Drug Delivery and Translational Research

, Volume 5, Issue 2, pp 116–124

Evans blue nanocarriers visually demarcate margins of invasive gliomas

Authors

  • Benjamin T. Roller
    • Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory School of Medicine
  • Jennifer M. Munson
    • Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory School of Medicine
  • Barunashish Brahma
    • Department of NeurosurgeryChildren’s Healthcare of Atlanta, and Emory University School of Medicine
    • Department of NeurosurgeryEmory University School of Medicine
  • Philip J. Santangelo
    • Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory School of Medicine
  • S. Balakrishna Pai
    • Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory School of Medicine
    • Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory School of Medicine
    • Wallace H Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory School of Medicine
Research Article

DOI: 10.1007/s13346-013-0139-x

Cite this article as:
Roller, B.T., Munson, J.M., Brahma, B. et al. Drug Deliv. and Transl. Res. (2015) 5: 116. doi:10.1007/s13346-013-0139-x

Abstract

Aggressive surgical resection is the primary therapy for glioma. However, aggressive resection may compromise functional healthy brain tissue. Currently, there are no objective cues for surgeons to distinguish healthy tissue from tumor and determine tumor borders; surgeons skillfully rely on subjective means such as tactile feedback. This often results in incomplete resection and recurrence. The objective of the present study was to design, develop, and evaluate, in vitro and in vivo, a nanoencapsulated visible dye for intraoperative, visual delineation of tumor margins in an invasive tumor model. Liposomal nanocarriers containing Evans blue dye (nano-EB) were developed, characterized, and tested for safety in vitro and in vivo. 3RT1RT2A glioma cells were implanted into brains of Fischer 344 rats. Nano-EB or EB solution was injected via tail vein into tumor-bearing animals. To assess tumor staining, tissue samples were analyzed visibly and using fluorescence microscopy. Area, perimeter ratios, and Manders overlap coefficients were calculated to quantify extent of staining. Nano-EB clearly marked tumor margins in the invasive tumor model. Area ratio of nano-EB staining to tumor was 0.89 ± 0.05, perimeter ratio was 0.94 ± 0.04, Manders R was 0.51 ± 0.08, and M1 was 0.97 ± 0.06. Microscopic tumor border inspection under high magnification verified that nano-EB did not stain healthy tissue. Nano-EB clearly aids in distinguishing tumor tissue from healthy tissue in an invasive tumor model, while injection of unencapsulated EB results in false identification of healthy tissue as tumor due to diffusion of dye from the tumor into healthy tissue.

Keywords

NanocarriersLiposomesTumor marginTumor borderIntraoperative MRITumor resection

Introduction

Currently, aggressive surgical resection is used to extend survival for patients suffering from high-grade glioma [15]. Even with the currently available treatment options, prognosis is poor, resulting in a mean survival time as low as 11 to 14 months [6]. However, patient life expectancy increases with an increase in extent of resection [6]. Concomitantly, minimizing removal of healthy neural tissue is critical to ensure quality of life. As a consequence, total and complete surgical resection of tumors is rare, and a vast majority of brain tumors recur, often in close proximity to the resection site [7, 8].

A major impediment to achieving greater accuracy in tumor resection stems from the absence of clear visual cues demarcating the tumor margin. Therefore, neurosurgeons currently rely on subjective criteria, such as tactile feedback, to assess whether a particular tissue is tumor or healthy. Presurgical magnetic resonance imaging (MRI) or computed tomography (CT) can provide relevant information to plan the surgical procedure, but does not provide real-time information during resection. Intraoperative histology can provide surgeons with information about the margins, but this method is time consuming. Further, by this methodology, assessment of the nature of the tissue (healthy vs. tumor) can be made only after its removal from the patient. Intraoperative CT or MRI are possible alternatives, providing non-invasive information about the extent of residual tumor, but are cost limited and special training is often needed, making these techniques available only in more advanced surgical environments. Use of fluorescent probes that stain tumors require special equipment for excitation and detection, as well as low light conditions, neither of which are optimal in a surgical environment [913]. Thus, a method to demarcate tumor margins without a significant change to surgical conditions is desired.

Ozawa et al. and Orringer et al. have suggested the use of a small molecule visible dye to address this challenge [14, 15]. This is an elegantly simple solution, however, the demarcation is temporally transient and the margin blurs, necessitating multiple dye injections during the course of the surgical procedure. More importantly, the technique stains healthy tissue in invasive tumors, where a strong need for demarcation of tumor exists. A non-invasive tumor such as the 9 L glioma used by Ozawa and others merely “pushes” healthy tissue out of the way as it grows so the tumor margin is already fairly distinguished; the tumor extracellular space and vasculature do not interact with the healthy tissue [16]. Clearly, this is not the case with most invasive tumors; diffusion through extracellular space would cause staining of healthy tissue, as evident from injections of Evans blue (EB) dye in sterile saline. Here we test the hypothesis that a nanoencapsulated dye, due to size-dependent diffusion limitations, would clearly demarcate tumor margins after extravasation via the enhanced permeation and retention effect, even in an invasive cancer model.

In previous studies, our lab has shown that a nanoencapsulated X-ray contrast agent will passively but selectively accumulate at the periphery of the tumor [17]. Due to their relatively large size (∼100–200 nm), these nanoparticles mark the margin stably without diffusing away, moving no more than 50 μm from the vessel from which they exited [18]. Further, liposomal nanocarriers are FDA approved for use in cancer therapy for delivery of chemotherapeutics, such as Doxil®, yielding predictable circulation times and biodistribution, as well as reducing systemic toxicity [1922]. Here, we have developed a liposomally nanoencapsulated Evans Blue dye (nano-EB) to provide accurate visual cues for the surgeon to intraoperatively delineate the tumor margins without the need for MRI, fluorescence, or other equipment. Demarcation of the tumor margin will allow more accurate and complete removal of tumor tissue, conceivably leading to extended survival times for patients.

Materials and methods

Choosing a dye using perceived luminance

Dyes were evaluated for perceived luminance, which estimates the darkness of a color. Dyes were dissolved in phosphate-buffered saline (PBS) at the same concentration (m/v), dilute enough for all dyes to remain translucent when contained in a 1.5-mL microcentrifuge tube. An image was taken of all the dyes together to ensure consistent lighting conditions. RGB values were measured in three places for each dye using the color picker tool in Photoshop (Adobe, San Jose, CA) and averaged. Perceived luminance was calculated using the following equation: 0.299*R + 0.587*G + 0.114*B [23].

Preparation of liposomal nanocarriers

Liposomal nanocarriers were prepared using a mixture of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Genzyme, Cambridge, MA), cholesterol (Sigma-Aldrich, St Louis, MO), and N-(carbonyl-methoxypolyethyleneglycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG-2000-DSPE) (Genzyme) in a 55:40:5 molar ratio dissolved in ethanol. Nano-EB particles were made by hydrating the lipid mixture using 75 mg/ml of Evans blue (Alfa Aesar, Ward Hill, MA) in PBS while blank liposomes were made by hydrating with PBS alone. The solution was stirred at 60 °C for 120 min then dialyzed overnight against iso-osmolar phosphate-buffered saline to remove ethanol. The liposomes were resized by placing the suspension in a Branson 1510 bath sonicator (Branson Ultrasonics, Danbury, CT) for 120 min at 60 °C. Nano-EB particles were then run through a Sepharose CL-4B (GE Healthcare, Pittsburgh, PA) size exclusion column chromatography to remove excess, unencapsulated dye. The filtrate was run through a 500 kD MWCO MicroKros diafiltration cartridge (Spectrum Laboratories, Rancho Dominguez, CA) to remove volume gained during chromatography.

Liposomal nanocarrier analysis and characterization

Liposomal nanocarriers were analyzed to obtain size distribution and lipid to dye ratio, as well as for stability in conditions similar to those encountered in vivo. Size distribution of the liposomal nanocarriers was determined using a Zetasizer Nano ZS90 dynamic light scattering particle sizer (Malvern Instruments Ltd, Worcestershire, UK). By using the same instrumentation, the zeta potential of the nano-EB was assessed. The particles were suspended in PBS and three estimations were made. Mean values ± SD were calculated from the values obtained.

For morphological characterization of nano-EB, the liposomes were frozen at −20 °C and then freeze dried using a Lacbconoco FreeZone® freeze dryer. The particles were then processed for scanning electron microscopy (SEM) and images obtained using a Zeiss Ultra-60 Field Emission Scanning Electron Microscope (FE-SEM).

Lipid to dye ratio is presented as amount of dye per unit of lipid (mg/mg). First, the amount of encapsulated dye in the liposome solution was determined by measurement of absorbance of EB. Liposomes were lysed in 0.8 % Triton X100 to release their contents and absorbance was measured at 610 nm using a Synergy HT Plate Reader (BIO-TEK, Winooski, VT) and compared to a standard curve.

Final lipid concentration in the liposomal nanocarrier suspension was obtained by quantifying the fluorescence of a reporter molecule included in the original liposome mixture. β-DPH-HPC (Invitrogen, Carlsbad, CA) was included in the liposome mixture at a concentration of 0.01 mol%. After liposome preparation was complete, lipid and dye in a 100-μL sample were separated via Folch extraction [24]. Dried lipid extract was resuspended in ethanol and fluorescence was measured in a Synergy HT Plate Reader (excitation 360 nm; emission 460 nm). A standard curve of known concentrations of β-DPH-HPC was used to calculate the amount of lipid present in the mixture.

Liposomal nanocarrier stability measurement was adapted from methods used for stability of doxorubicin-containing liposomes [25]. Briefly, liposomal nanocarriers were placed in 50 % fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA) in PBS solution to imitate blood serum. Nanocarriers were present at a concentration low enough to eliminate self quenching of the dye. The solution was placed in a 37 °C water bath and samples were taken at 0, 24, and 48 h. Leak of dye from the nanocarriers was assessed by measuring fluorescence of the samples at excitation 620 and emission 680 nm. Percent leak was calculated as follows:
$$ {{{\%\ \mathrm{leak} = \left[ {{{{\left( {\mathrm{sample\;intensity}} \right)}}_T}\text{-}\ {{{\left( {\mathrm{sample\;intensity}} \right)}}_{T=0 }}} \right]}} \left/ {{\left[ {{{{\left( {\mathrm{sample\;intensity}} \right)}}_{\mathrm{lysed}}}\text{-}\ {{{\left( {\mathrm{sample\;intensity}} \right)}}_{T=0 }}} \right]}} \right.} $$
where T is the time point, T = 0 is the initial time point (representing zero leak), and lysed indicates a sample lysed using 0.8 % Triton X-100 (Sigma-Aldrich, St Louis, MO) representing 100 % leak.

Cell culture

3RT1RT2A (rat glioma cell line, stably expressing green fluorescent protein, GFP) was generously donated by Helen L. Fillmore (Virginia Commonwealth University, Richmond, VA). Cells were cultured in Dulbecco’s Modification of Eagle’s Medium with 4.5 g/L glucose without L-glutamine and sodium pyruvate (Mediatech, Manassas, VA), supplemented with 10 % FBS, 1 % penicillin/streptomycin (Mediatech), 1 % non-essential amino acids (Mediatech), 1 % L-glutamine (HyClone Thermo Scientific, Logan, UT), and 1 mg/ml G418 (Gemini Bio-Products) as selective pressure for the GFP marker. Primary cortical rat astrocytes were obtained and cultured as described previously [26].

In vitro cell viability

Primary astrocytes, representing normal brain tissue, and 3RT1RT2A cells, representing aggressive glioblastoma, were used to assess viability of cells treated with nano-EB in vivo. Astrocytes were plated at 10,000 cells per well, while 3RT1RT2A cells were plated at 25,000 cells/well, in tissue culture-treated 24-well plates. Cells were treated for 24 h with serum-free media containing either nano-EB, unencapsulated EB in saline, blank liposomes, or sterile saline as a control. EB dose was 0.2 mg/ml in both nano-EB and unencapsulated EB solutions; the dose was chosen under the assumption that a maximum of 2 % of the liposomes delivered to an animal would reach the tumor based on previous work in the lab with similar liposomal nanocarriers [22]. The lipid dose was equal in the two liposomal groups. After 24 h, the treatments were removed and fresh appropriate standard serum containing cell culture medium was added. Cell viability counts were taken at 0, 24, 48, and 72 h using the CCK-8 counting kit (Dojindo, Rockville, MD) as per the manufacturer’s instructions. Cell counts were normalized to the untreated groups at each time point and reported as percent viable cells. Statistical significance was determined using a two-way ANOVA with Tukey’s post test using GraphPad Prism (Graphpad, La Jolla, CA). Data is presented as average ± standard deviation.

Ethical use of animals

All animal protocols were approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee. Fisher 344 male rats 8–10 weeks of age were purchased from Harlan Laboratories. Animals were housed in a temperature-controlled environment with a 12-h light/dark cycle, and were provided food and water for the entire duration of their housing.

In vivo toxicity testing

Non-tumor-bearing male Fisher 344 rats were injected via tail vein with sterile solutions of nano-EB, blank liposomes, or unencapsulated EB dye dissolved in saline. To ensure safety and sterility, all solutions were analyzed for correct pH and osmolarity, as well as tested for endotoxins (Lonza, Walkersville, MD), then filtered through a 0.2-μm sterile filter. Dye dose was 25 mg/kg in the two Evans blue formulations and lipid dose was kept equal in the two liposomal treatment groups. Total body weights and overall condition of the animals were monitored over a period of 4 weeks.

Tumor implantation

Tumor implantation was adapted from Fillmore and colleagues [27]. Rats were anesthetized initially using 5 % isofluorane and maintained at 2–3 % during the entire procedure. Briefly, animals were placed in a stereotactic frame, an incision was made to expose the skull, and a burr hole was made at the coordinates of 2 mm anterior and 2 mm left lateral from lambda. 3RT1RT2A GFP expressing rat glioma cells were slowly injected 3 mm below the surface of the skull at this position over the course of 3 min (200,000 cells in 10 μL Liebovitz L-15 medium (Invitrogen Life Technologies/GIBCO, Carlsbad, CA)). Tumors were present in all animals that were injected in this way. Tumor-implanted animals typically died by day 12–14; for this reason, animal experiments were constrained to 10 days.

Tumor staining in vivo

On day 8 of tumor growth, animals were injected via tail vein with sterile, non-pyrogenic solutions of 40 mg/kg EB, either as nano-EB or EB in sterile saline solution. Animals were euthanized 48 h later and brains were removed and preserved in a 4 % paraformaldehyde solution.

Visual and histological analysis of tumor staining

Brains of animals were removed and preserved in 4 % paraformaldehyde in PBS. Brains were sliced coronally through the tumor and tumors were visually inspected to confirm presence of blue staining. Tissue was then cryoprotected by submersion in a 30 % w/v solution of sucrose in PBS. Sections of 16 μm thickness were obtained using a Leica CM 300 cryostat (Leica, Bannockburn, IL). Sections were stained with anti-GFP monoclonal antibody conjugated with Alexa Fluor 488 (Invitrogen) to enhance green fluorescence and DAPI (Invitrogen) to indicate nuclei. Microscopic images of the entire tumor (×5 magnification) were obtained and tiled together using an Axiovert 200 M microscope (Zeiss, Thornwood, NY) with a ORCA-ER camera (Hamamatsu Photonics, Bridgewater, NJ), Sedat-ET filter set (Chroma Technology Corp., Bellows Falls, VT), and Volocity acquisition software (PerkinElmer, Walthamm, MA).

Images were imported to ImageJ (NIH) where background subtraction was performed using the included rolling ball background subtraction algorithm. Images were converted to 8-bit and Manders Coefficients plugin was used to find Manders colocalization R and M1 (red/green, indicates how much red staining is also green) [28]. Additionally, the 8-bit images were used to trace the tumor border as well as the EB staining border in the nano-EB-treated samples. The measure function was used to obtain area and perimeter of the tracing. A ratio of red area to green area and red perimeter to green perimeter were calculated. Data is presented as average ± standard deviation.

Higher magnification fluorescence images (×10 magnification) of the tumor border were taken at 0°, 90°, 180°, and 270° around the border of the tumor to visually confirm that there was no healthy tissue staining.

Results

Liposomal nanocarrier fabrication and characterization

For nanocarrier fabrication, a visible dye that would produce a high contrast to inherent brain tissue color was desired. Color wheel analysis showed that, other than black, blue and dark green had the potential for highest contrast to tissue encountered during surgery, including red (blood) and yellow/grey (healthy brain tissue). At identical concentrations, EB was visibly darker than any of the other dyes considered (Fig. 1a). This was confirmed by calculating perceived luminance of each dye, where a lower perceived luminance correlates with a darker color (values superimposed on Fig. 1a). EB had a perceived luminance value of 27, approximately 21 % lower than the next darkest dye, Coomassie blue. Further, EB was more soluble than any of the other dyes, being seven times more soluble than the next most soluble dye (EB 280 g/L vs. methylene blue 40 g/L). The liposomal nanocarrier core is aqueous, so high solubility in water is desirable for maximum loading of dye.
https://static-content.springer.com/image/art%3A10.1007%2Fs13346-013-0139-x/MediaObjects/13346_2013_139_Fig1_HTML.gif
Fig. 1

a Candidate dyes in solution at equal concentrations (m/v) and physiological pH. From left to right: Evans Blue (chosen for use in our experiments), Coomassie blue, methylene blue, bromophenol blue, and lissamine green. Perceived luminance values are provided above each dye. Lower luminance correlates with darker appearance. b SEM images of the nano-EB liposomes

Nano-EB particles were developed and characterized, assessing size, drug to lipid ratio, and stability. After processing was complete, the ratio of dye to lipid was 0.08 mg EB to 1 mg lipid. Average hydrodynamic diameter of nanocarriers was 173 nm with a polydispersity index of 0.1. The morphological characteristics of the nano-EB particles as characterized by SEM are depicted in Fig. 1b. Nano-EB particles had a zeta potential of −8.84 ± 0.17. Nano-EB particles were stable at body temperature (37 °C), leaking only 13.3 % (SD = 0.49 %) of their contents over a 48-h period.

In vitro and in vivo toxicity assessment

To ensure nano-EB had no significant cytotoxic effects against healthy cells, it was tested against both a glioma cell line and non-malignant primary astrocytes. Nano-EB treatment resulted in no significant changes in cell viability compared to blank stealth liposomes in either the 3RT1RT2A (Fig. 2a) or the healthy primary astrocytes (Fig. 2b).
https://static-content.springer.com/image/art%3A10.1007%2Fs13346-013-0139-x/MediaObjects/13346_2013_139_Fig2_HTML.gif
Fig. 2

Cell viability of a 3RT1RT2A cells and b primary astrocytes, after treatment with either 0.2 mg/ml nano-EB or EB, or blank liposomes. Lipid dose of blank liposomes matched that of the nanoencapsulated Evans blue. All values normalized to PBS-treated control

Non-tumor-bearing animals were injected with either nano-EB, EB in saline, or blank liposomes and body weight and general health of the animals were monitored for 4 weeks. All animals consistently gained weight after injection (Fig. 3) and showed no visible signs of distress over the monitoring period. Animals injected with nano-EB remained slightly tinted blue for an average of 2 weeks post injection, while those injected with unencapsulated EB remained tinted blue for the entire span of the experiment. During the testing period, no discernible signs of toxicities were observed. The lack of toxicity is consistent with the earlier published reports [29, 30].
https://static-content.springer.com/image/art%3A10.1007%2Fs13346-013-0139-x/MediaObjects/13346_2013_139_Fig3_HTML.gif
Fig. 3

In vivo animal growth after treatment. Non-tumor-bearing animals were injected with either nano-EB (a), unencapsulated EB (b), or blank liposomes (c). Evans blue dose was equal in both EB treatments, lipid dose was equal in both liposomal treatments. Lines represent data on individual animal body weight in each group (N = 3)

Tumor staining/visualization in vivo

Brains were sliced coronally through the center of the tumor to macroscopically show the extent of tumor staining evident to the naked eye (Fig. 4a). Brains of animals given unencapsulated EB showed diffuse staining in and around the tumor, with dye diffusing from the tumor into the healthy tissue (Fig. 4b). It should be noted that EB does not cross an intact blood–brain barrier. Brains of animals given nano-EB only had staining in the region of the tumor (Fig. 4a).
https://static-content.springer.com/image/art%3A10.1007%2Fs13346-013-0139-x/MediaObjects/13346_2013_139_Fig4_HTML.gif
Fig. 4

Coronal slice of brains of tumor-bearing animals treated with nano-EB (a) or EB (b). Scale bars = 1 mm

Besides the visible blue color, EB also exhibits fluorescence in the deep red wavelengths similar to the organic fluorophore Cy5, making high magnification microscopic analysis possible. Fluorescence microscopy was performed to confirm that nano-EB did not stain healthy tissue (Fig. 5). Manders coefficient analysis was performed on tiled whole tumor images, comparing green fluorescence from GFP expressing tumor cells with red fluorescence of the nano-EB (results in Table 1). Average overall Manders R was found to be 0.51 ± 0.08 (n = 3 animals, three slices per animal). M1 (red/green) was found to be 0.97 ± 0.06, indicating that 97 % of the red fluorescence co-localized with green fluorescence.
https://static-content.springer.com/image/art%3A10.1007%2Fs13346-013-0139-x/MediaObjects/13346_2013_139_Fig5_HTML.gif
Fig. 5

Microscopic analysis of staining. Whole tumor stitch, ×5. a Tumor of nano-EB-treated animal. Green indicates tumor cells (GFP). Red indicates EB. Arrows indicate invasive clusters of cells marked by nano-EB. b Tumor of free EB-treated animal. Scale bars = 500 μm

Table 1

Border quantification of tumor-bearing animals treated with nano-EB

Area ratioa

Perimeter ratiob

Manders Rc

M1 (red/green)d

0.89 ± 0.05

0.94 ± 0.04

0.51 ± 0.08

0.97 ± 0.06

N = 3 animals, three tissue sections per animal. Data presented as average ± standard deviation

aArea ratio = area red (nano-EB)/area green (3RT1RT2A glioma cells, GFP)

bPerimeter ratio = perimeter red/perimeter green

cManders R indicates the amount of colocalization of red and green throughout the tissue

dM1 indicates how much tissue stained red is also stained green

Further analysis compared the total area of the tumor to that of the nano-EB-stained tissue to get an indication of how much of the total tumor was stained. Ratio of red area to green area averaged 0.89 ± 0.05. The ratio of perimeter of the same slices was 0.94 ± 0.04. Numbers close to but not greater than 1 indicate that the nanocarrier staining of tissue always stayed within the tumor tissue. Lack of healthy tissue staining was confirmed by visually inspecting higher magnification (×10) images of the tumor border in nanocarrier-treated animals (Fig. 6).
https://static-content.springer.com/image/art%3A10.1007%2Fs13346-013-0139-x/MediaObjects/13346_2013_139_Fig6_HTML.gif
Fig. 6

Tumor border images of nano-EB-treated animals (×10 magnification). a Tumor (GFP), b nano-EB (red fluorescence). c Composite image. Scale bars = 100 μm

Discussion

Accurate, complete resection of tumor is essential for successful treatment of gliomas. Currently, during surgery, the surgeon has limited ability to distinguish the margin of the tumor, relying on subjective criteria such as tactile feedback and marginal, often indistinguishable, color differences between healthy and tumor tissue. Intraoperative MRI has been deployed, but it remains an expensive and therefore inaccessible tool in a majority of hospitals. Thus, it would be beneficial to have an objective means to distinguish the tumor margin visually for the neurosurgeons when performing the surgery. Other approaches use fluorescent molecules, but these require extra equipment and training, as well as altered lighting conditions, since the halogen lights typically used in surgical environments often emit radiation in the wavelengths that causes interference with fluorescence detection [913]. In response, non-fluorescent dyes have been evaluated, though these compounds are freely injected small molecules and were only examined in non-invasive tumor models to date, which may not adequately represent the use in the clinic [14, 15].

In this study, we demonstrate that freely circulating small molecule compounds, such as Evans blue dye, diffuse out of the tumor and into healthy tissue, complicating their ability to help demarcate the margins between tumor and healthy tissues. On the contrary, intravenous injection of liposomal nanocarriers containing Evans blue dye clearly marked the tumor and its margins, as well as small nodules of invasive cells that had invaded beyond the bulk tumor border. A Manders coefficient of 0.6 or higher is generally accepted as indicating true colocalization in fluorescence microscopy [31]. In this study, the Manders overlap coefficient average is slightly lower than ideal at 0.51 ± 0.08, but this was not unexpected. As Manders coefficients measure overlap, actually, intracellular green fluorescent protein does not overlap with extracellular liposomal Evans blue as little endocytosis of the nanocarriers occurs at 48 h.

Further, more importantly, in the overlap coefficient analysis, the value of M1 (red/green) was 0.97, indicating that nearly all area that was stained red was also stained green. This indicates that nanocarriers were localized specifically to tumor tissue. The area and perimeter ratio analysis further demonstrated that the nanocarriers only stained tumor tissue. Higher magnification border inspection again confirmed that only tumor tissue was stained, with nanocarriers slightly and consistently underestimating the true margin on the order of tens to hundreds of micrometers. This is sufficient, considering surgeons often remove tissue in sections with thicknesses on the order of millimeters, several orders of magnitude greater than the underestimation [32]. It is important to note that healthy tissue was not stained, preventing false positives and removal of healthy tissue by the surgeon which facilitates sparing the healthy tissue. This, in turn, has positive implications for maintaining function.

The amount of leak of dye from the liposomes over the 48-h period after injection is consistent with previously reported clinically used liposomal nanocarrier formulations [3335]. It should be noted that even with this slight leak, the nano-EB did not stain healthy tissue. Evans blue is a strongly polar molecule; leaked dye quickly binds to blood serum proteins, limiting its diffusion [36].

Evans blue dye was initially FDA approved for use in blood volume measurement. It was later found to be toxic in repeated use or large doses [29, 30]. However, the negative side effects of molecules are usually reduced and their pharmacokinetics are drastically altered when encapsulated in liposomal nanocarriers [1922].

In vitro, nano-EB did not have a negative effect on the viability of either the tumor cell line (3RT1RT2A) or its healthy counterpart, primary astrocytes. Intravenous injections of either nano-EB or EB had no observable negative effect on the growth of animals treated, nor did it cause any visible signs of distress in the animals. This was to be expected since the doses used were much lower than those seen as toxic in previous studies [29, 30]. Further, studies using other types of brain tumors are ongoing to test the utility and generality of this methodology for effective demarcation of tumors for aiding the surgical removal of brain cancers.

Conclusions

This study demonstrates that size-mediated diffusion limitation made possible by nanocarriers is critical to maintaining tumor staining without ‘leak’ of dye into normal brain tissue. This methodology thus allows specific staining of the tumor as well as reduces or eliminates false positive staining thereby preventing the removal of healthy tissue. Avoiding the unnecessary removal of healthy tissue allows for preservation of function and thus the quality of life of the patient after resection.

Funding

This work was funded by support from the Georgia Cancer Coalition and Ian’s Friends Foundation to RVB.

Conflict of interest

The authors have no financial interests relevant to the technologies or data published in this manuscript.

Copyright information

© Controlled Release Society 2013