Photoactivated Polymersome Nanomotors: Traversing Biological Barriers

Abstract Synthetic nanomotors are appealing delivery vehicles for the dynamic transport of functional cargo. Their translation toward biological applications is limited owing to the use of non‐degradable components. Furthermore, size has been an impediment owing to the importance of achieving nanoscale (ca. 100 nm) dimensions, as opposed to microscale examples that are prevalent. Herein, we present a hybrid nanomotor that can be activated by near‐infrared (NIR)‐irradiation for the triggered delivery of internal cargo and facilitated transport of external agents to the cell. Utilizing biodegradable poly(ethylene glycol)‐b‐poly(d,l‐lactide) (PEG‐PDLLA) block copolymers, with the two blocks connected via a pH sensitive imine bond, we generate nanoscopic polymersomes that are then modified with a hemispherical gold nanocoat. This Janus morphology allows such hybrid polymersomes to undergoing photothermal motility in response to thermal gradients generated by plasmonic absorbance of NIR irradiation, with velocities ranging up to 6.2±1.10 μm s−1. These polymersome nanomotors (PNMs) are capable of traversing cellular membranes allowing intracellular delivery of molecular and macromolecular cargo.


Nuclear Magnetic Resonance Spectroscopy (NMR):
Routine proton nuclear magnetic resonance ( 1 H-NMR) measurements were conducted on a Bruker Avance 400 MHz Ultrashield TM spectrometer equipped with a Bruker SampleCase auto-sampler, using CDCl3 as the solvent and TMS as the internal standard.

Gel Permeation Chromatography (GPC):
The molecular weights and dispersity index of the copolymers were characterized by using a Prominence-I GPC system (Shimadzu) with a PL gel 5 μm mixed D (Polymer Laboratories) and equipped with a RID-20A differential refractive index detector. Polystyrene standards were used for calibration. THF was used as an eluent, with a flow rate of 1 mL per minute.

Dynamic Light Scattering (DLS):
The hydrodynamic size of the polymersomes was measured using a Malvern Instruments Zetasizer (model Nano ZSP) equipped with a 633 nm He-Ne laser and an avalanche photodiode detector. Zetasizer software was used to process and analyze the data.

pH meter:
FiveEasy Plus TM FEP20 pH Meter (METTLER TOLEDO) was used to monitor the pH. 2.8 UV-vis spectroscopy: Cumulative Dox release at 37 o C in PBS buffer was characterized using UV-vis spectroscopy (V-650, JASCO). 2.9 NanoSight: NanoSight LM10HS instrument (Malvern Instruments) equipped with an Electron Multiplication Charge Coupled Device camera and external laser source (660 nm, BeamQ Lasers) was utilized to track the motion of PNMs, polymersomes, and gold shells.

Synthesis of PEG44-CHO:
The synthesis of aldehyde modified PEG was performed according to a previously reported literature procedure. [1] Firstly, PEG (4 g, 2 mmol), 4-carboxybenzaldehyde (1.5 g, 10 mmol), EDC· HCl (3.85 g, 20 mmol) and DMAP (0.06 g, 0.5 mmol) were dissolved in 100 mL dichloromethane (DCM) and stirred for 48 h at 25 °C. After the reaction was completed, the solution was concentrated by rotary evaporation, and then the mixture was washed with saturated NaCl solution (3 times). Subsequently, the organic layer was collected and dried with anhydrous magnesium sulfate. After filtration, the filtrate was concentrated and precipitated twice in excess cold diethyl ether. The final product was dried under vacuum at room temperature overnight and obtained as a white solid with a yield of 56%. 1  Synthesis of PEG44-benzoic-imine-OH: The synthesis of pH responsive PEG was performed according to a previously reported literature procedure. [2] 6-aminohexan-1-ol (1.17 g, 10 mmol) was added to a solution of PEG44-CHO (2 g, 1 mmol)) in a mixture of tetrahydrofuran and DMSO (8/1, 9 ml), and the mixture was stirred at 40 °C for 12 h. The solvent was evaporated under reduced pressure. The product was precipitated in anhydrous cold ethyl ether twice to remove impurities and dried under vacuum; it was obtained as a white solid with a 89% yield. 1  Synthesis of PEG44-benzoic-imine-PDLLA115 (PEG44-PDLLA115): The synthesis of pH responsive PEG-PDLLA was performed according to a previously reported literature procedure. [3] PEG44-benzoic-imine-OH macro-initiator (0.1 mmol) was weighed into a round bottom flask along with D,L-Lactide (12 mmol). Then, dry toluene was added to the flask and the solvent evaporated in order to dry the contents before polymerization. The dried reagents were then re-dissolved in dry DCM (25 mL, [monomer] = 0.5M) and DBU was added (0.5 equiv. with respect to [initiator]; 0.1 mmol = 8 μL) under argon. The reaction was stored at room temperature (RT) for around 3 hours, until there was no evidence of the monomer from the 1 H-NMR spectra. After completion was confirmed by 1 H-NMR, the reaction mixture was quenched with excess benzoic acid and then precipitated into ice cold diethyl ether (100 mL) twice and the remaining wax was partially dried under nitrogen before dissolving in dioxane and lyophilisation to yield a white powder with a yield of 73%. 1

Preparation and characterization of polymersomes and hemispherical gold-coated polymersome nanomotors (PNMs)
In a 15 mL vial PEG44-PDLLA115 (20 mg) was dissolved in 2 mL of a mixture of THF/dioxane (1:4, v/v). Thereafter, a magnetic stirring bar was added, and the vial was sealed with a rubber septum. Subsequently, 2 mL of ultrapure MilliQ water was added via a syringe pump (Chemyx Inc. Fusion 100 Syringe Pump) with a flow rate 1 mL h -1 . Afterwards, the obtained cloudy solution was transferred into a prehydrated dialysis bag (12-14 kDa, 2 mL cm -1 ) and dialyzed against a pre-cooled NaCl solution (50 mM) at 5 o C for at least 24 h with a dialysis solution change after 1 h. For preparation of Janus polymersomes, the sputter coating technique was used following a previously published protocol. [4] Briefly, a droplet of polymersome solution was drop-cast onto a clean silicon wafer, followed by sputter coating using a turbo sputter coater (Quorum Technologies, K575X). Thereafter, Janus polymersomes were redispersed into Milli-Q water through ultrasound treatment. The morphology of polymersomes, before and after coating, was characterized using SEM and DLS. For Dox loading, 1 mg Dox was dissolved together with the block copolymer (20 mg). Similarly, FDG loading was performed by dissolving it (1mg) together with the block copolymer.

pH-sensitive behaviors
PBS with two pH values (pH 6.5 and pH 7.4) was prepared by tuning with 0.1 M HCl. Size changes of polymersomes in the presence of PBS buffer were monitored by DLS for 10 hours. Morphological changes of polymersomes as a function of pH value was observed by SEM.

Doxorubicin (Dox) release as a function of pH value
The release profile of Dox towards PBS buffer (pH 6.5 and pH 7.4) at 37 o C was calculated by measuring the absorbance intensity of released Dox in buffer at each time point using UV-vis spectroscopy.

Near-infrared (NIR)-activated motility of PNMs
The autonomous motion of PNMs was observed and recorded by TP-CLSM (Zeiss LSM510 META NLO) equipped with a ×63 oil immersion microscope objective. PNMs were detected by the fluorescent signal of the encapsulated Dox. Movement trajectories were tracked and analyzed by using ImageJ and Origin softwares. Based on the extracted trajectories, the velocity of NIR propelled PNMs (V) was determined by measuring both the travelled distance (D) and duration time (t)following the formula: V = D/t. Corresponding mean square displacements (MSD) were then calculated following the reported equation: MSD= (x(△t)-x(0)) 2 + (y(△t)y(0)) 2 . [5] Nanoparticle tracking analysis (NTA) was also used to analyze the motion behavior of PNMs, polymersomes, and gold shells by using NanoSight. Samples were suspended in Milli-Q water to yield an approximate concentration of 10 7 and 10 8 particles per mL. 1 mL of samples was loaded in the NTA chamber via a syringe. Then, the motion of PNMs, polymersomes, and gold shells was recorded for 30 s. A 660 nm DPSS Red Diode Laser Max Output Power was utilized as external laser source to propel the particles. Two laser intensities were used during the whole experiment, namely 0 W (laser off) and 1W (laser on). The concentration of PNMs was 5.19 × 10 7 particles mL -1 (0 W) and 1.41 × 10 8 particles mL -1 (1 W), respectively. For gold nanoshells, 2.89 × 10 8 particles mL -1 (0 W) and 5.11 × 10 8 particles mL -1 (1 W). For polymersomes, the concentration was 3.31 × 10 8 particles mL -1 (0 W) and 3.89 × 10 8 particles mL -1 (1 W). The NTA 2.2 software allows the extraction and analysis of the trajectories of single particles. For each group, 30 nanoparticles were tracked for 30 seconds and MSDs and velocity were calculated according to the reported method. [6]

Cell culture
Human cervical cancer cells (HeLa) and mice embryonic fibroblast cells (NIR/3T3) were cultured in DMEM cell culture medium supplemented with 10% FBS and 1% penicillin-streptomycin in the cell incubator (ThermoFisher Scientific) at 37 o C with an atmosphere of 5% CO2 and 70% humidity.

PNMs-intracellular delivery of encapsulated doxorubicin (Dox)
HeLa cells were seeded in μ-Slide 8 wells (ibidi) and incubated in DMEM cell culture medium. The cell membrane was stained with Wheat Germ Agglutinin, Alexa Fluor TM 488 conjugate to show the boundary of the cells. PNMs / polymersomes and PI were added before TP-CLSM characterization (Zeiss LSM510 META NLO).

Intracellular localization of PNMs
Pre-seeded HeLa cells in μ-Slide 8 wells were stained with Hoechst 33342 and LysoTracker TM Green DND-26 for the observation of the cell nucleus and lysosomes, respectively. After co-culture with PNMs (50 μL well, 2 mg mL -1 ) for 4 h, HeLa cells were characterized by TP-CLSM (Leica TCS SP5X). The cells were washed thoroughly with PBS buffer for three times to remove free PNMs.

3D cellular spheroids penetration
3D spheroids better resemble the complex in vivo environment of cells, as compared to traditional 2D approaches, more realistically recapitulating the tumor microenvironment. As valuable tool for nanomedicine, 3D spheroid models could be used for (a) a more accurate drug screening, (b) a better understanding of molecular and cellular mechanisms, (c) the evaluation of potential therapeutic agents before further pre-clinical studies, (d) cell matrix interactions. [7] 3D HeLa cellular spheroids were prepared by seeding HeLa cells in agarose-coated 96-well plates, according to previously published protocols. [8] Briefly, 0.15 g agarose was added to 10 mL of low glucose DMEM (1.5% wt/vol) in an appropriate beaker, sealed with a lid and autoclaved at 120 o C for 20 min. Thereafter, the hot solution (~80 o C) was added to a 96-well plate (flat bottomed, 50 μL per well) under sterile conditions. The HeLa cell suspension in high glucose DMEM medium was seeded and cultured for the formation of 3D HeLa cellular spheroids. Before TP-CLSM (Zeiss LSM510 META NLO) measurement, 50 μL PNMs / polymersomes (2 mg mL -1 ) were introduced to the 3D spheroids and co-cultured for 2 h, followed by careful washing with pre-warmed PBS buffer to remove all the free polymersomes (without Au coat) and PNMs.

Therapeutic evaluation towards 3D cellular spheroids
The therapeutic effect of PNMs was evaluated using 3D HeLa cellular spheroids. 3D HeLa spheroids were prepared by seeding HeLa cells in agarose pre-coated 96-wells plates. When the diameter of HeLa spheroids reached ca. 500 μm, the spheroids were moved to ibidi-8 wells and divided into two groups, including PNMs with/without NIR laser irradiation, and spheroids with/without NIR laser irradiation. Before characterization by TP-CLSM, 50 μL PNMs (2 mg mL -1 ) were added to the 3D HeLa spheroids and cocultured for 2 h. Calcein-AM and PI were then used to co-stain the spheroids for assessing the cell viability after photothermal treatment by TP-CLSM.

PNM-mediated intracellular delivery of fluorescein isothiocyanate conjugate albumin from bovine serum (FITC-BSA)
To evaluate the PNM-mediated active intracellular delivery of macromolecules, pre-cultured cells (in μ-slide 8 wells) were randomly divided into five groups, which included PNMs with/without NIR irradiation, polymersomes (without Au coat) with/without NIR irradiation, and cells only. For both PNM and polymersome groups, 50 μL of a 2 mg mL -1 particle dispersion was added separately to each well before NIR irradiation. 50 μL FITC-BSA (10 μg μL -1 ) was then added to all the samples. TP-CLSM (Zeiss LSM510 META NLO) was used to observe and record the active intracellular transportation of FITC-BSA.