Inducing a Transient Increase in BloodâBrain Barrier Permeability

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Inducing a Transient Increase in Blood Brain Barrier Permeability for Improved Liposomal Drug Therapy of Glioblastoma Multiforme David J Lundy, Keng-Jung Lee, I-Chia Peng, Chia-Hsin Hsu, JenHao Lin, Kun-Hung Chen, Yu-Wen Tien, and Patrick C.H. Hsieh ACS Nano, Just Accepted Manuscript • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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Inducing a Transient Increase in Blood Brain Barrier Permeability for Improved Liposomal Drug Therapy of Glioblastoma Multiforme David J. Lundy†,‡, Keng-Jung Lee†, I-Chia Peng†, Chia-Hsin Hsu†, Jen-Hao Lin†, Kun-Hung Chen†, Yu-Wen Tien§ and Patrick C.H. Hsieh†,§,∥,⊥,*

†Institute

of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.

‡Graduate

Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University,

Taipei 110, Taiwan. §Department ∥

of Surgery, National Taiwan University and Hospital, Taipei 100, Taiwan.

Institute of Medical Genomics and Proteomics, National Taiwan University, Taipei 100,

Taiwan. ⊥Institute

of Clinical Medicine, National Taiwan University, Taipei 100, Taiwan.

Word count (excluding references): 10,396 Display: 6 figures Supporting Information: 2 methods, 3 tables, 13 figures

Correspondence: Patrick C.H. Hsieh, M.D., Ph.D. Institute of Biomedical Sciences, Academia Sinica 128 Academia Road, Sec. 2, Nankang, Taipei 115, Taiwan Phone: 886-2-27899074 E-mail: [email protected]

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ABSTRACT The blood brain barrier (BBB) selectively controls the passage of endogenous and exogenous molecules between systemic circulation and the brain parenchyma. Nano-carrier based drugs such as liposomes and nanoparticles are an attractive prospect for cancer therapy since they can carry a drug payload and be modified to improve targeting and retention at the desired site. However, the BBB prevents most therapeutic drugs from entering the brain, including physically restricting the passage of liposomes and nanoparticles. In this paper, we show that a low dose of systemically injected recombinant human vascular endothelial growth factor induces a short period of increased BBB permeability. We have shown increased delivery of a range of nanomedicines to the brain including contrast agents for imaging, varying sizes of nanoparticles, small molecule chemotherapeutics, tracer dyes, and liposomal chemotherapeutics. However, this effect was not uniform across all brain regions, and permeability varied depending on the drug or molecule measured. We have found that this window of BBB permeability effect is transient, with normal BBB integrity restored within four hours. This strategy, combined with liposomal doxorubicin, was able to significantly extend survival in a mouse model of human glioblastoma. We have found no evidence of systemic toxicity, and the technique was replicated in pigs, demonstrating that this technique could be scaled up and potentially be translated to the clinic, thus allowing the use of nanocarrier-based therapies for brain disorders. KEYWORDS glioblastoma, doxorubicin, nanomedicine, drug delivery, xenograft model

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Glioblastoma Multiforme (GBM) is an aggressive primary cancer of the brain with a life expectancy of less than two years following diagnosis.1,2 Upon diagnosis, a combination of surgical resection, intensive radiotherapy and chemotherapy is attempted, achieving an overall median survival of 15 months.3–5 Unfortunately, 45-70 % of tumours develop resistance to Temozolomide (TMZ), the first line chemotherapeutic for GBM, and many other chemotherapeutic drugs have been trialled, but ultimately failed to demonstrate significant improvements in survival.6–8 The blood-brain barrier (BBB) is a highly selective two-way barrier system which separates systemic circulation from the brain parenchyma. The BBB preserves homeostasis of the brain by maintaining ion and neurotransmitter compartmentalisation, and controlling the transport of peptides, metabolites, cells and cytokines. Much of the physical barrier property results from tight junctions between the endothelial cells of the brain vasculature, but it is known that cells (astrocytes, microglia, pericytes, neurones) and an enormous array of selective transporters and efflux pumps all contribute towards a biochemical barrier.9 As a result, the BBB prevents the majority of therapeutic drugs from passing into the brain following intravenous or oral administration.10 In particular, larger substances such as nanoparticles or liposomes are unable to enter the brain parenchyma. Although solid tumours often have defective, “leaky” vasculature, allowing enhanced permeability and retention of drugs (so-called EPR effect), many types of glioma maintain a blood brain tumour barrier (BBTB) comparable to the normal BBB, rendering them resistant to chemotherapy.11,12 Although GBM tumours often show sporadic increased permeability, thus allowing their demarcation by contrast-enhanced MRI, drugs may still need to cross intact, healthy areas of BBB in order to access the entirety of a tumour. Intranasal, intracerebroventricular and intrathecal delivery can bypass the BBB to some extent, but not all therapeutics can be safely or adequately delivered by these routes. In order to bypass the BBB entirely, direct injection of free drugs, or drug-infused hydrogels, polymers, wafers, catheters etc. have been used to successfully deliver substances directly into the brain.13–19 However, these procedures are invasive and carry risks such as infection, haemorrhage, or damage to healthy brain tissue.10,19 Low molecular weight, highly lipid soluble molecules, such as TMZ, are more

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likely to penetrate the BBTB, as are those packaged into more favourable carriers such as stealth liposomes.20–22 It is well established that the physicochemical properties of nano-carriers affect their uptake and distribution in the body, including their ability to cross the BBB. A size of between 50 and 200 nm is generally accepted as optimal, as is a neutral or slightly negative zeta potential. Indeed, studies have shown that a high zeta potential is toxic to the BBB.23 In addition, surface modification with PEG has also been shown to improve passage through brain tissue, in addition to pharmacokinetic benefits such as extending half-life, reducing opsonisation and reducing immune sequestration.24,25 A recent study demonstrated greatly improved survival in a mouse GBM model using a TMZ-carrying functionalised nanoparticle, clearly demonstrating the potential of nanomedicine-based therapy for GBM.26 BBTB disruption has been attempted in the clinic using intraarterial infusion of hyperosmotic solutions, but this is unpredictable and has multiple side effects.27,28 Focused ultrasound combined with intravenous microbubble contrast agents have proven capable of weakening the BBB in a selectively targeted area and have already been used in humans.29–34 Although disrupting or bypassing the BBB/BBTB is desirable for therapeutic purposes, it is also potentially pathogenic, leading to dysregulation of ion, protein and neurotransmitter concentrations, seizures, intracranial oedema, infection, or neuroinflammation.35,36 Therefore, the ability to induce a transient window of permeability, with restoration of normal function, is highly desirable. Vascular endothelial growth factor (VEGF) is a soluble homodimeric protein responsible for the normal formation of new blood vessels, as well as promoting cell growth and survival. Five forms of VEGF are found in humans, with VEGF165A being the predominant form found in normal cells and tissues.37 VEGF acts through binding to VEGFR-1 and VEGFR-2 receptors present on endothelial cells, and has been long-known to affect vascular permeability.38–40 Given its role in angiogenesis, VEGF has undergone human clinical trials for use in ischaemic diseases, where it was found to be well tolerated, although not particularly efficacious.41 VEGF is also known to play a role in pathophysiological angiogenesis, and therapies focusing on reducing free circulating VEGF (Bevacizumab) or interfering with VEGFR activity (Cediranib), have been successfully used to slow tumour progression by reducing nutrient delivery and interfering with

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cell survival pathways.7,42–44 These drugs may also normalise tumour vasculature, resulting in more effective drug delivery to tumours.45 The role of VEGF in the brain has been studied extensively in the context of brain tumours, cerebrovascular ischaemia and traumatic brain injury.46–49 During our previous work using VEGF for cardiac regeneration, we incidentally observed that intramyocardial injection of VEGF allowed increased accumulation of systemically injected polystyrene nanoparticles in the brain.50 We pursued this further and found that low-dose intravenous VEGF can induce a moderate, but significant, increase in BBB permeability. In this study, we aim to demonstrate that this effect is transient, and that this short window of opportunity can be exploited to deliver a variety of nanomedicines to the brain tissue, including the delivery of a liposomal anti-cancer drug for glioblastoma therapy.

RESULTS The basic experimental design is shown in Figure 1A. Mice were intravenously injected with VEGF or vehicle control, followed by an agent either 45 minutes or 4 hours later. Figure 1B shows the half-life of human VEGF in the mouse blood stream to be approximately 18.67 minutes. Penetration of contrast agent into brain parenchyma, measured by magnetic resonance imaging (MRI), is often used to demonstrate BBB integrity in live animals.31,51,52 Under normal conditions, only a small amount of Gadolinium-based agent (Gd) will enter the brain parenchyma, providing little contrast enhancement. As shown in Figure 1C and 1D, controladministered animals showed an average increase of only 3.5 % in the signal to noise ratio (SNR) of cortex tissue in T1-weighted post-contrast images compared to pre-contrast images. However, if Gd was given 45 minutes following VEGF administration, there was a significant increase (16 %, p < 0.001) in the average SNR of the cortex, indicating that VEGF pre-treatment increased the penetration of Gd into the brain tissue. Importantly, if Gd was given 4 hours after VEGF, the SNR enhancement remained less than 5 %, indicating that BBB integrity had normalised (p = 0.6150 vs. control, p < 0.001 vs. VEGF 45 mins). Analysis of the area surrounding the central cerebral sinus (yellow boundary) showed a large signal enhancement in 5


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all groups due to contrast agent present in the sinus, with no significant difference between the groups. Example images are shown, indicating the regions of interest (ROIs) of random noise (red boundary), central cerebral sinus (yellow boundary), and cortex (blue boundary). Difference images are shown in Supporting Information Figure S1A. The dye Evans blue rapidly binds to serum albumin and does not cross the intact BBB.32,53,54 Evans blue was injected either 45 minutes or 4 hours after VEGF, and allowed to circulate for 30 minutes. The results, shown in Figure 1E, show that VEGF pre-treated mice had a 4.85-fold higher concentration of Evans blue in the brain tissue compared to control-treated animals (p = 0.0069), whereas if Evans blue was injected 4 hours following VEGF, no increase was detected (p = 0.9405 vs. control). This again indicates that the increase in BBB permeability appears to be temporary. The kidney also showed an increase in Evans blue uptake at 45 minutes. Representative sections of the brain cortex, shown alongside Figure 1F, show increased Evans blue staining (red) outside of isolectin-stained blood vessels (green). A positive control was carried out by causing local damage to the BBB using cryolesion prior to Evans blue injection. The lesioned area showed strong Evans blue signal in the parenchyma. It is well established that smaller nanoparticles pass more readily into the brain than larger nanoparticles.50,55,56 45 minutes following VEGF administration, PEG-modified polystyrene nanoparticles containing fluorescent dye were administered by tail vein injection. The nanoparticles had solid core diameters of 20 nm, 100 nm and 500 nm, with hydrodynamic diameters of 52, 120 and 512 nm respectively, and neutral zeta potentials. Nanoparticle properties are shown in Table S1. After allowing 30 minutes for nanoparticle circulation, mice were perfused with 50 ml saline and brain nanoparticle content was quantified by IVIS. As expected, 20 nm nanoparticles displayed more penetration into the brain than 100 nm or 500 nm nanoparticles under normal conditions. In VEGF-primed animals, a significant increase in 20 nm nanoparticle retention (3.5-fold vs. control, p = 0.0002) by the brain was detected, as shown in Figure 1G. A significant increase in 100 nm nanoparticle penetration (8-fold vs. control, p = 0.0182) was also detected, but there was no significant change in the retention of 500 nm nanoparticles (p = 0.9762 vs. control). In addition, we also have preliminary evidence that VEGF pre-treatment allows the passage of systemically injected IgG antibody into the brain

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(Supporting Information Figure S1B), which could be beneficial for antibody-based chemotherapy. In summary, these data show that low-dose intravenous VEGF may increase BBB permeability and allow penetration of small molecules or nanoparticles into the brain. This effect appears transient, with restoration of BBB function after 4 hours. Next, we sought to determine whether this same approach could be used to deliver therapeutic compounds to brain tissue. Temozolomide (TMZ) is the first line drug therapy for treatment of GBM. We hypothesized that combining TMZ with VEGF could further increase the intra-brain concentration of TMZ. Surprisingly, as shown in Figure 2A, we failed to observe any significant increase in TMZ concentration in the brain after VEGF priming, even using a 10-fold higher concentration of VEGF. Increasing the systemic dose of TMZ to 20 mg/kg increased the amount in the brain, but this was not further increased by pre-treatment with VEGF. As a small (MW = 194.15), highly lipid-soluble molecule, TMZ shows excellent penetration into the brain, hence its clinical use.3 The standard curve and sample high performance liquid chromatography (HPLC) peaks for TMZ quantification are shown in Supporting Information Figure S2B. Given this negative result, we theorized that a larger, water soluble molecule, more similar in properties to Evans blue, may be appropriate. Doxorubicin hydrochloride (MW = 579.98), has extremely poor entry into the brain following systemic administration, and many attempts have been made to deliver Doxorubicin to brain tumours due to its potent efficacy against other solid tumours.29,30,57 Mice were injected with VEGF or control followed by Doxorubicin (8 mg/kg) 45 minutes later. The drug was allowed to circulate for two hours before the animal was perfused with saline. Doxorubicin was then extracted from the vital organs and quantified by HPLC (Supporting information Figure S2C). Biodistribution results, shown in Figure 2B, confirm that less than 0.1 % of systemic Doxorubicin entered the brain of healthy control mice, which is expected. Although the addition of VEGF did result in a statistically significant increase (p = 0.0180 vs. control) in the Doxorubicin concentration into the brain, this dose is too low to be considered therapeutically useful.

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We hypothesised that PEG-modified liposomal doxorubicin (LipoDox) may be more amenable to brain delivery. We determined that these liposomes are neutrally charged (-1.53 mV), with an average hydrodynamic diameter of 95.55 nm (Table S2). These are similar properties to the PEG-modified nanoparticles which successfully entered the brain (Figure 1E) and in line with previously published papers showing that a diameter between 50-200 nm, PEG surface modification, and a neutral or slightly negative zeta potential may all aid in BBB passage.23–25,58 Results in Figure 2C show a significant increase (6.4-fold vs. control, p = 0.0037) in LipoDox entry into the brain following VEGF administration, showing that LipoDox was able to cross the healthy BBB. Figure 2D shows the data normalised against the blood plasma LipoDox concentration of each individual mouse at the time of sample collection, thus correcting for individual differences in drug metabolism and excretion. There were no significant differences detected in the concentration of LipoDox in any peripheral organs. Supporting information Figure S3A-E shows the HPLC method for LipoDox quantification. Using an MTT assay (Figure 2E) we found that LipoDox had a 25-fold lower IC50 than TMZ when cultured with the human glioblastoma cell line DBTRG-05MG. In addition, we determined the circulatory half-life of LipoDox to be 44.72 hours in mice, following systemic administration of a 5 mg/kg dose (Figure 2F). This is significantly longer than the published half-life of TMZ (1.8 hours) or Doxorubicin (11 hours).59,60 We also found that VEGF did not affect DBTRG-05MG cell viability at any given concentration (Supporting information Figure S3F). Next, we sought to carry out a proof-of-principle study in large animals to determine whether these findings could be scaled up to more clinically relevant drug doses. Lanyu mini pigs (n = 3 per group) were administered VEGF (0.2 µg/kg) or vehicle control via the carotid artery. Gd SNR enhancement was used to determine BBB integrity in multiple brain regions by MRI, as shown in Figure 3A. Quantification of the normalised increase in signal intensity (Figure 3B) revealed statistically significant increases in the ROIs encompassing the cortex, hippocampal formation, thalamus and white matter. Other brain regions showed a trend towards increased SNR, but these were not statistically significant (assessed by ANOVA). This may be due to the distribution of VEGF and/or Gd through the brain vasculature, or the degree of BBB

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integrity. For example, it is known that capillary density varies between brain regions, as does tight junction density, p-glycoprotein expression, pericyte coverage, and VEGF receptor expression. The average SNR across all regions (Figure 3C) was four-fold increased (p = 0.0035) in VEGF pre-treated pigs, which is similar to the change we observed in mice (Figure 1C-D). A heat map showing the change between normalised post and pre signal intensity is also shown. Analysis of standard deviation of the brain parenchyma is shown in Figure S4. A biodistribution study was also carried out in pigs using PEG-modified polystyrene nanoparticles (100 nm core diameter) and LipoDox as model drugs, as shown in Figure 3D. Examination of brain tissue by IVIS (Figure 3E) appeared to show a slight increase in total nanoparticle accumulation in the brain tissue of VEGF pre-treated pigs. Precise HPLC-based quantification of systemic nanoparticle biodistribution (Figure 3F) found that the majority of the particles accumulated in the lung, and comparison of specific brain regions (Figure 3G) found an overall trend towards more nanoparticle retention after VEGF pre-treatment. Averaging all brain regions (Figure 3H) showed a small, but statistically significant increase (2.4-fold, p = 0.0258) in nanoparticle retention in the brain. Analysis of systemic LipoDox (Figure 3I) biodistribution by HPLC revealed a similar pattern to that observed in mice (Figure 2C), with most LipoDox remaining in circulation, and the spleen being the major organ of LipoDox retention. Examination of region-specific LipoDox accumulation in the brain (Figure 3J) found that no specific region showed a significant increase (as analysed by ANOVA). However, there was an overall trend towards more LipoDox in VEGF-pre-treated animals. The fold change ranged from 1.1-fold (thalamus) to 2.1-fold (hypothalamus). Averaging the LipoDox concentration of the whole brain revealed a slight increase (1.69-fold) in LipoDox accumulation (Figure 3K). One limitation of the drug distribution experiments, aside from the small number of pigs per group, is that animals were not systemically perfused before tissue collection which would lead to higher baseline drug concentrations. Due to technical difficulties, uncontaminated cerebrospinal fluid (CSF) could only be collected from three pigs. Nevertheless, the two VEGF pre-treated animals both showed a higher LipoDox concentration in the CSF than the control treated animal (Figure 3L). Overall,

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these results demonstrate that VEGF-induced BBB permeability could be scaled up and induced in a large animal which is more clinically relevant to human patients. To shed light upon the possible mechanism underlying the transient effect we have observed, mouse brains were collected 45 minutes or 4 hours following VEGF or saline injection. BBB permeability may be characterised by many changes including tight junction protein expression or altered localisation, pericyte detachment from endothelial cells, astrocyte loss, as well as changes in the activity of BBB transporters and efflux pumps. As an initial screen, the expression of key genes related to BBB integrity was measured.61 Interestingly, the results show changes in the expression of several genes, including an increase in BBB transporters such as Slc2a1 (GLUT-1, glucose transporter) and Slc6a8 (CRT, creatinine transporter) and a decrease in tight junction components such as Tjp2 (ZO-2) and Cldn5 (Claudin 5), as shown in Figure 4A. Brains were taken from healthy mice at 45 minutes, 90 minutes and 4 hours following VEGF injection, then frozen sectioned, and stained for key indicators of BBB integrity. Transmission electron microscopy (TEM) was used to examine the morphology of brain blood vessels following VEGF treatment. Representative images (Figure 4B) show that in control animals, pericytes were present adjacent to endothelial cells, separated by a basement membrane, as normal. However, at 15 minutes following VEGF injection, many vessels appeared slightly dilated and lacking adjacent pericytes. At 45 minutes, most vessels were no longer dilated, and at four hours following VEGF treatment, both vessels and pericytes appeared normal. To further investigate this finding, we took frozen samples from GBM-bearing mice at 45 minutes following VEGF injection. Pericytes were stained using antibodies against platelet-derived growth factor receptor beta (PDGFRβ), which has been previously used to visualise BBB integrity.62 The length of pericyte coverage of blood vessels, stained with antibodies against CD31, was quantified, as shown in Figure 4C. In control animals, PDGFRβ staining was observed outside of CD31+ blood vessels (91.1 % coverage). However, at 15 minutes pericyte coverage was reduced (57.6 %) and returned to normal after 45 minutes (86.2 %) and 4 hours (83.0 %), in line with observations from TEM. In the tumour region, blood vessels were highly

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variable in size and morphology, and showed little coverage with pericytes (30.8 %). Pretreatment with VEGF did not affect pericyte coverage (28.9 %) within the tumour region. GFAP, a marker of astrocytes, was also examined, as shown in Figure 4D. No obvious change in astrocyte morphology was apparent between treatment groups. Few astrocytes were present in the tumour region. Claudin 5, a component of endothelial cell tight junctions, was costained with the endothelial cell marker CD31.56 The results show strong colocalisation (> 95 %) of claudin 5 and CD31 in control mice, which decreased at 45 minutes (55.8 %) and 4 hours (42.7 %) following VEGF administration, as shown in Figure 4E. This agrees with gene expression data shown in Figure 4A which showed downregulation of Cldn5. In addition, a Western blot for Claudin-5 showed a trend towards reduced protein expression after VEGF administration (Supporting information Figure S5). Interestingly, the tumour region still showed a large presence of tight junctions (80.0 %) in control-treated mice. Following VEGF pre-treatment, this decreased to 48.5 %. P-glycoprotein, the predominant efflux pump on the BBB, also appeared uniformly on the membrane of blood vessels at all time points, as shown in Supporting information Figure S5B.63 Based on these results, we carried out an experimental therapy of glioblastoma, as outlined in Figure 5A. Owing to the long circulatory half-life of LipoDox (44.72 hours), and the transient nature of VEGF-induced BBB opening, we theorised that administration of multiple doses of VEGF (MV) after LipoDox administration could provide multiple windows of therapeutic delivery to the brain. MV mice were given VEGF, followed 45 minutes later by LipoDox, with further doses of VEGF at three hours and six hours after LipoDox administration. Biodistribution of LipoDox in MV+LD mice is shown in Supporting information Figure S6. We utilised a human glioblastoma cell line, DBTRG-05MG, engineered to express luciferase, to form a xenograft glioblastoma model in BALB/c NU mice, as shown in Supporting information Figure S7A-A. Tumour progression was monitored by weekly IVIS and mice were assigned randomly to receive treatments of either VEGF + control (V+Ctrl), control + LipoDox (Ctrl+LD), VEGF + LipoDox (V+LD), or Multi-VEGF + LipoDox (MV+LD). Sham mice were intracranially injected with saline rather than tumour cells and received the MV+LD treatment

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course. LipoDox was given at a dose of 5 mg/kg, and treatments were given on Day 21, 25 and 28. We quantified the delivery of LipoDox to GBM xenografts following VEGF pre-treatment (Figure 5B). The results show that intratumoural LipoDox was 7.8-fold higher than the contralateral side following VEGF pre-treatment. This was 13.6-fold more than the intratumoural concentration in control pre-treated mice. Importantly, the concentration detected in the tumour region of Ctrl+LD mice was only slightly higher (2.3-fold) than in the contralateral side, suggesting a small EPR effect from the tumour itself. We also found that the sham injection procedure did not affect LipoDox accumulation (Supporting information Figure S8A), and a single injection of VEGF (V+LD) also increased intratumoural LipoDox, though to a lesser degree than MV+LD (Supporting information Figure S8B). A Kaplan-Meier survival curve, shown in Figure 5C, shows an improved median survival of V+LD (67 days, p = 0.0271) and MV+LD (79 days, p = 0.0042) groups compared to mice receiving Ctrl+LD (60 days). The difference between V+LD and MV+LD was also significant (p = 0.0483). No sham-operated mice died during the course of the experiment. A weekly examination of tumour luminescence, shown in Figure 5D, reveals no differences between the groups before the commencement of treatment (day 21). However, two weeks following completion of treatment (day 42), mice receiving V+LD and MV+LD treatments had significantly smaller tumours than Ctrl+LD mice (p = 0.0425 and p = 0.0417 respectively). This same trend continued at day 49 and day 56. By day 63 the MV+LD treated mice had significantly smaller tumours than the other groups (p = 0.0029 vs. Ctrl+LD, p = 0.01273 vs. V+LD). Representative IVIS images of mice from each group are shown above the corresponding graphs. Five mice each from the Ctrl+LD and V+LD treatment groups were randomly selected on day 45 to confirm tumour volume by MRI, as shown in Figure 5E. Image slices were captured and analysed by a blinded MRI technician. The results confirm that tumours in the V+LD treated mice were significantly smaller (p = 0.0358) in total volume and were present in less MRI slices (p = 0.0303) than Ctrl+LD treated mice, indicating delayed tumour progression. Representative image slices, with the tumour outlined, are shown. Supporting

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information Figure S9A shows that the correlation between IVIS luminescence and tumour volume confirmed by MRI is excellent (r-squared = 0.7884). Mouse body weights are shown in Supporting information Figure S9B. Samples from animals which died between days 60-70 were selected for staining. V+Ctrl samples are included for reference, although these animals died before 60 days and so cannot be directly compared. Figure 5F shows significant reductions in Ki67+ tumour cells in V+LD (p = 0.0061) and MV+LD (p = 0.0001) treated mice compared to Ctrl+LD treated mice, and between MV+LD and V+LD treated mice (p = 0.0208). The overall cell density of the tumour determined by DAPI staining (Figure 5G) was also slightly reduced in the MV+LD treated group (p = 0.0478 vs. Ctrl+LD). V+Ctrl treated mice show less cell proliferation, likely due to the earlier time point of sample collection. Given that VEGF is a potent stimulator of vasculogenesis, sections were stained with isolectin and blood vessels in the tumour were counted. Tumours from mice in the MV+LD treatment groups showed less blood vessels than mice in the Ctrl+LD group (p = 0.02) (Figure 5H). Immunohistochemical staining for the microglial/macrophage marker Iba1 revealed no significant difference in the number of Iba1+ cells in the tumours of the various treatment groups, as shown in Figure 5I. V+Ctrl treated mice showed less immune infiltration, again likely due to the earlier time point analysed. Example images of Iba1-stained tumours are shown in Supporting information Figure S9C. Furthermore, since VEGF may increase interstitial fluid retention, H&E stained images were used to identify areas of oedema and haemorrhage within the tumour using ImageJ. Example H&E stained images are shown in Supporting information Figure S9D. Interestingly, there was a trend towards V/MV+LD treated animals showing less oedema than control treated animals (Figure 5J). There was no significant difference in haemorrhage between groups, although it was highly variable between individual animals (Figure 5K). To examine whether VEGF may act on other malignant tumours, we quantified LipoDox uptake in pancreatic ductal adenocarcinoma (PDAC) model (Supporting information Figure S10A-C) following the Ctrl/MB+LD protocol, and compared uptake by the normal pancreas and the tumour xenograft. The results (Supporting information Figure S10D) show that PDAC xenografts took up ~3-fold more LipoDox than the sham-operated pancreas. This suggests some EPR effect is present in this model, although the overall concentration of LipoDox is still low. The addition of VEGF pre-treatment did not change uptake in either the

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normal pancreas or the PDAC xenograft. This agrees with previous data showing that VEGF at this dose does not cause changes in vascular permeability of peripheral organs. We also examined the effect of VEGF pre-treatment on LipoDox accumulation in GBM xenografts placed subcutaneously, as shown in Supporting information Figure S11A-C. We found no significant change in LipoDox accumulation in the tumour following the MV+LD treatment protocol. Of note, the LipoDox concentration in control-treated tumours was 25.8x higher in subcutaneous than in orthotopic xenografts, since the tumours are no longer sheltered by the BBB. Finally, we wished to consider some of the fundamental questions which would be necessary before using this technique in the clinic. One important question is whether administration of VEGF disrupts compartmentalisation of the brain, resulting in the escape of brain components into systemic circulation. The presence of calcium-binding protein S100 beta (S100) in blood has been previously shown to serve as a peripheral marker of brain injury and loss of BBB integrity.64,65 The results, shown in Figure 6A show no significant change in mouse plasma S100 two hours following administration of VEGF, or a 10-fold higher dose of VEGF. Lipopolysaccharide (LPS), a potent inducer of neuroinflammation which increases BBB permeability, was used as a positive control and caused a significant elevation of plasma S100 two hours following administration. In human clinical trials, VEGF induced temporary systemic hypotension during infusion. 41,66

To investigate whether our given bolus dose could cause the same effect, mice were injected

with VEGF, or a ten-fold higher dose, and blood pressure was measured every 30 minutes using a BP-2000 Series II Blood Pressure Analysis System. The results, shown in Figure 6B, show no notable change in blood pressure over a four-hour period following VEGF administration. Similarly, no clear changes in blood pressure were seen in the pigs which received VEGF compared to control (Figure 6C), although an overall trend towards decreased blood pressure was seen in both groups, likely due to anaesthesia and surgery. Interestingly, we did observe a rapid, but temporary, flushing reaction in one pig which received VEGF – a phenomenon which has also been observed in humans.67

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Endogenous VEGF is known as an inducer of neuroinflammation following brain injury.47 However, the effects of exogenous intravenous VEGF on the brain are unclear, given that many VEGF receptors are present on the abluminal, brain-facing side of brain endothelial cells.68 Therefore, to gain insight into whether intravenous VEGF may also induce neuroinflammation, we used real time quantitative PCR to screen for changes in the gene expression of several major cytokines related to neuroinflammation.69,70 Animals were perfused at four hours or 24 hours following administration of the VEGF and LipoDox treatment groups used in this study. Cryolesion and LPS were both used as positive controls. The results, shown in Figure 6D, show that VEGF administration did indeed moderately increase the expression of a number of neuroinflammation-related genes. Expression of Tnfa, Ccl2 and Cxcl1 was found to be unchanged four hours after VEGF treatment, but was moderately increased 24 hours after treatment. The gene expression of the acute inflammation marker Il6 was increased after 4 hours in treatment groups utilising multi-VEGF, but not single VEGF. No treatment group significantly increased Il1b or Gfap expression, although both were raised by cryolesion or LPS. Gene expression data for additional inflammation markers is shown in Supporting information Figure S12A, and a list of all primers used is in shown in Table S3. Measurements taken 45 minutes following VEGF administration (Supporting information Figure S12B) show no elevation of these same genes compared to controls, indicating that inflammation may be a delayed response – potentially a response to enhanced BBB permeability. In addition, blood chemistry results for liver and kidney function (Supporting information Figure S13) showed no adverse changes following treatment. These results demonstrate that the given dose of VEGF appears safe, although a full safety evaluation would clearly need to be carried out in relevant patients prior to clinical use.

DISCUSSION We have shown that a low dose of intravenous VEGF is sufficient to induce a short-lived increase in BBB permeability which could be used to deliver nanomedicines to the brain in both small and large animals. The combination of VEGF and liposomal doxorubicin was able to greatly extend survival in a mouse model of human glioblastoma. The low dose of VEGF used did not increase tumour vasculogenesis, induce hypotension or cause any obvious adverse effects. 15


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The concept of using a drug pre-treatment to induce a temporary window of enhanced BBB/BBTB permeability for cancer treatment has been shown previously. For example, an A2A adenosine receptor agonist, Regadenoson, has been shown to temporarily downregulate pglycoprotein expression and function, which was exploited to increase Epirubicin delivery to the brain. Similarly, Ondansetron pre-treatment has been shown to allow increased Doxorubicin delivery to the brain.63,71 A phase I human clinical trial (NCT02389738) is now investigating the use of Regadenoson pre-treatment in the delivery of Temozolomide to the brain. Trials using focused ultrasound to temporarily open the BBB prior to chemotherapy are also underway (NCT02253212, NCT02343991). Two previously published papers have shown that the intravenous administration of VEGF can affect BBB permeability in animals. However, in both cases, the authors used a dose of VEGF more than 100-fold higher than our own, with effects lasting for several hours.49,51 This greatly increases the chance of harmful side effects arising from BBB dysfunction, whereas in our experiments we found that normal BBB function appeared to be restored by four hours after VEGF administration. It is interesting that VEGF did not increase penetration of all types of molecule through the BBB. For example, Temozolomide (TMZ) entrance into the brain was unaffected by VEGF pretreatment, likely because TMZ takes a transcellular pathway, essentially bypassing the BBB. It has also been shown that TMZ itself may down regulate p-glycoprotein efflux pumps.72 Doxorubicin, unable to travel by the transcellular route, is also a known substrate for the pglycoprotein efflux pump, and so any Doxorubicin which did enter the brain following VEGF pre-treatment would be quickly removed by efflux pumps. 500 nm diameter polystyrene nanoparticles were also unaffected by VEGF pre-treatment, most likely due to physical size limits. We did not carry out an analysis of liposome size-dependent BBB passage, which may be different to the passage of solid polystyrene nanoparticles. Nanoparticles of 20 nm and 100 nm diameter, and LipoDox (~95 nm diameter), all readily passed into the brain following VEGF pretreatment. LipoDox is currently used for treatment of solid tumours in the breast and ovary but is not currently FDA-approved for use in GBM. One small human clinical trial found that it was well tolerated and slightly delayed brain tumour progression when combined with TMZ.73 LipoDox may be more effective than Doxorubicin in patients whose tumours express p-

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glycoprotein, since PEG modification shields the drug molecule from efflux, and may allow it to pass more easily within the brain tissue.24 MRI analysis based on gadolinium contrast enhancement showed very similar results in pigs (~4-fold increase in SNR) to those we observed in mice. This is encouraging, given that the MRI is measuring the real-time signal in the living brain, whereas other methods rely on post-mortem collection of tissues, drug extraction and quantification. MRI also allows for before-after comparisons from the same animal, countering inherent heterogeneity between animals. However, one limitation of our pig biodistribution study is that we were unable to perform systemic perfusion as we did in mice. This would result in a higher baseline drug concentration in the control samples, thus masking the increased delivery following VEGF pre-treatment. Cerebrospinal fluid (CSF) samples can serve as a proxy for the central nervous system drug concentration, however we were unfortunately unable to gather enough CSF samples to make a statistical comparison. VEGF has been well demonstrated, both in vitro and in vivo, to affect brain endothelial cell tight junctions and sub-cellular organisation.46 Our own results are in agreement, showing decreased gene expression of Tjp2 (ZO-2) and Cldn5 in the brain soon after VEGF administration. Staining of brain sections following VEGF also confirmed these findings. The tumour model is slow-growing (median survival 50-60 days without treatment) and still showed a high degree of tight junction colocalization with endothelial cells, indicating that the BBTB is relatively intact. Indeed, we found that only 2.3-fold more LipoDox entered the tumour compared to the contralateral healthy side. When the same tumour cell line was used to establish subcutaneous GBM xenografts, the LipoDox concentration in the tumour was 25 times higher than for orthotopic xenografts, clearly demonstrating how the BBB prevents effective drug delivery to the brain. Endogenous VEGF is known to modulate astrocyte activation, which in turn mediates BBB integrity. This is particularly relevant during the response to injury such as ischaemia, where astrocyte-secreted VEGF locally increases BBB permeability.47 However, we observed no change in astrocyte morphology or Gfap gene expression under the conditions we analysed.

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Previous studies have found that exogenous VEGF can modulate p-glycoprotein activity in isolated brain capillaries and in situ rat brains.74 However, we found no change in p-glycoprotein gene expression (Abcb1a), or the morphological appearance of p-glycoprotein staining in frozen sections following VEGF administration at our given dose (Supporting information Figure S5B). VEGF also increases BBB permeability through the calveoli-mediated transcellular pathway when injected directly into the brain.48 Interestingly, by TEM we observed what appeared to be vessel dilation in the brain 15 to 45 minutes following VEGF injection which, along with pericyte detachment and tight junction weakening, could explain the increased BBB permeability which we observed in healthy brains.75 However, in tumour xenografts, pericyte coverage of blood vessels was already significantly reduced compared to healthy brain, and was not further reduced by VEGF pre-treatment. Thus, we speculate that intravenous VEGF increases BBB permeability through transient degradation of brain endothelial cell tight junctions, although we cannot rule out other mechanisms. In terms of safety, we found that intravenous VEGF increased the expression of a number of neuroinflammation-related genes in the brains in otherwise healthy mice. This is certainly something which requires further investigation, but it is important to note that patients with GBM would already be experiencing profound neuroinflammation.76 Neuroinflammation is a complex multi-faceted process involving local production of cytokines as well as increased activity of BBB cytokine transporters which allow more externally produced cytokines into the brain.35 These issues would require further investigation before translation to the clinic. Inhibition of VEGF signalling is an important therapeutic target in cancer treatment.42 Therefore, administering exogenous VEGF to cancer patients may seem counter-intuitive. However, tumour vasculogenesis relies on sustained, local, signalling from multiple growth factors, not only VEGF. Data from human patients indicates that VEGF has a circulatory halflife of less than 30 minutes after systemic infusion.66 In addition, previous trials have found that single administrations of VEGF have very little effect on blood vessel formation, and that newly formed vessels regress upon VEGF withdrawal.41,77 In fact, we found that treatment using MV+LD slightly reduced tumour blood vessel density. Under this regimen VEGF would always

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be coupled with administration of a potent chemotherapeutic such as liposomal Doxorubicin which inhibits tumour angiogenesis and has direct cytotoxic activity. The main limitations of the described approach are that the overall effect is modest and that it is heterogenous. Although our results for Evans blue, Doxorubicin and Gd are in line with published data for other methods of BBB opening, VEGF appears to act in a heterogenous manner over the entire brain tissue, whereas techniques such as focused ultrasound can be accurately targeted to specific regions. This may be due to reliance on delivery by brain capillaries, which are known to vary in density and permeability through different brain regions.78 We speculate that this regimen may be useful for diffuse brain tumours which cannot be sufficiently targeted by radiotherapy, and where the margin of tumour and healthy brain cannot be clearly seen. Indeed, in these areas the BBB would be mostly intact, inhibiting access of chemotherapeutics. For GBM treatment, this paper showed delayed tumour progression and improved survival in mice, but never regression or cure. This is in line with other studies, but within the context of glioblastoma, which has an extraordinarily poor prognosis, this may still be meaningful. For example, Temozolomide has become a first line drug therapy for human patients, yet only provides a two-month increase in progression-free survival. Bevacizumab is often used in GBM patients even though it provides no overall increase in survival, but a moderate increase in quality of life. This is indicative of the dire need for more effective GBM drug therapies.79 Finally, it is known that the human BBB and rodent BBB differ, particularly in the proportions of transporters and efflux pumps present on brain vasculature.80 Therefore, all rodent model data must be interpreted with this limitation in mind. However, given that recombinant human VEGF was used in this study, and that the effect was preserved in pigs, we are optimistic that this method should also be relevant to human patients.

CONCLUSION

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In summary, we have shown that a low dose of intravenous VEGF improved the delivery of nanomedicine therapeutics to the brain. We believe that this finding has potential for translation to the clinic in order to enable improved therapy of brain tumours, which is an unmet clinical need of upmost urgency. Future studies will need to be done in order to narrow down the ideal doses, drug timing and safety parameters in the target patient population.

MATERIALS AND METHODS Animal Experimentation. Animals used for drug biodistribution studies were 8-10-week-old male Friend leukaemia virus B (FVB) mice, weighing approximately 25 g. 6-8 week old male BALB/c NU mice, approximately 21 g, were used for human GBM tumour xenograft experiments. For PDAC tumour xenografts, 8-week-old NOD/SCID mice weighing 25-30g were used, and 8-10 week old female ICR mice, approximately 30 g, were used for mechanistic and safety studies. All mice were purchased from BioLasco, Taiwan. Mice were housed in a 12 hour day-night cycle with free access to food and water. For large animal studies, Lanyu minipigs, 1924 kg were used. All mouse experiments were approved by Academia Sinica Institutional Animal Care and Use Committee (IACUC) and pigs were used in accordance with approved protocols from Taiwan National Laboratory Animal Center, under supervision of veterinary staff. Agent Administration. In mice, drugs were administered as a bolus injection by tail vein using a .30G insulin needle, unless otherwise stated. Recombinant human VEGF165A (Peprotech, Taiwan) was suspended in 0.1 % w/v bovine serum albumin and administered via lateral tail vein at a dose of 1.5 ng/g body weight, unless stated otherwise. Evans blue (Sigma E2129) was suspended at 4 % w/v in normal saline and administered at a dose of 4 ml/kg. Doxorubicin Hydrochloride (Sigma D1515) suspended in saline, or LipoDox (TTY Bio, Taiwan) was administered slowly at doses between 2-8 mg/kg by lateral tail vein. Temozolomide (Sigma T2577) was administered at either 5 mg/kg or 20 mg/kg. Fluorescent PEG-modified yellowgreen polystyrene microspheres with 20, 100 and 500 nm solid core diameters (Life Technologies, Thermo Fisher) were administered at a dose of 3 mg/kg. Lipopolysaccharide (Sigma L4391), used to induce neuroinflammation, was given at a dose of 5 mg/kg. In pigs, rhVEGF165A (0.2 µg/kg, 2 µg/ml) or vehicle control was injected into the right common carotid 20


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artery. LipoDox (TTY Bio, Taiwan), diluted to 0.35 mg/ml in 5 % w/v dextrose, was administered by intravenous infusion by syringe pump at a dose of 1.5 mg/kg at a rate of approximately 3.0 ml/min. Yellow-green PEG-modified polystyrene nanoparticles (100 nm core diameter) were administered by bolus injection at a dose of 3 mg/kg. Nanoparticle PEGylation. Fluorescent nanoparticles were PEG-modified using mPEG amine (5 kD, Nanocs, Taiwan) and Carbodiimide (Sigma) and characterised using a Malvern ZetaSizer ZS, as previously described.81 Contrast Magnetic Resonance Imaging (MRI). For mice, a Pharmascan 7T 16-cm bore horizontal system was operated by a technician. FVB mice, anaesthetised with inhaled isoflurane, were injected with VEGF or an equal volume of vehicle control. Pre-contrast T1weighted spin echo images were taken (repetition time (TR) = 400ms, echo time (TE) = 10.8 ms, field of view (FOV) = 2x2 cm, number of excitations (NEX) = 8, slice thickness 0.8 mm). 45 minutes, or 4 hours, after VEGF administration, contrast agent (Gadovist, Bayer) was administered at 0.2 mmol/kg by catheterised tail vein. Post-contrast T1-weighted images were acquired one minute after contrast agent injection. The SNR was calculated by dividing the signal of a ROI (mean pixel intensity) by the standard deviation of the background noise. All image acquisition, SNR measurement and tumour volume measurement was performed by two MRI operating technicians, who were blinded to the study groups. For pigs, VEGF or vehicle control was administered and two pre-contrast T1-weighted turbo spin echo images were taken (TR = 600 ms, TE = 10 ms, FOV = 20x20 cm, slice thickness = 3 mm) using a Philips Achieva X 3.0 3T MRI machine. 45 minutes following VEGF administration, Gadodiamide (Omniscan, GE Healthcare) was administered intravenously by power injector at a dose of 0.1 mmol/kg (approximately 5 ml). One minute later, a series of three post-contrast images were taken at the same settings. The two pre-contrast images were averaged, and the post contrast image with the highest SNR from each animal was selected for analysis. Evans Blue Quantification. After 30 minutes circulation, 50 ml phosphate buffered saline (PBS) was perfused through the abdominal aorta. Organs were removed, cut into multiple smaller pieces, rinsed, dried, weighed, homogenised in 500 µl formamide, heated at 60 ˚C

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overnight and centrifuged at 21,000x g for 15 minutes. Supernatant absorbance was measured at 620 nm with background correction at 740 nm, and unknowns quantified by standard curve. Organs from mice which did not receive Evans blue injection were used to establish blank absorbance readings.82 Concentrations from multiple pieces of the same organ were averaged. The standard curve for this method is shown in Supporting information Figure S2A. For imaging of Evans blue extravasation, we adapted a method from del Valle and colleagues.83 Mice were intracardially perfused with 50 ml PBS followed by 50 ml of Evans blue (1 % w/v) in paraformaldehyde (4 % w/v). Cryolesion injury, to induce a local area of BBB damage, was used as a positive control. The brain was embedded in optimal cutting temperature compound (OCT) and cryosectioned into 20 µm thick sections for analysis. Fluorescent PEG-Modified Polystyrene Nanoparticle Quantification. In mice, nanoparticles were allowed to circulate for 30 minutes before animals were perfused, as described above. IVIS was used to quantify nanoparticle retention (ex 485, em 530 nm). A brain from a mouse which did not receive nanoparticle injection was used to correct for background. In pigs, HPLC was used to quantify nanoparticle retention.84 Briefly, nanoparticle fluorescent dye was extracted into o-xylene, and quantified using a Waters e2695 separation module and X-bridge C18 (250 x 4.6 mm, 5 µm) column with a mobile phase of 77:23 methanol:water, flow rate 1 ml/min. Detection used a Waters 2475 FLR detector with excitation at 505 nm and emission at 515 nm. HPLC Quantification of Temozolomide (TMZ). Approximately 100 mg tissue was homogenised in acidified ammonium acetate (200 µl, 10 mM pH 3.5), zinc sulphate (200 µl, 100 mM) and methanol (400 µl), followed by centrifugation at 10,000 x g for 30 minutes at 4˚C. Supernatant was taken for HPLC analysis. Separation was carried out with a Water e2695 separation module using 80:20 ratio of acetic acid (0.1 % v/v) to methanol at a flow rate of 0.8 ml/min in an Atlantis T3 3 µm HPLC column at 35˚C. Detection was performed using a Waters 2489 UV/vis detector at 316 nm. Theophylline was used as an internal standard, measured at 275 nm, and results calculated as the peak ratio of TMZ to theophylline. Unknowns were calculated from a standard curve of TMZ dissolved in lysis buffer, as shown in Supporting information Figure S2B.

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HPLC Quantification of Doxorubicin and Liposomal Doxorubicin. Organs were removed, dried, weighed, cut into multiple smaller pieces (approximately 100 mg tissue or 100 µl plasma) then thoroughly homogenised with 1 ml lysis buffer (0.25 M sucrose, 5 mM Tris-HCl, 1 mM MgSO4, 1 mM CaCl2, pH 6.7). 200 µl homogenate was then mixed with 1 ml acidified alcohol (70% ethanol, 0.3 N HCl), left overnight at -20 ˚C, then centrifuged at 10,000x g for 30 minutes at 4˚C. Supernatant was taken for HPLC analysis. Separation was via a Waters e2695 separation module, mobile phase 35 % 10 mM KH2PO4, 65 % methanol, flow rate 1 ml/min in an XBridge 5 µm column at 40 ˚C. Detection used a Waters e2475 module (ex 480, em 600 nm). For LipoDox extraction, the protocol was modified to include 30 minutes of sonication, 2 rounds of freeze thaw, and the addition of Triton-X100 (1 % v/v) to the lysis buffer, adapted from previous publications.30,85 Standard curves, example HPLC peaks and recovery rates are shown in Supporting information Figure S3. Xenograft Models. DBTRG-05MG human glioblastoma cells, engineered to express luciferase, were a gift from Dr. Chi-Huey Wong, Academia Sinica, Taiwan. Evidence of luciferase expression is shown in Supporting information Figure S7A. DBTRG-05MG cells were routinely cultured at 37 ˚C in RPMI 1640 media supplemented with 10 % FBS, 1 mM sodium pyruvate and 1 % penicillin/streptomycin. MTT assay was carried out in accordance with the manufacturer protocol. For GBM tumour generation, 300,000 live DBTRG-05MG cells, suspended in 6 µl sterile saline, were administered to 6-week-old BALB/c NU mice by stereotactic injection, 0.5 ml/min. The location was 2 mm posterior to the bregma, 1.5 mm laterally in the right cerebral hemisphere, and 2.5 mm deep from the dura, thus delivering the cells into the thalamus.86 Bone wax was applied to the skull and the skin was closed with sutures. The success rate of xenograft tumour formation was approximately 90 %. Animal deaths were recorded when the animal succumbed from tumour progression, or upon sacrifice if they met pre-determined criteria including severe cachexia (loss of > 25 % starting body weight), inability to feed, and lack of response to toe-pinch stimulus.87 Further characterisation of the tumour model is shown in Supporting information Figure S7. For subcutaneous GBM xenografts, 1x106 DBTRG-05MG cells were injected into each flank of balb/c NU mice and allowed to grow for 58 days. For orthotopic model of pancreatic cancer (PDAC), luciferase-expressing AsPC1 human pancreatic cancer cells were routinely cultured at 37 ˚C in RPMI 1640 with 10 % FBS,

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and 1 % penicillin/streptomycin.88 5x105 live AsPC1 cells, suspended in 10 µl sterile PBS, were administered into the pancreas.89 After returning the pancreas to the abdominal cavity, the incision was closed in two layers using a continuous suture for the peritoneum and an interrupted suture for the skin. Further information is available in Supporting information Figure S10. IVIS Assessment of Tumour Progression. Luciferin substrate, 75 µg/g, (Monolight, BD Bioscience) was given by intraperitoneal injection. Mice were anaesthetised with inhaled isoflurane and repeated IVIS images were acquired at five-minute intervals using a Perkin Elmer IVIS Spectrum. The time point presenting the strongest luminescent signal was selected for analysis. Background readings from a sham mouse present in every frame were subtracted. Immunofluorescence Staining. Brain tumours from mice which died between days 60-70 were selected for immunofluorescence staining. VEGF+Ctrl samples were included for reference, although those mice died earlier than day 60. Primary antibodies and dilutions used were antiKi67 (1:500 GeneTex GTX16667), Isolectin IB4-AlexaFluor 647, anti-GFAP (1:500 Abcam ab68428), anti-Iba1 (1:1000 Wako 019-19741), anti-p-glycoprotein (1:100 Abcam ab170904), anti-pdgfrβ (1:100, ab32570 Abcam), anti-claudin-5 (1:50 34-1600 Thermo Fisher Scientific), anti-CD31 (1:100 550274, BD Pharmingen). Samples were fixed in 4 % w/v paraformaldehyde (PFA) pH 6.8 overnight, embedded in paraffin and sectioned. Slides were de-waxed with xylene, rehydrated through graded alcohols to water, then subjected to antigen retrieval, permeabilisation and blocking in accordance with the manufacturers’ instructions. Primary antibody was applied overnight at 4 ˚C diluted in blocking buffer, apart from isolectin, which was applied for one hour at room temperature. Slides were washed 3x with PBS, before the second primary antibody was added and left overnight at 4 ˚C. Secondary antibodies used were goat anti-rabbit IgGAlexaFluor 568 (Invitrogen A-11011) and goat anti-rat IgG-AlexaFluor 488 (Invitrogen A11006), applied for one hour at room temperature. At least three separate sections were examined, and at least three images were captured per section. For frozen sections, samples were perfusion fixed then submerged in 4 % paraformaldehyde (PFA), cryoprotected in 30 % w/v sucrose solution, then frozen in OCT, sectioned and stained as above, continuing on from blocking. H&E staining (Mayers) followed standard lab protocols. Images were captured using a

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Zeiss AxioScop microscope with AxioFluor objective lenses and AxioVision software, or a Model-Zeiss LSM 700 Stage confocal microscope. Transmission Electron Microscopy. Mice were anaesthetised and perfused with PBS followed by 100 ml 4 % PFA in 0.1 mM phosphate buffer, pH 7.4. The brain was removed and post-fixed in 4 % PFA (overnight, 4 ˚C) and washed in PBS. Coronal brain sections (100 µm thick) were cut on the same day with a cryomicrotome and processed free floating. Sections were immersed in 4 % PFA, 2.5 % glutaraldehye in PBS (overnight, 4 ˚C), washed with PBS for 5 minutes for 3 times. The specimens were immersed in 1 % osmium tetroxide for 45 minutes, dehydrated and embedded with Spurr’s low viscosity resin. The sample was then trimmed and sectioned using a Leica EM UC6 ultramicrotome. The ultrathin sections were then double stained with uranyl acetate and lead citrate. Images were acquired using a Jeol JEM 1200EX TEM with an acceleration voltage of 80KV. Enzyme Linked Immunosorbent Assay (ELISA). For plasma S100β, mouse plasma was separated by 15 minutes centrifugation at 1,500 x g and the ELISA was carried out according to the manufacturer’s instructions (Elabscience, E-EL-M1033). Diluted brain homogenate in saline was used as a positive control. To measure plasma rhVEGF, an anti-human VEGF ELISA kit (Boster, EK0539) was used, following the manufacturer protocol. Samples from the same mice prior to VEGF administration were used as blanks. Real Time Quantitative PCR. Samples of mouse cerebral cortex weighing approximately 50 mg were homogenised in Trizol, and total RNA extracted via the manufacturer’s protocol, then quantified by Nanodrop. Samples were reverse transcribed to cDNA using a SuperScript III Reverse-Transcriptase Kit (Life Technology). OmicsGreen qPCR SYBR Green master mix (Omics Bio, Taiwan) was used to monitor amplification using an Applied Biosystems 7500 RealTime PCR system. GAPDH was used an internal control. Primers used are shown in Table S3. Software and Statistics. GraphPad Prism 7.0b (Mac) was used for all statistical analysis and graph generation. Statistical tests are described in the figure legends. For before-after analyses, paired t-test was used, and for grouped analyses one or two-way ANOVA (analysis of variance)

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with Tukey’s post-test to correct for multiple comparisons were used. For tumour survival analyses, deaths were recorded and used to generate Kaplan-Meier survival curves which were compared using Mantel-Cox log rank tests. IVIS images of tumour luminescence and nanoparticle fluorescence were quantified using Living Image 4.0 software for Mac. MRI DICOM images were sorted in MicroDicom (Windows) and SNR calculation was performed in FIJI/ImageJ (Mac) using the measure tool. For heatmap generation, voxels within the animal were compared to the average of a 64*64 voxel region in the corner of the frame and the difference was scaled from 0 to 100, using Python. Adjustments to immunofluorescence image brightness and contrast were made to improve visual clarity and were applied equally to all images within a series. For colocalisation analysis, the raw images were analysed in Zeiss ZEN software. The threshold for no colocalisation was established using the DAPI/CD31 channels, and that threshold was then applied to the other channels. Pericyte coverage analysis was carried out using the freehand line selection tool in ImageJ. Figures were assembled in Affinity Designer (Mac).

ASSOCIATED CONTENT Supporting Information Please see the supporting information file for additional biodistribution studies, HPLC method validation, western blot of Claudin 5, supplementary tumour model information, representative H&E/IHC/IF images, additional gene expression data, and results of mouse blood chemistry analysis. The supporting information file is available online. Table S1. Properties of polystyrene nanoparticles. Table S2. Properties of LipoDox. Table S3. Primer sequences used in quantitative real-time PCR. Fig. S1. Supporting MRI images and penetration of IgG antibody into the brain. Fig. S2. Standard curves for Evans blue, TMZ and Doxorubicin. Fig. S3. LipoDox and nanoparticle HPLC quantification, MTT assay. Fig. S4. Quantification of variation in pig MRI signal enhancement. Fig. S5. Western blot of claudin 5. P-glycoprotein staining. Fig. S6. Biodistribution of LipoDox following MV+LD treatment in mice. Fig. S7. Further GBM model information. 26


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Fig. S8. Effect of sham injections on drug retention. Intratumoral Lipodox following V+LD treatment. Fig. S9. Mouse body weights, Iba1 tumour staining, tumour H&E images. Fig. S10. PDAC model information. Fig. S11. Subcutaneous GBM model information. Fig. S12. Supplementary 45-minute, 4 hr and 24 hr inflammation gene expression. Fig. S13. Mouse serum blood chemistry.

P.C.C.H. and D.J.L. have applied for protection of intellectual property related to the results in the study. There are no other conflicts of interest to declare.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Patrick C.H. Hsieh: 0000-0002-8910-3596

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. P.C.H.H. contributed the idea and overall project supervision. D.J.L., Y.W.T. and P.C.H.H. contributed experimental design; D.J.L, K.J.L, I.C.P, C.H.H, R.H.L and K.H.C performed the experiments and analysed the data. D.J.L., K.J.L and P.C.H.H. wrote the manuscript. Funding Sources This work was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2811B-001-036, 106-2319-B-001-003 and 107-2314-B-004), the National Health Research Institutes grant EX106-10512SI and the Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis (AS-KPQ-106-TSPA), Thematic Research Program (AS-107-TP-B12) and Summit Research Program (MOST 1070210-01-19-01).

ACKNOWLEDGMENTS We thank the Taiwan Mouse Clinic (MOST 105-2325-B-001-010) which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the Ministry of Science and Technology (MOST), Taiwan for technical support in mouse blood pressure measurement and blood chemistry analyses. In addition, we are grateful to the Academia Sinica MRI, electron microscopy core, and biophysics core facilities, as well as the National Laboratory Animal Center (NLAC) for technical assistance. We thank Instrument Technology Research Center (ITRC), Hsinchu, Taiwan, for technical support in porcine MRI, and we also thank Dr. Kenneth Chien (Karolinska Institute, Sweden) and Dr. Yi-Chuang Chen (Academia Sinica, Taiwan) for helpful discussions and feedback. We also thank Dr. Niall Duncan (Taipei Medical University, Taiwan) for assistance with MRI analysis.

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Figure 1. Low-dose VEGF induces a transient increase in BBB permeability. (A) Schematic diagram showing experimental design. (B) Calculation of VEGF circulatory halflife following i.v. administration in mice, n = 5 animals. (C) Representative T1-weighted pre and post gadolinium (Gd)-enhanced MRI images of mouse brains, 45 minutes or 4 hours following VEGF or control administration. The regions of interest (ROI) of the cortex (blue), sinus (yellow) and noise (red) are shown. (D) Quantification of signal to noise ratio in selected regions. Analysis by ANOVA with Tukey’s HSD. (E) Biodistribution of Evans blue 45 minutes

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or 4 hours following VEGF pre-treatment. Analysis by ANOVA with Tukey’s HSD. (F) Representative images showing isolectin (green) and Evans blue (red) in the cerebral cortex. Nuclei were stained with DAPI (blue). Cryolesion was used as a positive control. Scale bar 100 µm. (G) Quantification of differently sized fluorescent PEG-modified polystyrene nanoparticles in the brain following control or VEGF pre-treatment. Analysis of each size control vs. VEGF by t-test. Error bars show standard error of the mean. Inset numbers indicate the number of animals. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. ### p < 0.001 compared to 4 hours. ns indicates not significant.

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Figure 2. VEGF enables enhanced delivery of selected anti-cancer drugs to the brain. (A) Quantification of Temozolomide (TMZ) in the brain of mice following pre-treatment with control (Ctrl + TMZ), VEGF (V + TMZ), or a ten-fold higher dose of VEGF (10xV + TMZ). TMZ was given at either 5 mg/kg or 20 mg/kg and circulated for one hour. Analysed by t-test vs. Ctrl + TMZ. (B) Doxorubicin (Dox) biodistribution 45 minutes following control or VEGF pretreatment. Dox circulated for two hours before sample collection. Analysed by ANOVA with Tukey’s HSD. (C) Percentage biodistribution of LipoDox, given 45 minutes following pretreatment with control (Ctrl + LD) or VEGF (V + 45m LD) based on LipoDox recovered from organs. LipoDox was allowed to circulate for 4 hours before sample collection. Analysed by ANOVA with Tukey’s HSD. (D) Organ concentrations of LipoDox normalised against the plasma concentration per mouse. Analysed by ANOVA with Tukey’s HSD. (E) Effect of LipoDox (LD) and TMZ on DBTRG-05MG human glioblastoma cell viability, as determined by

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MTT assay. n = 4. (F) LipoDox circulatory half-life following i.v. injection of 5 mg/kg by tail vein. n = 5 animals. Error bars show standard error of the mean. Inset numbers indicate the number of animals. * p < 0.05

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Figure 3. VEGF enhances drug delivery to the brain in a large animal model. (A) Schematic diagram showing experimental design of MRI studies in pigs. TSE, turbo spin echo. Pig brain slice showing regions of interest. CTX, cerebral cortex; G, grey matter; W, white matter; HPF, hippocampal formation; TH, thalamus; STR - striatum (cerebral nuclei area); HY, hypothalamus; PIR, piriform area. (B) Representative T1-weighted MRI images pre and post contrast in control and VEGF pre-treated pigs, and subtracted images showing a heatmap of the difference in normalised signal intensity scaled from 0 to 100. Quantification of SNR

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enhancement in selected brain regions. Compared by ANOVA with Tukey’s HSD. (C) Average increase in SNR across all brain regions. Compared by unpaired t-test. (D) Schematic diagram showing experimental design of pig drug biodistribution studies. (E) IVIS images showing nanoparticle fluorescence and pig brain accumulation. (F) HPLC-based quantification of nanoparticle systemic biodistribution. (G) nanoparticle distribution throughout brain areas. Analysed by ANOVA with Tukey’s HSD. (H) Average brain retention of nanoparticles. Analysed by unpaired t-test. (I) LipoDox systemic biodistribution and (J) brain distribution. Analysed by ANOVA with Tukey’s HSD. (K) Average brain retention of LipoDox. Analysed by unpaired two-way t-test. (L) LipoDox concentration in CSF. Error bars show standard error of the mean. Inset numbers indicate the number of animals. * p < 0.05, ** p < 0.01 compared to control. ns indicates not significant.

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Figure 4. VEGF affects multiple aspects of BBB permeability. (A) Quantitative real-time PCR for key BBB genes 45 minutes and four hours following VEGF administration. n = 4. Analysed vs. Control with Tukey’s HSD. (B) TEM imaging of brain blood vessels following VEGF administration. EC, endothelial cell; L, lumen; Er, erythrocyte; P, pericyte. Embedded scale bars 1 µm. (C) Staining of pericyte marker PDGFRβ (red) and endothelial cell marker CD31 (green) in healthy brains and GBM xenografts. The average degree

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of pericyte coverage is shown in the upper right corner of each image. Scale bar 100 µm. (D) Staining of astrocyte marker GFAP (red) and endothelial cell marker CD31 (green) in healthy brains and GBM xenografts. Scale bar 100 µm. (E) Immunofluorescence images of tight junction protein claudin 5 (red) and endothelial cell marker CD31 (green) in healthy brains and GBM xenografts. Separate channels and a merged image are shown. The colocalisation coefficient is shown in the upper right of each image. Scale bar 40 µm. (F) Average pericyte coverage. (G) Average claudin 5 colocalisation. Error bars show standard error of the mean. Inset numbers indicate number of animals. * p < 0.05, ** p < 0.01, *** p < 0.001 , **** p < 0.0001 compared to control. ns indicates not significant.

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Figure 5. VEGF combined with LipoDox extends survival in a mouse model of glioblastoma. (A) Schematic diagram showing experimental time course and explanation of VEGF (V) and multiple VEGF (MV) treatment courses. (B) Quantification of intratumoural LipoDox concentration in tumour-bearing mice. GBM xenografts and the contralateral region from the same animal were analysed. Analysed by t-test. (C) Kaplan-Meier survival curve. Pairs of curves compared by Log-rank (Mantel-Cox) test. (D) Weekly summary of tumour luminescence in each treatment group. The number of animals at each time point are inset and representative IVIS images are shown. Analysed by ANOVA with Tukey’s HSD. (E) Tumour volume analysis, as determined by MRI at day 45. Representative 1 mm thick slices (slices 12, 13 and 14) are shown, with the tumour area marked by a white boundary. Analysed by unpaired t-test. (F) Ki67 analysis of tumour sections from mice which died between days 60 and 70. Representative images show Ki67 (green) and DAPI (blue). Scale bar 100 µm. Analysed by ANOVA with Tukey’s HSD. (G) Intratumoural cell density determined by DAPI staining. Analysed by ANOVA with Tukey’s HSD. (H) Tumour blood vessel density per 400 x magnification field, as determined by isolectin staining. For sham mice, the injected region was imaged. Analysed by ANOVA with Tukey’s HSD. (I) Quantification of Iba1 positive cell content in brain tumour. Analysed by ANOVA with Tukey’s HSD. (J) Quantification of intratumoural oedema, as determined by H&E staining. For sham, an equal-sized area of normal brain was analysed. Analysed by ANOVA with Tukey’s HSD. (K) Quantification of intratumoural haemorrhage, as determined by H&E staining. For sham, an equal-sized area of normal brain was analysed. Analysed by ANOVA with Tukey’s HSD. Error bars show standard error of the mean. Inset numbers indicate the number of animals analysed. * p < 0.05, ** p < 0.01, *** p < 0.001. ns indicates not significant.

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Figure 6. Analysis of safety. (A) Quantification of plasma S100 concentration in mice. Lipopolysaccharide (LPS) to induce BBB disruption was used as positive control. Brain lysate was used as a second positive control. Before and after samples were analysed by paired two-way t-test. n ≥ 4 per group. (B) Mouse systolic and diastolic blood pressure measured every 30 minutes for four hours following VEGF or a ten-fold dose. The first sample (0 minutes) was taken immediately prior to VEGF administration. (C) Changes in pig blood systolic and diastolic blood pressure after VEGF administration. Analysed by paired t-test. (D) Gene expression of key neuroinflammation markers 45 minutes and four hours following VEGF administration. n ≥ 5. Cryolesion injury (cryo) and LPS were used to induce neuroinflammation. Each sample was normalised against Gapdh. Each group analysed vs. PBS, and 4 hrs vs. 24 hrs by two-way ANOVA with Tukey’s

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HSD. Average threshold cycle numbers (CT) for the PBS group are shown for reference. Error bars show standard error of the mean. Inset numbers indicate the number of animals. * p < 0.05, ** p < 0.01, *** p < 0.001 , **** p < 0.0001 compared to control. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to 4 hours. ns indicates not significant.

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Inducing a Transient Increase in BloodâBrain Barrier Permeability

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