Functionalized NPs can cross the intact BBB in mice

We first performed biodistribution studies in non-tumor-bearing mice using NPs that were functionalized with a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG 2K ) linker conjugated with Cy5.5-Transferrin (Tf-NP) or DSPE-PEG 2K -Cy5.5-Folate (Fol-NP) to assess their ability to cross the intact BBB. Non-functionalized PEGylated NPs (PEG-NP) or NPs functionalized with DSPE-PEG 2K -Cy5.5-Hemagglutinin (Hg-NP) served as negative controls. We chose to use Cy5.5 fluorescence imaging, understanding that it was a semi-quantitative technique for assessing biodistribution of NPs compared to radio-isotope labeling, to simply demonstrate as proof of concept that the conjugated NPs were able to be transported across intact BBB. Mice given I.V. Tf-NPs demonstrated 1.7% total uptake of the injected dose in the brain, compared to 0.9% in Fol-NP-treated mice, respectively, with negligible accumulation in mice injected with PEG-NP or Hg-NPs, 24 h following the injections (Supplementary Fig. 1A). Quantification of confocal microscopy images through fresh frozen coronal brain sections showed highest accumulations of Tf-NPs in the brain compared to Fol-NPs, with no significant increases in Cy5.5 fluorescence signal detected using Hg-NPs compared to unconjugated PEG-NPs (Supplementary Fig. 1B,C). This provided further preliminary evidence that the functionalized NPs can cross the intact BBB in mice.

We then performed multiphoton intravital live imaging through a cranial window (Fig. 1b) to assess the ability of these NPs to cross the BBB in non-tumor bearing mice. To control for vessel leakiness, we injected 70 kDa FITC-Dextran intravenously to ensure vessel integrity following the cranial window procedure (Supplementary Movie 1). Autofluorescence was controlled by increasing signal to noise ratio, thus all Cy5.5 red fluorescence seen in this and subsequent intravital images represents the presence of Cy5.5-labeled NPs only. As Tf-NPs demonstrated the highest percent uptake in the brain and had the smallest average diameter (Z avg d h ~ 137 nm) (Table 1), we decided to conduct the remainder of our experiments using Tf-NPs. As a negative control, we injected Hg-NPs intravenously and failed to demonstrate uptake by the endothelium of brain microvessels (Fig. 1c). In contrast, Tf-NPs demonstrated transport across the endothelium of microvessels into the surrounding subarachnoid space (Fig. 1d). This was further appreciated upon imaging 500 μm deeper into the cortical mantle, where diffusion of Tf-NPs was observed across an isolated section of a microvessel, forming a diffusion gradient of NPs away from the blood vessel (Fig. 1e).

Table 1 Average diameter, polydispersity index, zeta potential, and drug-loading properties of liposomes Full size table

Gliomas take up transferrin-functionalized NPs

We next assessed the ability of Tf-NPs to achieve receptor-mediated transcytosis in two intracranial orthotopic mouse models of gliomas: the human U87MG and murine GL261 glioma models. Immunohistochemistry (IHC) staining demonstrated transferrin receptor expression in the endothelium of tumor-associated blood vessels and on tumor cells in both U87MG and GL261 tumor-bearing mice, with relatively higher intensity of staining in U87MG compared to GL261 tumors (Fig. 2a; α-Tf Receptor). This increased uniform staining for the transferrin receptor throughout the tumor was not seen in serial sections stained using control IgG antibody (Fig. 2a; IgG Control). Western blot analysis also further demonstrated ~1.4-fold increased expression of transferrin receptor in U87MG compared to GL261 cells, consistent with the relative differences in staining intensity observed between tumor types on IHC (Fig. 2b). Our findings are consistent with studies demonstrating transferrin receptor expression in U87MG12 and GL26113 cells, suggesting that transferrin would be suitable as a ligand for our NPs for assessing receptor-mediated transcytosis and tumor targeting as previously reported in the literature14, 15. We first assessed the ability of U87MG and GL261 cells to internalize Tf-NPs in vitro. Cells incubated with Cy5.5-Tf-NPs showed increased intracellular Cy5.5 signal which co-localized to late endosomal/lysosomal compartments compared to cells incubated with Cy5.5-PEG-NPs, visualized using immunofluorescence microscopy over the course of 24 h (Fig. 2c). This intracellular uptake was then quantified using flow cytometry, demonstrating an average of 13% Cy5.5-positive cells after 24 h of incubation with Cy5.5-Tf-NPs, compared to <1% Cy5.5-positive cells after incubation with Cy5.5-PEG-NPs (Fig. 2d). These results suggest that functionalization with transferrin is required for cellular uptake of NPs in U87MG and GL261 glioma cells.

Fig. 2 Transferrin-functionalized liposomes achieve receptor-mediated transcytosis and delivery to intracranial models of GBM. a Immunohistochemistry demonstrates expression of transferrin receptor (α-Tf receptor) in the endothelium of tumor-associated blood vessels and in tumor tissue of U87MG and GL261 glioma brain tumors. Mouse IgG served as a negative control for non-specific antibody staining (IgG control). Scale bar = 20 μm. b Representative western blot and quantification shows ~1.4-fold increased expression of transferrin receptor in U87MG compared to GL261 cells. Data presented as mean ± SEM of three separate experiments. c Immunofluorescence staining demonstrates time-dependent intracellular uptake of Tf-NPs but not PEG 2K -Cy5.5 (PEG-NP) liposomes in U87MG and GL261 cells in vitro. Tf-NPs co-localize to late endosomal/lysosomal compartments (LAMP-1). Nuclei were visualized using DAPI counterstain (DAPI). Scale bar = 10 μm. d Flow cytometry plots and quantification of cellular PEG-NP or Tf-NP signal in U87MG and GL261 cells. Data presented as mean ± SEM of three separate experiments. Statistical analysis performed using Student’s t-test (***p < 0.001). e Multiphoton images of PEG-NP or Tf-NPs (red) at the site of GFP-expressing U87MG and GL261 intracranial gliomas (green). Scale bars = 6.25 μm or 12.5 μm Full size image

We then assessed the ability of Tf-NPs to achieve delivery to brain tumors in vivo. Tumors were allowed to grow for 14 days post implantation. Tumor-bearing mice were treated with an I.V. injection of Tf-NPs on day 14 post-tumor induction and their tumors subjected to multiphoton imaging 7 days following I.V. injection (tumor induction day 21). Multiphoton imaging demonstrated accumulation and uptake of Tf-NPs on the surface of U87MG (Fig. 2e, upper right panels & Supplementary Movie 2) and GL261 tumors (Fig. 2e, lower right panels & Supplementary Movie 3), demonstrating the ability of Tf-NPs to achieve accumulation and retention on the surface of intracranial gliomas. This was in stark contrast to non-functionalized NPs (PEG-NPs) which failed to demonstrate accumulation or uptake by U87MG and GL261 tumors (Fig. 2e, left panels), suggesting that transferrin functionalization is required for transport across the BBB and delivery of NPs to glioma-bearing mice.

TMZ and bromodomain inhibitor therapy is additive in gliomas

Current gold standard of care treatment for gliomas in humans includes the DNA damage-inducing alkylating agent TMZ5. We recently published the novel observation that inhibition of BET bromodomain proteins with the potent small molecule inhibitor JQ1 regulates the DNA damage response in multiple cancer cell lines in vitro, including U87MG cells16, and others have also reported antitumor effects following BET bromodomain inhibition in mouse models of glioma17. As gliomas show rapid resistance to TMZ18, we hypothesized that the addition of a bromodomain inhibitor may further sensitize gliomas to TMZ therapy. To demonstrate sensitivity of the glioma cell lines to bromodomain inhibition, we first treated U87MG and GL261 cells with 500 nM JQ1 for 48 h and observed increased γH2AX DNA damage foci formation in cells (Fig. 3a). DNA damage foci were also seen in cells treated with 150 μM TMZ, with additive effects when cells were treated with both JQ1 and TMZ (Fig. 3a).

Fig. 3 The bromodomain inhibitor JQ1 and temozolomide have additive effects in U87MG and GL261 glioma cells. a Representative immunofluorescence images of γH2AX DNA damage foci in U87MG and GL261 cells treated with 500 nM JQ1 and/or 150 μM TMZ for 72 h. Scale bar = 10 μm. b Cell viability plots demonstrating combinatorial effects of JQ1 and TMZ. Combinatorial index (C.I.) values determined using the Chou–Talalay method. Data presented as mean ± SEM of three separate experiments Full size image

To further explore the combinatorial effects of TMZ and JQ1, we performed conventional IC 50 analyses in U87MG and GL261 cells and calculated combinatorial indexes using these two drugs. Cells treated with incremental log-fold increases in JQ1 in the presence of 150 μM TMZ demonstrated decreased viability quantified using the CellTiter-Glo® viability assay (Fig. 3b, red lines) compared to cells treated with log-fold increases of JQ1 alone (Fig. 3b, blue line). Calculation of JQ1 and TMZ combinatorial index (C.I.) values using the Chou–Talalay method19 generated C.I. values of 0.95 and 0.94 for U87MG and GL261 cells, respectively, suggesting that the combination of JQ1 and TMZ achieved additive cytotoxic effects. This data was in line with our observation that single agent JQ1 or TMZ treatment was able to elicit DNA damage in glioma cells, and that the combination of JQ1 and TMZ increased DNA damage over either drug alone (Fig. 3a).

Tf-NP encapsulated drugs have superior therapeutic efficacy

We next interrogated the therapeutic efficacy of drug-loaded NPs compared to free drug dosing in our mouse models of glioma. Despite TMZ and JQ1 both having good penetration across the BBB20, 21, only 20% of total serum drug levels of TMZ are achieved in the CSF at best20 (a paucity of data regarding the neuropharmacology for JQ1 exists), thus we hypothesized that the ability to package both drugs in NPs for targeted delivery to the site of the tumor would combat CSF washout effects and increase treatment efficacy. To test this, we first packaged TMZ and JQ1 into NPs (Fig. 4a, schematic) and characterized the kinetics of drug release from the NPs. Single or dual drug-loaded NPs were incubated in float-a-lyzer® devices with a 100,000 MW cut-off at 37 °C in normal saline with agitation. Samples were removed from the devices at multiple time points over a course of 72 h and analyzed using high-performance liquid chromatography to quantify drug release from the NPs over time. NPs loaded with JQ1 demonstrated ~90% release of drug by 24 h (Fig. 4b) while NPs loaded with TMZ demonstrated an attenuated release profile with ~90% release by 48 h (Fig. 4c). Dual drug-loaded NPs demonstrated similar release kinetics with relatively more rapid release of JQ1 compared to TMZ and complete release of both drugs by 72 h (Fig. 4d).

Fig. 4 Characterization of drug release from JQ1 and TMZ liposomes. a Schematic of liposomes loaded with JQ1 and TMZ. Kinetics of drug release from liposomes loaded with b JQ1 alone, c TMZ alone, or d both JQ1 and TMZ. Data presented as mean ± SEM of three separate experiments Full size image

Tumor-bearing mice were then treated daily with I.V. injections of vehicle, JQ1, TMZ, or JQ1 + TMZ in free drug form, or drugs packaged in either non-functionalized PEG-NPs or Tf-NPs at equivalent doses of 2 mg kg−1 per drug for 5 days and tumor signal evaluated using luciferase bioluminescence. Both U87MG (Fig. 5a) and GL261 (Fig. 5b) tumors showed decreases in tumor signal following 5 days of free JQ1 or TMZ compared to vehicle-treated mice, with additive effects when both drugs were combined. Mice treated with drug-loaded PEG-NPs demonstrated similar decreases in tumor signal compared to mice treated with free-drug, however, mice treated with equivalent doses of drugs packaged in Tf-NPs experienced slower longitudinal tumor growth quantified using bioluminescence imaging compared to mice treated with free drug or PEG-NP drug regimens across all treatment arms (Fig. 5c, d). U87MG mice treated with Tf-NP encapsulated JQ1 and TMZ demonstrated a 99.1% decrease in tumor signal compared to an 82% and 79% decrease in signal when treated with free JQ1 and TMZ or dual drug-loaded PEG-NPs, respectively, after 7 days of treatment (Table 2). Similarly, GL261 mice treated with Tf-NP encapsulated combination therapies demonstrated a 99.3% decrease in tumor signal compared to a 97% and 96% decrease in signal in mice treated with either free drug combinations or dual drug-loaded PEG-NPs, respectively (Table 2). These results suggest that functionalization of drug-loaded NPs with transferrin allows for targeted delivery of drugs across the BBB to tumor cells in vivo to achieve significant reductions in tumor burden compared to untargeted drug-loaded NPs, which only achieve reductions in tumor burden similar to free drug regimens.

Fig. 5 Tf-NPs loaded with JQ1 and TMZ have superior pharmacodynamic effects in intracranial orthotopic models of glioblastoma. Representative bioluminescent images of a U87MG and b GL261 mice taken on day 0 and day 5 following initiation of treatment with free drug formulations (free drug), drugs loaded in PEG-Cy5.5 liposomes (PEG-NP drug), or transferrin-PEG-Cy5.5 liposomes (Tf-NP drug). Quantification of average tumor bioluminescence values in the different treatment arms throughout the course of treatment for c U87MG and d GL261 mice. Kaplan–Meier survival plots of e U87MG and f GL261 glioma mice in the different treatment arms. Study powered with eight mice per treatment arm for statistical significance. Log-rank (Mantel–Cox) test performed on survival plots. Student t-test used to quantify differences in treatment arms (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) Full size image

Table 2 Relative fold change in tumor bioluminescence over the course of drug treatment Full size table

Finally, treatment with Tf-NP therapies translated into significantly prolonged survival of U87MG (Fig. 5e; solid lines) and GL261 (Fig. 5f; solid lines) mice compared to mice treated with either free drug (Fig. 5e, f; dashed lines) or drug loaded in PEG-NPs (Fig. 5e, f; dotted lines). These results suggest that the ability of Tf-NPs to achieve receptor-mediated transcytosis across the BBB and accumulate at the site of glioma tumors (Fig. 2e) allows for superior delivery of drugs into tumor cells, compared to free drugs or drugs encapsulated in non-functionalized NPs.

Tf-NP therapies have increased pharmacodynamic effects in vivo

To test the hypothesis that Tf-NPs achieve increased delivery of drugs to tumors compared to free drug regimens, we performed IHC staining of brain sections of U87MG and GL261 mice to quantify the amount of DNA damage and apoptosis in tumors between the different treatment arms. U87MG and GL261 mice were sacrificed 1 week following treatment initiation and brains were sectioned and stained for markers of DNA damage (γH2AX), apoptosis (cleaved caspase 3), and proliferation (Ki-67). Mice treated with drug-loaded Tf-NPs had brain tumors that showed increased staining for γH2AX and CC3 compared to mice that received free drug (Fig. 6 & Supplementary Fig. 2). These increases in markers of DNA damage and apoptosis corresponded with decreased numbers of cells that stained positive for the proliferative marker Ki-67 in both U87MG and GL261 tumors (Fig. 6 & Supplementary Fig. 2), suggesting that our observed binding of Tf-NPs on the surface of tumors led to effective release of drugs into the parenchyma of U87MG and GL261 tumors. Furthermore, tumors from U87MG and GL261 mice treated with dual drug-loaded Tf-NPs showed significantly increased DNA damage and apoptosis with decreased proliferation compared to mice treated with single drug-loaded Tf-NPs (Fig. 6 & Supplementary Fig. 2). These results demonstrate the superior efficacy of combined TMZ and bromodomain inhibitor therapy when packaged in functionalized dual drug-loaded NPs in achieving tumor control and survival in mice with gliomas compared to treatment with equivalent combination free drug dosing.

Fig. 6 U87MG and GL261 mice treated with dual drug-loaded Tf-NPs demonstrate increased DNA damage and apoptosis in tumors. Quantification of number of tumor cells that stained positive for markers of DNA damage (γH2AX), apoptosis (CC3), and proliferation (Ki67) in a U87MG and b GL261 mice that received free drug vs liposome-loaded drug (Tf-NP). Signal intensity was quantified from >300 cells in tumors of three mice per treatment condition using ImageJ. Data presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Full size image

NP therapies protect from systemic drug toxicity

Finally, we hypothesized that the protective PEGylated packaging and imbued stealth capability of the NPs would offer relative shielding from the known systemic toxicities of JQ122 and TMZ23. To test this, we performed serial daily blood monitoring of red blood cell (RBC), white blood cell (WBC), and platelet (PLT) levels in immunocompetent BL6 mice throughout the treatment period comparing values between mice treated with free drug versus Tf-NP-loaded drug. Mice treated with free JQ1, TMZ, or JQ1 + TMZ had progressive leukopenia (Fig. 7a) and thrombocytopenia (Fig. 7b) over the 5-day course of treatment. In contrast, mice treated with drug-loaded Tf-NPs had significantly more stable WBC and PLT levels over the treatment course, suggesting that the PEGylated exterior of the NPs protected the mice from drug toxicity effects. There were no significant changes in the red blood cell counts in either treatment arms (Fig. 7c). Furthermore, mice treated with drug-loaded Tf-NPs maintained their body weight and body conditioning scores compared to mice treated with free drug, which demonstrated significant weight loss and poor body conditioning (Fig. 7d). Although we did not perform liver function tests, we extrapolated that the lack of weight loss and maintained body conditioning in mice treated with drug-loaded NPs was an indication of normal liver function despite the NPs having a high degree of tropism for the liver as shown in our biodistribution studies. This data supports the ability of these PEGylated NPs to compartmentalize drug and retain it in the circulatory system in stealth mode while achieving targeted therapeutic effects in the brain.