PLGA/PEG particle production

Thermosensitive particles were fabricated from blends of 53kDa P_DLLGA (85:15 DLG 4CA) (Lakeshore Biomaterials, USA) and PEG 400 (Sigma Aldrich, UK) as previously described [26]. Briefly, a mixture of 93.5%:6.5% PLGA:PEG (w/v) was blended at 80-90°C on a hotplate. The melted PLGA and PEG were mixed together by hand using a PTFE-coated spatula and allowed to cool. Polymer blend sheets were then ground into particles in a bench-top mill (Krups Mill F203) and the particles were sieved to obtain the 100-200µm particle size fraction.

Matrix preparation

Composite matrices were prepared in PTFE moulds. For in vitro release experiments cylindrical scaffolds of 12mm length and 6mm diameter were produced. The PLGA/PEG particles were mixed manually with drug solution (trichostatin A, etoposide or methotrexate) or water (negative controls). A ratio of 1:0.6 of particles to solution was used. The drug loaded matrices contained 50µg of drug per matrix. The particle paste was then packed into the mould which was placed at 37°C for 2 hours to allow matrix formation (sintering). For in vitro biocompatibility and toxicity assays cylindrical scaffolds 1mm thick and 4mm diameter were produced. The PLGA/PEG particles were mixed manually with drug solution (trichostatin A, etoposide or methotrexate) at a ratio of 1:0.6 of particles to solution. The drug loaded matrices contained 50µg of drug per matrix. The particle paste was then packed into the mould which was placed at 37°C for 2 hours to allow matrix formation.

Experimental cell lines and culture

PFSK-1 (childhood central nervous system primitive neuroectodermal tumor) (CNS PNET)), DAOY (childhood medulloblastoma), U87 (adult glioblastoma multiforme) and C6 (rat glioma) cell lines were previously characterized by and purchased from ATCC. Monolayer cells were cultured in DMEM (Sigma, UK), supplemented with 10% fetal bovine serum (PAA Labs, UK), 5mM sodium pyruvate, 5mM L-Glutamine and maintained in a humidified incubator at 37°C and 5% CO₂. To assess in vitro biocompatibility and toxicity of PLGA/PEG matrices cells were seeded in triplicate. Matrices were placed in single wells of a 24-well plate and pre-treated with 50µl of culture media. A 30µl suspension containing 1x10⁵ cells were placed onto each matrix and plates incubated for 2 hours at 37°C to allow cells to adhere to the polymeric microparticles. Fresh culture media (1ml) was added to each well ensuring matrices were submerged, and incubated at 37°C until required for assaying.

Ex vivo application of PLGA/PEG matrices

Sheep heads were obtained from a local abattoir after permission was granted to use these animal parts (C Brumpton Butchers Ltd, Nottingham). Heads were fixed in a vice and bilateral skin and muscle flaps raised. Three burrholes were performed with a Hudson brace on each fronto-temporal region of the cranium and joined with a Gigli saw to raise craniotomies. The dura was incised and flapped back, and secured with vicryl stay sutures. Incisions were made through the pia and the brain parenchyma excised to give a cavity of dimensions 2.5x2.5x2.5cm. PLGA/PEG blended microparticles were mixed with PBS in a 1:0.6 ratio to form polymer-based paste and applied to the ovine pseudo-resection cavity. Cavities were either filled completely with PLGA/PEG or lined all around to an approximate depth of 2mm.

MRI and CT scanning of ex vivo brain

MR imaging was performed using a clinical 3 T Achieva MR scanner (Philips Medical, Best, The Netherlands) with the specimen placed inside the 8 channel receive-only head coil. Routine fat-suppressed 3D turbo spin echo T1- and T2-weighted whole brain imaging was performed with acquisition resolution of 1x1x1 mm, 192x192x160 matrix, echo train length of 133, echo time of 262 ms, repetition time of 2500 ms, and 464 s overall scan time. The CT scans were acquired using an adult head protocol on a clinical Mx8000 IDT 16 scanner (Philips Medical, Best, The Netherlands). 144 slices, 1.5 mm thick, were acquired at 120 kV with 512x512 matrix and 0.45 mm in-pixel pixel size.

Chemotherapeutic and experimental agents

TSA and ETOP were purchased from Sigma, UK and resuspended in dimethyl sulfoxide (DMSO) to give 5mM and 50mM working concentrations respectively. MTX was purchased from Sigma, UK and resuspended in phosphate buffered saline to give a 100mg/ml stock solution.

Radiotherapy dosing regime

A 12mm x 6mm sample of the biomaterial was placed within a cavity of the head region of an anthropomorphic phantom (Rando^TM phantom, The Phantom Laboratory, Salem, NY, USA). The head area was then CT scanned to give a set of contiguous 3 mm slices (Aquilion LB CT scanner, Toshiba, Crawley, UK). These CTs were transferred to a radiotherapy treatment planning system (Oncentra version 3.3 SP3, Nucletron, UK) using a DICOM protocol. Within the planning system the sample material was outlined and a region of interest 1 cm in all directions was grown to provide a target volume. An experienced radiotherapy planner then devised a treatment plan to irradiate the target volume as uniformly as possible to 60 Gy using a 6 MV beam. A standard isocentric 3 field arrangement was used, which gave a mean target dose of 60 Gy (range 59.3 Gy to 60.7 Gy). The plan was delivered as 30 daily 2 Gy fractions using a clinical linear accelerator (Elekta Precise, Elekta, Crawley, UK). The fractionation schedule was as routine clinical practice, i.e. one fraction per day Monday to Friday, no irradiations at the weekends.

Live/Dead viability assay

Brain tumor cells were seeded onto PLGA/PEG matrices as described previously and Live/Dead™ viability/cytotoxicity assay (Invitrogen, UK) conducted on days 1,2 and 3 post-seeding. 5µl of 2mM ethidium homodimer-1 and 2.5µl of 4mM calcein AM were added to 10ml DMEM and 1ml of the final solution was used to stain and completely submerge one cell-seeded PLGA/PEG matrix (one matrix per well of 24-well plate). Following incubation for one hour at 37°C, matrices were rinsed three times with DMEM to remove background staining and a further three times with PBS. Each matrix was carefully placed on a microscope slide and fluorescence signals (green = viable; red = dead) visualized using a Leica DMRB upright fluorescent microscope. Matrices were placed back into culture to ensure tracking of the cells present on the same matrix over 3 days with respect to viability observations and quantitation of fluorescent cells. Three matrices were used for each cell line and 100 cells were counted for each matrix to determine the proportion of live:dead cells.

Alamar Blue proliferation assay

Brain tumor cells were seeded onto PLGA/PEG matrices in 24-well plates as described above. On days 1, 2 and 3 post-seeding, matrices were washed three times with warm PBS and finally placed in 1ml PBS. 100µl Alamar Blue indicator dye (Invitrogen, UK) was added to each well ensuring matrices were submerged. Following incubation for 90 minutes at 37°C, 100µl aliquots were transferred to single wells of a 96-well black-bottom plate in triplicate and fluorescence emission measured at 585nm using a plate reader (Tecan, Switzerland). Three matrices were used for each cell line and fluorescent readings were measured for each matrix in triplicate. Matrices were placed back into a single well of a 24-well plate and fresh culture media added to ensure tracking of the same matrix over 3 days with respect to cell proliferation. Fluorescence intensity over a 3-day culture period was compared to counterpart monolayer cells. Data is presented as the average relative fluorescence units of three independent experiments with error bars indicating standard error of the mean values.

Scanning electron microscopy

Cell-seeded PLGA/PEG matrices were washed three times in fresh PBS and fixed in 3% glutaraldehyde for 12 hours. Fixed samples were then washed three times in PBS and dehydrated for a further 12 hours by exposure to air at room temperature. Fixed and dehydrated cell-seeded matrices were mounted on aluminium stubs and sputter-coated with gold at an argon current rate of 30mA for 3 minutes. Cell attachment and morphology on the matrices was visualized using a scanning electron microscope (SEM) (JEOL JSM-6060LV) at 10 kV.

In vitro drug release from PLGA/PEG matrices

50µg or 100µg drug-loaded matrices were placed in 4ml fresh PBS buffer pH 7.4 and incubated at 37°C. At various time intervals, the entire volume of PBS was replaced with 4ml fresh release buffer and 100µl of buffer containing released drug was sampled in triplicate at a relevant UV wavelength (TSA, 342nm, ETOP, 295nm and MTX, 324nm) and concentration of drug measured using a standard curve determined at the same time. Three matrices were used for each drug and blank PBS loaded PLGA/PEG matrices were used as a control for background absorbance values. Assays were terminated when 80% or more of the drug had been released and when no drug release was detected for two days or more. Data is presented as cumulative drug release as a function of time.

Cytotoxicity of released agents

50µg drug-loaded matrices were placed in 2ml of culture media in single wells of a 24-well plate. After either 24 hours or 14 days, media was removed and brain tumor cells were seeded onto matrices using a 30µl suspension containing 1x10⁵ cells. Cells were allowed to adhere onto matrices for two hours prior to adding 2ml fresh culture media to the wells. Alamar Blue proliferation assay (Invitrogen, UK) was conducted 72 hours post-cell seeding and percentage viability of cells relative to untreated cells was measured. Data is presented as the average relative fluorescence units of three independent experiments with error bars indicating standard error of the mean values.

In vivo etoposide release from PLGA/PEG matrices

This study was approved by the University of Nottingham local Ethical Review Committee and granted by the UK Home Office (License No. PPL 40/3559), after consideration of the justification of animal research and good animal welfare. Six 4-6 week old male MF-1 nude mice (3 mice per arm) were maintained under standard conditions as detailed in the UK Home Office Animals (Scientific Procedures) Act 1986 and studies conducted and reported in compliance with the 2010 NC3R ARRIVE guidelines. Animals U87 GBM cells tagged with a bioluminescent marker (DLuX) were injected subcutaneously into the left flank and the tumour grown for 15 days whilst monitoring using the IVIS Spectrum bioluminescent imaging system (PerkinElmer, UK). Mice with satisfactory tumour take and growth rates underwent partial tumour resection. The previous flank incision was re-opened and a biopsy punch/fine suction tip used to resect tumour back to the tumour/tissue interface, thus mimicking the surgical technique utilized in human patients undergoing comparable surgery for GBM. Etoposide-loaded PLGA/PEG matrices (experimental arm) or blank PLGA/PEG matrices (control arm) were moulded around the resection cavity. Animals were weighed daily by an experienced technician, any adverse effects noted, and sacrificed using cervical dislocation once their clinical condition deteriorated, in order to ameliorate suffering. Mice were sacrificed 3 days post-implantation and tumour tissue sectioned prior to staining with hematoxylin and eosin or glial fibrillary acidic protein (GFAP) (anti-GFAP (Abcam, ab726; 1:1000).

Statistics

Pearsons correlation coefficient was used to determine the concordance of drug release rates between PLGA/PEG formulations with two different drug concentrations. Unpaired student t-test was used to determine whether proliferation rates of cells cultured on matrices differed significantly from corresponding cells cultured as monolayers with a p-value < 0.05 deemed statistically significant. Statistical analyses were conducted using SPSS software version 6.

PLGA/PEG microparticle-based matrix can be molded around a resection cavity wall

The PLGA/PEG paste was easily applied to and readily molded around, an irregular-shaped pseudo-resection cavity wall ex vivo (Figure 1a and Movie S1). The polymer formulation fully maintained its shape after 15 minutes sintering at 37°C, with close apposition to the cavity wall (Figure 1b and Movie S2).

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Figure 1. Application of PLGA/PEG microparticle-based matrices to a surgical resection cavity ex vivo.

(A) When mixed with a saline carrier solution, PLGA/PEG microparticles form a paste that can be molded around an ovine pseudo-resection cavity ex vivo. (B) Within 15 minutes at 37°C, particles solidify to form a matrix that retains close apposition to the resection cavity wall. (C-D) Computerized tomography scans of ex vivo ovine brain demonstrating clearly distinguishable pseudo-resection cavity filled with PLGA/PEG (black arrows) and second cavity lined with PLGA/PEG (black arrowheads). (E-F) T2 and T1 weighted magnetic resonance imaging scan of ex vivo ovine brain with clearly distinguishable pseudo-resection cavity filled with PLGA/PEG (white arrows).

https://doi.org/10.1371/journal.pone.0077435.g001

PLGA/PEG microparticle-based matrix can be distinguished by MRI and CT scanning

To establish whether PLGA/PEG interferes with MRI- and CT-based brain scans by causing image artifacts that obscure visualization of brain parenchyma and thereby potentially hampering identification of a recurrent tumor, an ovine head containing one polymer-filled and one polymer-lined pseudo-resection cavity was scanned ex vivo under standard clinical procedure. Cavities filled and lined with PLGA/PEG are distinguishable from the surrounding brain parenchyma using standard CT scanning (Figure 1 c-d). Similarly, cavities filled with PLGA/PEG are distinguishable from surrounding brain parenchyma using T2- and T1- weighted MRI scans (Figure 1 e-f respectively). Edges of the cavity are clearly defined with no additional image artifacts observed from the PLGA/PEG, indicating insignificant spatial distortion and good contrast with brain parenchyma on T2 and T1 MR (Figure 1c). Therefore application of PLGA/PEG matrices does not interfere with clinical scanning modalities used as standard procedures for the detection of brain tumors.

Radiotherapy does not alter PLGA/PEG matrix microstructure

In anticipation of radiotherapy treatment for patients with PLGA/PEG intra-cavity implants, it is important to determine effects of radiation on the biomaterial and subsequent drug release. Moreover the effect of radiation on the microstructure of the PLGA/PEG matrix is unknown. PLGA/PEG matrices, placed in the brain area of an anthropomorphic phantom were subjected to a standard isocentric high dose radiotherapy regime of 60 Gy (2 Gy fractions daily for 30 days) (Figures 2a-b). Irradiated matrices show no visible difference in terms of microparticle size, morphology and distribution throughout the matrices when compared to control non-irradiated matrices stored under similar conditions. Microparticles appear structurally intact with visible pores between microparticles in both cases (Figure 2c-d).

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Figure 2. Effect of clinically-relevant radiotherapy regime on PLGA/PEG microparticle-based matrices.

(A) PLGA/PEG matrices (12mm x 6mm) were placed within cavities in the brain area of an anthropomorphic phantom. (B) A head computerized tomography was obtained (3mm slices) using a standard radiotherapy protocol and the computerized tomography transferred to a radiotherapy planning system to give a uniform standard isocentric three field 60 Gy dose (30 daily 2 Gy fractions) of radiation to the biomaterial and surrounding area. (C) Scanning electron microscopy of control non-irradiated matrices kept at room temperature for 30 days, showing distinct and structurally intact microparticles with visible pores between particles. (D) Scanning electron microscopy of irradiated matrices showing no obvious difference in matrix microstructure (scale bar 100µm).

https://doi.org/10.1371/journal.pone.0077435.g002

PLGA/PEG matrices are non-toxic to normal and cancerous cells

To address whether PLGA/PEG matrices exert cytotoxicity, PFSK-1, DAOY, C6 and U87 brain tumor cell lines were seeded directly onto matrices and cultured for three days. All seeded tumor cells show distinct 3D morphology both on and between polymer microparticles, with visible extracellular matrix laid on the particle surface (Figures 3a-e). The Live/Dead assay confirmed that ~85% of tumor cells seeded onto matrices remain viable after three days (Figure 3f-j). When regarding a three day culture period, there is no significant difference in metabolic activity of tumor cells grown on matrices compared to the same cell lines cultured as 2D monolayers (PFSK-1 p<0.8, DAOY p<0.4, C6 p<0.2 and U87 p<0.6), thus indirectly demonstrating a comparable proliferation rate over a three day culture period (Figure 3k-n). To verify that the observed lack of cellular toxicity due to PLGA/PEG matrices is not observed exclusively in cancerous cells, normal brain endothelial cells (HBMEC) were seeded onto matrices. Over a three day culture period, endothelial cells were also observed to proliferate with 100% viability (Figure 3o-p). Thus these results demonstrate that PLGA/PEG microparticle-based matrices are non-toxic to neoplastic and normal brain cells.

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Figure 3. Assessment of PLGA/PEG microparticle-based matrix toxicity to normal and cancerous cells.

To address toxicity of polymer matrices, brain cancer cells were grown on PLGA/PEG matrices. (A) Scanning electron microscopy images of blank PLGA/PEG matrices with distinct microparticles (m) and pores (p). (B-E) PFSK-1 (CNS PNET), DAOY (medulloblastoma), C6 (rat glioma) and U87 (glioblastoma multiforme) brain tumor cells cultured on PLGA/PEG matrices for 3 days. Cells exhibit 3D morphology both across microparticle faces and between microparticles, with extra cellular matrix visible. Representative tumor cells are denoted with an asterisk. Magnifications x1000. (F) Live/Dead assay of PFSK-1 cells demonstrates that ~85% of cells are viable. (G) Image of PFSK-1 cells without adjustment of background fluorescence, with visible microparticles (background fluorescence from particles is reddish-brown). (H-J) Live/Dead assay of DAOY, C6 and U87 cells demonstrates that 85%-100% of cells are viable. All images are shown on culture day 3 post-seeding onto matrices. Green = viable; Red = non-viable. Scale bar = 100µm. (K-N) Alamar Blue proliferation assay for PFSK-1, DAOY, C6 and U87 respectively, over a 3-day culture period post-seeding onto PLGA/PEG matrices and compared to 2D monolayer cultures of the same cell lines. The proportion of proliferating cells was inferred indirectly from a measurement of metabolic activity. Error bars represent the standard error of the mean from three independent matrices and over three independent experiments. Unpaired t-test analyses reveals no significant differences between the two culture systems (O-P) Viability and proliferation of brain endothelial cells (HBMEC) over a 3-day culture period post-seeding onto PLGA/PEG matrices Green = viable; Red = non-viable. Scale bar = 100µm.

https://doi.org/10.1371/journal.pone.0077435.g003

PLGA/PEG matrices permit sustained release of chemotherapeutic agents in vitro

To evaluate the capability of PLGA/PEG matrices to release standard of care and experimental chemotherapeutic agents, TSA, ETOP and MTX were loaded onto polymer matrices and drug release profiles measured in vitro. Release of TSA, ETOP and MTX followed a similar profile, exhibiting an initial burst phase followed by slower, sustained release profiles (Figure 4a-c). An initial burst release of 44% TSA was observed, caused by drug on the surface of the scaffolds being released into the PBS immediately. This was followed by an average daily release of 1.2% until day 18 (Figure 4, top). At this point the PLGA/PEG matrices stopped releasing drug, with a total of 94% TSA released. ETOP-loaded matrices exhibited an initial burst of 29% total drug loaded followed by an average daily release of 1.3% (Figure 4, middle). Drug release from the matrices stopped at day 26, with a total of 77% ETOP released. A burst release of 49% MTX was observed followed by an average of 0.7% release per day (Figure 4, bottom). The matrices stopped releasing MTX by day 28, at which point 78% total loaded MTX was released.

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Figure 4. In vitro cumulative release profiles of agents loaded onto PLGA/PEG matrices.

All drug release behaviors from the drug-loaded polymer matrices were investigated under simulated physiological conditions (PBS, pH 7.4) at 37°C. (Top) TSA exhibits an initial burst of 44% of total drug loaded with a total of 94% of drug released by day 18 and no drug detected thereafter. (Middle) ETOP exhibits an initial burst of 29% of total drug loaded with 77% of drug released by day 26. (Bottom) MTX exhibits an initial burst of 49% of total drug loaded with 78% of drug released by day 28. No drug release is detected thereafter. Three independent matrices were used for each experimental set-up with 50µg of drug loaded per matrix. Day 0 initial burst readings were taken 4h after drug-loaded matrices were placed in PBS. UV absorbance wavelengths were as follows: TSA, 342nm; ETOP, 295nm; MTX, 324nm. Error bars represent standard error of the mean from three independent matrices.

https://doi.org/10.1371/journal.pone.0077435.g004

To determine whether the amount of drug loaded influences the release profile, TSA was loaded onto PLGA/PEG matrices at 50µg and 100µg and drug release profiles directly compared. The initial burst on Day 0 is greater in matrices loaded with 100µg TSA (50%) compared to matrices loaded with 50µg TSA (28%); the release profiles thereafter however, are comparable with a high degree of correlation over an 8-day period (Pearsons correlation coefficient, 0.94). Therefore although the initial burst release of TSA is directly proportional to the amount of drug loaded, the slower sustained zero-order release is independent of the amount of drug loaded (Figure S1).

Released agents from PLGA/PEG matrices retain cytotoxic capabilities in vitro

To determine whether drugs released from PLGA/PEG microparticle-based matrices retain cytotoxic function, brain tumor cells were assessed for viability after seeding onto drug-loaded matrices 24 hours after drugs were incorporated and exposing to released drugs for 72 hours. PFSK-1 is sensitive to all three agents with approximately 20-70% viable cells remaining with drug potency in the order: TSA>ETOP>MTX (Figure 5a). DAOY is sensitive to all three agents with approximately 30-55% viable cells remaining with drug potency in the order: TSA/ETOP>MTX (Figure 5b). C6 is sensitive to all three agents with approximately 20-70% viable cells remaining with drug potency in the order: TSA>ETOP>MTX (Figure 5c). U87 is sensitive to TSA and ETOP with approximately 25-75% viable cells remaining with drug potency in the order: TSA>ETOP. U87 is insensitive to MTX under the experimental conditions described here (Figure 5d). Additionally, TSA cytotoxicity on brain tumor cells was assessed following a 2 week release period in vitro, to demonstrate that the released drug retains its activity and is capable of exerting cytotoxic effects after a prolonged period of time within the PLGA/PEG matrix. TSA-loaded PLGA/PEG matrices were placed in PBS for 14 days to allow drug release. On day 14 brain tumor cells were seeded onto matrices and assessed for cell viability after three days of culture. The results indicate that cumulative release of TSA on days 15-17 post drug-loading (~4% of total drug loaded), retains cytotoxic function as cell proliferation is impaired in all four cell lines investigated at IC₅₀ concentrations (Figure S2) These results indicate that chemical and physical interaction of TSA, ETOP and MTX with the PLGA/PEG polymer does not impair molecular structure integrity of the drug, as these agents retain cytotoxic function in vitro.

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Figure 5. Cytotoxicity of agents released from PLGA/PEG matrices.

Brain tumor cells were assessed for viability after seeding onto drug-loaded matrices one day after drugs were loaded, to determine cytotoxic capabilities of released agents. (A-C) PFSK-1, DAOY and C6 cells are sensitive to all three agents with approximately 20-70% viable cells remaining (D) U87 is sensitive to TSA and ETOP with approximately 25-75% viable cells remaining but is insensitive to MTX. Percentage cell viability was calculated relative to PBS-loaded control matrices for each cell line.

https://doi.org/10.1371/journal.pone.0077435.g005

PLGA/PEG matrices release active etoposide in vivo

Etoposide-loaded polymer was moulded around a flank GBM resection cavity after performing partial tumour resection. Mice were sacrificed 3 days after implantation of the drug-loaded polymer to demonstrate proof-of-concept for in vivo drug release and gauge short-term drug diffusion distance. Haematoxylin and eosin staining shows a clear kill-zone visible beyond the resection boundary consisting of necrotic tumour cells, demonstrating in vivo release of cytotoxic levels of etoposide from the polymer matrix. Etoposide drug diffusion after release from the polymer matrices was approximately 1mm after 3 days, based indirectly upon the region of the kill-zone (Figure 6).

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Figure 6. Efficacious release of etoposide-loaded PLGA/PEG in a mouse flank xenograft.

Mice were sacrificed 3 days after implantation of either blank (A-B) or etoposide-loaded (C-D) polymer. Haematoxylin and eosin staining shows a clear kill-zone is visible beyond the resection boundary (R) consisting of necrotic tumour cells (N), demonstrating in vivo release of cytotoxic levels of etoposide from the polymer matrix. A similar result is observed with GFAP staining of tumour xenograft cells. T, viable tumour cells; P, residual polymer implant. Scale bar = 400µm.

https://doi.org/10.1371/journal.pone.0077435.g006