamine and a biodistribution study was carried out in nude mice bearing HT1080 tumour xenografts 48 hrs after intravenous delivery. In this model we found that small spherical polystyrene core nanoparticles with a PEG corona (diameter 21 nm) have the highest tumour accumulation when compared to the larger spherical nanoparticles (diameter 33 nm) or rod-like (diameter 37 nm, contour length 350 – 500 nm) or worm-like counterparts (diameter 45 nm, contour length 1 – 2 µm). This finding has provided critical information on the biodistribution of polystyrene core nanoparticles with a PEG corona of different sizes and shapes prepared by the PISA technique and will inform their use in medical applications.

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Introduction Following the clinical approval of the first nanoparticle for cancer drug delivery in 1995, a myriad of nanoparticle platforms have been designed and produced.1,2 Nanoparticles improve the efficacy of delivering chemotherapeutic agents by enhancing in vivo solubility and stability, and increase tumour accumulation via the enhanced permeability and retention (EPR) effect.3 Simultaneously, drug-loaded nanoparticles also reduce peripheral toxicity and cardiotoxicity as compared to conventional chemotherapeutics.4 Although significant advances have been made in overcoming physiological barriers, such as PEGylation, specific targeting of cancer cells leading to enhanced cellular uptake, endosomal escape, and controlled drug release, many challenges still remain.5 For example, the majority of nanoparticles accumulate in the liver, spleen and kidney, and only a small percentage (i.e., 0.7% of injected dose) accumulates in tumour tissue.6 Therefore, a high tumour accumulation is arguably the most challenging goal in cancer drug delivery. There has been increasing recognition that the physical characteristics of nanoparticles such as stiffness, rigidity, size and shape can influence their use in nanomedical applications.7,8 Recent literature reports have indicated that worm-like nanoparticles hold great promise for improving the tumour accumulation of nanoparticles.9,10 In general, most of the studies conclude a beneficial effect of a filamentous morphology, as compared to spherical particles. For example, Discher and co-workers demonstrated that filamentous micelles were less readily taken up by phagocytes than spherical nanoparticles, enhancing blood circulation.11 Moreover, it was shown that worm-like nanoparticles penetrate dense tumour tissue more efficiently than spheres.12 The flexibility of rods was also important, as flexible rods cleared from plasma much more slowly than size-matched rigid rods.5

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Eisenberg and co-workers reported the first example of self-assembly of asymmetric block copolymers in solution to form worm-like micelles in 1995.13,14 Over the following years, block copolymers containing different physical and chemical properties have been increasingly employed to generate worm-like nanoparticles as well as other morphologies.15-17 The main limitations, such as low nanoparticle concentration and loading capacity, have been addressed by taking advantage of simultaneous polymerization and self-assembly to obtain polymeric nanoparticles. This polymerization-induced self-assembly (PISA) approach is based on the chain extension of soluble homopolymers with a co-monomer to give an insoluble second block that forces self-assembly in the polymerization solution.18 Davis and co-workers successfully showed the formation and simultaneous self-assembly of reversible addition–fragmentation chain transfer (RAFT) synthesized poly(oligo(ethylene glycol) methyl ether methacrylate)-bpolystyrene (POEGMA-b-PS) nanoparticles, yielding several morphologies: spherical micelles, rod-like micelles, worm-like micelles and spherical vesicles.19 In follow-up studies, this technique was extended to applications in drug delivery (e.g., by conjugating doxorubicin via a pH-sensitive bond)20 and magnetic resonance imaging.21,22 However, to the best of our knowledge no biodistribution study has been carried out to study the in vivo behavior of spherical and worm-like micelles synthesized by the PISA approach, and this limits the translation of these materials to downstream biomedical applications. To investigate the possible advantages of worm-like polymeric nanoparticles we used the PISA technique to prepare spherical micelles and worm-like micelles. We first synthesized a short poly(glycidyl methacrylate) (PGMA) block as functional segment using RAFT polymerization. The PGMA block was then chain extended with oligo(ethylene glycol) methyl ether methacrylate (OEGMA) to give PGMA-b-POEGMA copolymer. Polymerization-induced

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self-assembly was then performed using the diblock copolymer as macromolecular chain transfer agent (macro-CTA) and styrene (ST) as monomer in methanol to yield PGMA-b-POEGMA-bPST block copolymers. The insolubility of the polystyrene block in methanol enabled selfassembly of this triblock copolymer, resulting in nanoparticles with different morphologies such as spherical, rod-like and worm-like nanoparticles. The length of the PS block can then easily be tuned to yield different nanoparticle sizes and morphologies, while keeping the surface chemistry consistent. The PGMA block provided an additional reactive glycidyl moiety which enabled further functionalization of the nanoparticles for the biodistribution study. Specifically, the epoxide groups were ring opened using tritiated (3H) ethanolamine. After radiolabeling, residual epoxide groups were inactivated by hydrolysis in the presence of an acidic catalyst. Additionally, non-radiolabeled nanoparticle batches were prepared by epoxide inactivation for cytotoxicity experiments. The cytotoxicity of non-radiolabeled and inactive nanoparticles was then tested using an HT1080 (human fibrosarcoma cells) cell line. Finally, tumour biodistribution experiments of the radiolabeled and inactivated nanoparticles were performed in tumourburdened female nude mice. HT1080 cells were injected to the flank region of the mice for tumour growth, and tumour and organ samples were collected 48 hours after nanoparticle injection. The radioactivity of the samples was then measured using a liquid scintillation counter.

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Experimental Section Materials. Glycidyl methacrylate (GMA), styrene (S) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA, average Mn 300 g/mol), 4-cyanopentanoic acid dithiobenzoate (CPADB), 2,2′-azobisisobutyronitrile (AIBN) and 12-14K molecular weight cut-off (MWCO) dialysis membrane were purchased from Sigma-Aldrich. Soluene-350 and Starscint were purchased from Packard Biosciences (Meriden, CT). Heparin (10,000 U/mL) was purchased from Faulding (SA, Australia). Isopropyl alcohol was AR grade and purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Fetal bovine serum (FBS), Glutamax and 0.25% trypsin-EDTA were obtained from Invitrogen (VIC, Australia). RPMI and MEM media and 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (NSW, Australia). Tritiated ethanolamine (40-60 Ci/mmol (1.48-2.22 TBq/mmol): 1 mCi/ml in ethanol) was purchased from American Radiochemicals. All other solvents were HPLC grade, obtained from Merck and used without further purification. Instruments. All polymers were characterized using 1H NMR spectroscopy (Bruker Avance 300 at 300 MHz) and GPC-DMAc (Shimadzu Modular System). Size and morphology of nanoparticles were examined using Dynamic Light Scattering (DLS) (Malvern Zetasizer Nano ZS) and Transmission Electron Microscopy (TEM). Absorbance values for cytotoxicity assays were measured using a Fluorostar microplate reader (Bio-Strategy). Disintegrations per minute (DPM) for the animal samples were measured using liquid scintillation counter (Perkin Elmer Tri-Carb 2800TR). Animals. Athymic nude mice (female, 6 weeks of age) were purchased from the Australian Research Centre (Perth, WA). Mice were maintained at 21 °C on a 12-hour light/ dark cycle.

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Food and water was provided ad libitum. The animal study was approved by the institutional Animal Ethics Committee. Synthesis of PGMA. RAFT polymerization was used for the synthesis of the PGMA. To a solution of GMA (2038 mg, 14.33 mmol) and CPADB (200 mg, 0.717 mmol) in toluene (9.5 mL), was added AIBN (11.8 mg, 0.0717 mmol). The mixture was purged with N2 to remove O2 and polymerization was allowed to proceed for 90 minutes at 70 °C. Polymerization was stopped by cooling down in an ice bath and exposing to air. Thereafter approximately 3/4th of the toluene was evaporated by placing the reaction mixture under a stream of air. PGMA was then purified by precipitating three times in cold petroleum benzene:diethyl ether (3:1) mixture followed by evaporation in vacuum oven overnight (at 30 °C) to give polymer with an average of approximately 9 GMA repeating units per chain (30% yield). Synthesis of PGMA-b-POEGMA. PGMA was used as macromolecular chain transfer agent for the synthesis of PGMA-b-POEGMA block copolymer via RAFT polymerization. To a solution of OEGMA (1900 mg, 6.34 mmol) and PGMA (200 mg, 0.127 mmol) in acetonitrile (10 mL), was added AIBN (2.6 mg, 0.0158 mmol). The mixture was purged with N2 and polymerization was allowed to proceed for 4 hours at 70°C. Polymerization was stopped as above. Approximately 3/4th of acetonitrile was evaporated by air flow. PGMA-b-POEGMA was then purified by precipitating three times in cold petroleum benzene:diethyl ether (1:2) mixture followed by evaporation in vacuum oven overnight (30 °C) to give polymer with an average of approximately 26 OEGMA repeating units (51% yield). Nanoparticle Formation by Polymerization-Induced Self-Assembly (PISA). The PISA approach was used to generate polymeric nanoparticles. To a solution of styrene (19.7 g, 189 mmol) and PGMA-b-POEGMA (350 mg, 0.0378 mmol) in methanol (27.8 mL), was added 100

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µL AIBN (1.24 mg, 0.00756 mmol) stock solution (12.4 mg in 1 mL methanol). The mixture was the purged with N2. To obtain PGMA-b-POEGMA-b-PS block copolymer nanoparticles with different size and shape, solution samples (5 mL) were collected at different time points under N2. The turbid and milky white nanoparticle solution samples were further purified by dialyzing against methanol (12-14K MWCO) to remove unreacted styrene monomer. Spherical small micelles (21 nm, after 6 hours), spherical large micelles (33 nm, after 10 hours), rod-like micelles (after 24 h) and worm-like micelles (after 46 hours) were obtained with the final concentrations of 8, 10.1, 13.4 and 16.5 mg/mL, respectively (concentration values were calculated gravimetrically from 1 mL nanoparticle solution). Radiolabeling of Nanoparticles via Reaction with Tritiated Ethanolamine and Inactivation of Remaining Epoxide Groups for Biodistribution Study. Tritiated ethanolamine in 100 µL ethanol was diluted with 900 µL methanol and reacted with purified nanoparticle solutions (2 mL) at RT overnight. The reaction mixtures were then purified by dialyzing against methanol (12-14K MWCO) to remove unreacted tritiated ethanolamine. Radiolabeled nanoparticle solutions (1 mL) were further reacted with 0.5 mL 0.5 M H2SO4 solution to inactivate unreacted epoxide groups on the nanoparticle surface. After this reaction nanoparticle solutions were dialyzed against water and sterile PBS, respectively (12-14K MWCO). Thereafter, the concentration of nanoparticles was adjusted to 2 mg/mL in sterile PBS for the biodistribution study and atomic disintegrations per minute (DPM) were measured before injection. Inactivation of Epoxide Groups for In Vitro and In Vivo Studies. Prior to conducting cytotoxicity assays, the epoxide groups on the surface of non-radiolabeled nanoparticles were

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inactivated and the concentrations of the nanoparticle solutions were adjusted to 2 mg/mL in sterile PBS as described above. Cytotoxicity of Nanoparticles against HT1080 cells. The HT1080 (human adenocarcinoma) cell line was purchased from ECACC (Salisbury, UK) and confirmed to be free from mycoplasma. HT1080 cells were propagated in RPMI media containing 10% FBS, 1% glutamax and 0.25 µM penicillin/streptomycin. Cells were passaged twice per week via trypsin digestion and were used between passages 5 and 10. To evaluate the impact of the nanoparticles on cell viability, HT1080 cells were seeded in each well of a 96 well microplate in 100 µL of RPMI containing supplements as described above at a density of 5,000 cells per well. After overnight seeding, the cell media was replaced with 100 µL fresh media containing (i) no nanoparticles, (ii) spherical micelles, (iii) worm-like nanoparticles or (iv) rod-like nanoparticles (0.01 to 1000 µg/mL) in triplicate wells and incubated for 48 h in a humidified incubator at 37 °C and 5 % CO2. Cytotoxicity was then evaluated via the MTT assay as described previously,24 and the absorbance read at 540 nm in a Fluorostar microplate reader (Bio-Strategy, Auckland, NZ). Biodistribution of Nanoparticles in Mice Bearing HT080 Tumour Xenografts (Ethics Approval #2015.18). For tumour induction, HT1080 cells were resuspended in Ca+2/Mg+2 free PBS to a final density of 6 × 107 cells/mL. A 50 µL suspension of HT1080 cells (3 × 106 cells in total) was then injected subcutaneously into the right flank of mice (n=17) using a 25G needle. Tumour growth was monitored with calipers every 2 days. When tumours reached approximately 100 mm3, mice were dosed with one of the radiolabeled nanoparticles (0.1 mg in 50 µL of sterile PBS) intravenously via a lateral tail vein (n = 4 mice per group). Mice were then euthanized 48 h later and whole blood collected via cardiac puncture. Major organs (liver, spleen, kidneys, heart,

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lungs, pancreas, and thigh muscle) and tumour were harvested. Tissue and whole blood samples were then analyzed via scintillation counting as described previously.25

Results and Discussion Nanoparticle Formation with Different Sizes and Morphologies by PISA. Functional polymeric nanoparticles were synthesized by PISA using PGMA-b-POEGMA-b-PS block copolymers in methanol. Glycidyl methacrylate was chosen to form the functional segment of the block copolymer as its epoxide groups facilitated post-synthesis functionalization with radiolabeled ethanolamine. POEGMA was incorporated as the second block as it is hydrophilic, biocompatible and has anti-immunogenic properties.25 Finally the extremely hydrophobic PST segment was selected for its high glass transition temperature (Tg)26,27 ensuring stability of the polymeric nanoparticles and potentially enabling the loading of hydrophobic drugs.28 Together, this provided an appropriate model nanoparticle composition for drug delivery with surfacefunctionality. First, the RAFT polymerization of GMA was conducted in toluene at 70 °C using azobisisobutryonitrile (AIBN) as initiator and 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPADB) as chain transfer agent. The homopolymerization of GMA was stopped at a conversion of about 30%, yielding a short PGMA block with a number average molecular weight (Mn) of 2500 g mol−1 and a polydispersity index (PDI) of 1.14. PGMA was then used as the macro chain transfer agent for the polymerization of OEGMA to give a PGMA-b-POEGMA block copolymer with Mn = 8800 g mol−1 and PDI = 1.09 (54% yield). The chemical structures and 1H nuclear magnetic resonance (NMR) spectra of PGMA and PGMA-b-POEGMA are shown in Figure 1.

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Figure 1. Chemical structures and 1H NMR spectra of PGMA and PGMA-b-POEGMA polymers. Dispersion polymerization of PGMA-b-POEGMA copolymer with styrene resulted in PGMA-b-POEGMA-b-PS copolymers (Figure 2-a) and simultaneously the self-assembly into nanoparticles due to the insolubility of PS polymer block in methanol. RAFT-mediated PISA proceeded in a controlled manner as was confirmed by continuous chain extension with styrene at increasing polymerization times. Gel permeation chromatography (GPC) and 1H NMR results showed that the molecular weight increased linearly and that relatively low PDI values (~10 nm) is known to be long, often with half-lives longer than 24 h due to their ability to evade renal and hepatobiliary clearance.29 Furthermore, nanoparticles with these long circulation times typically take advantage of the EPR effect, resulting in greater tumour accumulation when compared to uptake in control tissues (such as muscle and fat).3 However, the heterogeneous tumour vasculature, high intratumoural pressures and presence of a dense collagen matrix within the tumour mass can also hinder tumour accumulation and lead to nanoparticle localization primarily in the peritumoural region of the tumour. Even taking this into account, smaller nanoparticles still tend to show better tumour penetration.9,30,31 The biodistribution of the four nanoparticles were then examined in HT1080 tumour bearing nude mice 2 days after intravenous dosing (Figure 6). In general, most of the nanoparticles were cleared from the blood circulation after IV administration as evidenced by the presence of