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[Cancer Research 62, 6831-6836, December 1, 2002]
© 2002 American Association for Cancer Research

Advances in Brief

Cationic Charge Determines the Distribution of Liposomes between the Vascular and Extravascular Compartments of Tumors1

Robert B. Campbell2, Dai Fukumura, Edward B. Brown, Laureen M. Mazzola, Yotaro Izumi, Rakesh K. Jain, Vladimir P. Torchilin and Lance L. Munn3

Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts 02114 [R. B. C., D. F., E. B. B., Y. I., R. K. J., L. L. M.], and Department of Pharmaceutical Sciences, Boston, Massachusetts 02115 [L. M. M., V. P. T.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Tumor vessels possess unique physiological features that might be exploited for improving drug delivery. In the present study, we investigate the possibility of modifying polyethylene glycol-ylated liposome cationic charge of polyethylene glycol coated liposomes to optimize delivery to tumor vessels using biodistribution studies and intravital microscopy. The majority of liposomes accumulated in the liver, and increasing charge resulted in lower retention in the spleen and blood. Although overall tumor uptake was not affected by charge in the biodistribution studies, intravital microscopy showed that increasing the charge content from 10 to 50 mol % doubled the accumulation of liposomes in tumor vessels, suggesting a change in intratumor distribution; no significant effect of charge on interstitial accumulation could be detected, possibly attributable to spatial heterogeneity. Increased vascular accumulation of cationic liposomes was similar in two different tumor types and sites. Our results suggest that optimizing physicochemical properties of liposomes that exploit physiological features of tumors and control the intratumor distribution of these drug carriers should improve vascular-specific delivery.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Physiological barriers hinder the effective delivery of drugs to tumors (1 , 2) . To target cancer cells, a blood borne therapeutic agent must first cross the vasculature and then travel through the interstitium. Recent strategies have attempted to avoid these barriers by attacking the blood supply instead of the cancer cells, either by suppressing the formation of new vessels (antiangiogenic therapy) or by abolishing established vascular networks (antivascular therapy). This approach has the advantage that the delivery vehicle, once in the blood stream, should have direct access to the target endothelial cells. Recent studies have shown that cationic liposomes have a propensity for localizing in tumor vessels, but the mechanism behind this selectivity and the optimal formulation to maximize this effect have not been defined. We propose that optimizing physicochemical properties of liposome vehicles should improve their interactions with tumor vessels and, more generally, control their intratumor distribution. As a first step in identifying the optimal liposome formulation for improving accumulation in tumors, we investigated the influence of cationic charge on the distribution of liposomes in tumors using bio-distribution studies and intravital microscopy (3) . We also performed these studies in two tumor types and two implantation sites to examine the role of host–tumor interactions.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Liposome Preparation.
In general, liposomes were prepared as described previously (4) . PEG4 -modified cationic and electroneutral liposomes were prepared from DOPC, cholesterol, DOTAP, PEG-PE, and rhodamine-PE stock obtained from Avanti Polar Lipids (Alabaster, AL). Doxorubicin hydrochloride was obtained from Sigma (St. Louis, MO). Lipids were stored at -70°C under argon. Solvents were obtained from Fisher Scientific (Pittsburgh, PA). The molar ratio of phospholipid to cationic lipids used to make liposomes varied as a function of total net charge. Typically, when preparing PEGylated electroneutral (DOPC:Chol:PEG-distearoylphosphatidylethanolamine:label) and cationic (DOPC:DOTAP:Chol:PEG-distearoylphosphatidylethanolamine:label) liposomes, the components responsible for lending the desired physicochemical feature were added at the expense of DOPC. The fluorescent label concentration (1 mol %) and PEG content (5 mol %) were similar for all liposomes. Percentage of charge was 0, 10, 25, or 50 mol %. Large multilamellar vesicles were extruded 15 times through a 100-nm polycarbonate membrane using an Avanti Extruder (Avanti Polar Lipids) to produce smaller unilamellar vesicles. Liposome sizes were estimated with a Coulter N4 plus sub-micron particle sizer (Miami, FL). {zeta} potentials for PEGylated cationic liposomes were measured at 25°C in double distilled water using the {zeta}-PLUS {zeta} potential analyzer (Brookhaven Instruments, Holtsville, NY).

Animals and Tumors.
SCID mice were bred and maintained in our defined flora gnotobiotic animal facility (Massachusetts General Hospital, Boston, MA). The two tumor cell lines (LS174T and MCAIV) were maintained in Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum at 37°C in a humidified CO2 atmosphere. Tumors grown s.c. in 8–10-week-old SCID mice were resected aseptically, and all necrotic tissue was removed. The viable tumor was cut into 1-mm-size pieces. Mice were anesthetized with a mixture of ketamine (90 mg/kg body weight) and xylazine (9 mg/kg body weight). Tumor pieces were implanted in the s.c. space in the cranial or dorsal window chambers described previously (5) .

Biodistribution Studies.
Biodistribution studies were performed in tumor (LS174T human colon adenocarcinoma)-bearing SCID mice (~25 grams). Tumor pieces were implanted s.c. in mice. Mice were used for experimental purposes when the tumor was between 8 and 10 mm in size. Twenty-four hours postinjection, the mice were anesthetized and sacrificed only after blood had been removed with a Pasteur pipet via retro-orbital sinus. Percentage of recovery of radioactivity was measured in liver, spleen, kidneys, lungs, blood, and tumor by a Beckman Gamma 5500B counter (Fullerton, CA), and data are expressed as a percentage of injected dose accumulated per organ. Weight of mouse blood was assumed to be 7.3% of the body weight (6) .

Intravital Fluorescence Microscopy.
Anesthetized mice bearing dorsal and cranial windows were restrained on a custom-designed stage and observed with an intravital fluorescence microscope (Axioplan; Zeiss, Oberkochen, Germany). FITC-Dextran (10 mg/ml) was injected via tail vein to assess microvascular function before measuring interactions of liposomes with tumor vessels. Fluorescently labeled liposomes (0.2 ml) were injected to measure the accumulation of liposomes. Five random regions were selected in every tumor from the FITC channel, as follows: in the FITC channel, we centered the tumor in the FOV using a low-magnification (x1.25) lens. Once a field was selected, we then switched to the high-magnification lens without disturbing the sample on the stage; this was marked as FOV #1. The next four FOVs were taken in the same way using FOV #1 under low magnification as a point of reference, ultimately resulting in five FOVs, in a cross (+) pattern. To standardize focus, we focused on the uppermost vessel in the FOV.

Image Analysis.
Images from FITC and rhodamine channels were analyzed using a space-filling routine written in NIH Image. Square ROIs were placed on the image forming a grid, and only those ROIs that overlapped the vascular compartment were selected using a thresholding process. The fraction of ROIs containing at least one liposome was then calculated. This procedure was repeated for ROI sizes of 1–100 pixels. Fitting the resulting plot of percentage filled versus ROI size to an exponential function [Y = 1 - (1 - a) x exp (-b x x)] gives an indication of both the overall extent of liposome localization and uniformity of coverage. We also analyzed interstitial accumulation in the same image set by selecting boxes that did not overlap the vascular compartment.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Characterization of Liposomes.
We first determined liposome sizes and {zeta} potentials for each liposomal formulation before in vivo studies were performed. The size of our PEGylated cationic liposomes (0, 10, 25, and 50 mol % cationic lipid content) did not change as a function of composition; the mean diameter was ~150 nm (+/- 40) for all liposomes. The {zeta} potentials (mv) estimated for each were as follows: -20 mv (0% charge), 20 mv (10% charge), 24 mv (25% charge), and 31 mv (50% charge). A positive {zeta} potential should facilitate interactions with the negative charge of the vascular glycocalyx.

Effect of Cationic Lipid Content on the Biodistribution of Liposomes in Vivo.
We next investigated the effect of cationic lipid content on the whole body distribution of liposomes by injecting 111In-labeled liposomes in LS174T-bearing mice. Cationic lipid content ranged from 0 to 50 mol % (Fig. 1)Citation .



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  		Fig. 1. Whole body liposome distribution in mice. Mice were injected with 111In-DTPA-PE-labeled PEGylated liposomes varying in cationic lipid content (consisting of 0, 10, 25, or 50 mol %). No effect of charge content (0–50 mol %) on uptake of PEGylated liposomes by the liver was observed (A). Significant lowering in the uptake of labeled liposomes by spleen was observed (P < 0.01; n = 5) with increasing charge content (B). There was no effect of charge content on uptake by kidneys (C). PEGylated cationic liposomes (50%) were taken up by the lungs significantly (P < 0.01; n = 5) over other percentages of PEGylated cationic liposomes (D). PEGylated cationic liposomes (50%) circulated in blood for the shortest period compared with others (P < 0.01; n = 5; E). There was no effect on whole tumor uptake with increasing percentage of cationic lipid content (F). Data are mean ± SE. *, P < 0.05.

 
The most significant uptake was found in the liver regardless of composition (Fig. 1A)Citation . Increasing cationic charge content of PEGylated liposomes (0% compared with 10 mol %) lowered uptake by the spleen; increasing charge content from 10 to 50 mol % had no additional benefit (Fig. 1B)Citation . Total distribution in lungs increased significantly between the 25 and 50 mol % samples but was the same for other formulations (Fig. 1D)Citation . Increasing charge resulted in significantly lower retention in blood (Fig. 1E)Citation but did not affect uptake by the kidneys or tumor (Fig. 1, C and F)Citation .

Heterogeneous Accumulation of Cationic Liposomes in Vessels.
The biodistribution studies provide information at the organ level and indicate that the overall tumor uptake is not affected by cationic charge. We next tested the hypothesis that charge affects the partitioning between the vascular and extravascular compartments using intravital microscopy. The possibility of controlling liposome localization by modifying charge has direct implications for targeting specific cell populations within a tumor (e.g., endothelial versus cancer cells). We monitored delivery of cationic liposomes to the tumor using dorsal window chambers in SCID mice (Fig. 2)Citation . The data showed that liposomes containing 10 and 50 mol % of cationic lipid associated with 14 and 27%, respectively, (P < 0.05) of the vascular area (Fig. 2B)Citation . Percentage of charge did not alter interstitial localization significantly (Fig. 2CCitation ; P > 0.05).



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  		Fig. 2. Intratumor liposome distribution profiles. In A, data were acquired by intravital microscopy image analysis using a space-filling analysis and fit to the following formula: Y = 1 - (1 - a) x exp (-b x x). a indicates overall extent of vessels targeted, and b indicates uniformity of distribution. *, P < 0.05; **, P < 0.01; NS, not significant. Quantification of vascular and interstitial accumulation of liposomes. Liposomes were injected i.v. via lateral tail vein in SCID mice to observe tumor vascular (B) and interstitial (C) accumulation as a function of low (10 mol %) and high (50 mol %) charge content. Data are mean ± SD

 
Increasing charge content enhanced vascular coverage; cationic liposomes interacted more selectively with tumor vessels (compared with the electroneutral variety). To confirm that our observations are also valid in deeper regions of the tumor rather than just the surface vessels, we investigated cationic lipid-mediated vascular interactions deeper (>250 µm) in tumors using multiphoton laser scanning microscopy (7) . Fig. 3Citation shows four random areas within the same tumor (LS174T). Several tumor vessels were targeted considerably (noteworthy are the undetectable levels in the interstitial compartments). Uptake was heterogeneous, because some areas with many liposomes were in close proximity to vessels with few liposomes. In some areas, liposomes were clustered along vessels (Fig. 3D)Citation , occasionally accumulating at branch points adjoining tumor vessels together (Fig. 3D)Citation . Because the green label in Fig. 3Citation marks perfused vessels, it is evident that not all well-perfused vessels of tumors attract cationized liposomes.



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  		Fig. 3. Heterogeneous liposome accumulation in tumor vessels. A–D, Multiphoton microscopic images of tumor (LS174T) vessels are shown in green, and rhodamine-labeled PEGylated cationic liposomes (50% charge) are shown in red. The figure depicts four images of tumor vessels taken 24 h post-i.v. injection of liposomes. Tumor vessels were highlighted with FITC-dextran (2 million molecular weight). Arrows show areas of no liposome accumulation (A and B) and clusters (D) of liposomes, which often occur at branch points. Image size is 250 µm across.

 
Effect of Tumor Type and Microenvironment.
Previous studies have shown that both vascular permeability and interstitial diffusion can be influenced by the site of tumor growth (2 , 8) . Suspecting that the microenvironment may also influence liposome localization, we repeated our dorsal window studies in the CRW model. We found no significant differences between interactions in the CRW compared with the DSC for LS174T (Fig. 4A)Citation , indicating that the extent of tumor vessels targeted in the two environments was similar for this cell line.



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  		Fig. 4. Effect of microenvironment and tumor type on vascular accumulation of liposomes. PEGylated cationic liposomes (50% charge) were used to evaluate targeting in LS174T- and McaIV-bearing mice. In A, PEGylated cationic liposomes accumulated more in tumor vessels compared with normal (nontumor) vessels in the DSC (*P < 0.01; n = 5). No significant differences in the accumulation of these liposomes were observed between two tumors (LS174T versus MCaIV) grown in the same location (P > 0.05; n = 5) or when the same tumor (LS174T) was grown in different sites (DSC versus CRW; P > 0.05). Data are mean ± SD (B–D) fluorescence images of cationic liposomes (red) and blood vessels (green) in the LS174T grown in the CRW (B) or in the DSC (C) and MCaIV in the DSC (D). Asterisks in B and C indicate interstitial regions without detectable liposomes. Magnification: x20.

 
The interactions of tumor vessels with cationic liposomes were compared with vascular interactions in normal tissues in the DSC. The data revealed significantly lower accumulation of liposomes in normal s.c. tissue (P < 0.01; Fig. 4ACitation ).

We next investigated the effect of tumor type on liposome accumulation by comparing MCaIV with LS174T tumors in the DSC. The vascular area associated with liposomes (50% charge) was 28% (Fig. 4A)Citation , similar to LS174T in the dorsal chamber (27.5%), suggesting that the extent of vascular accumulation of cationic liposomes is similar in these two tumors.

In general, liposomes remained associated with the vessel wall and did not travel far into the interstitium (Fig. 4, B and C)Citation ; thus, cationic liposomes may be effective vehicles for delivering drugs to endothelial cells.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Because liposomes are effective drug delivery systems and their physicochemical features can be manipulated with relative ease (4) , tumor vessel-specific liposomes would be an effective strategy for delivery of antiangiogenic and antivascular drugs to tumor endothelial cells. It is known that physicochemical features, such as macromolecular size (1 , 3 , 8) and charge (9 , 10) , can regulate accumulation of carriers in tumors. For example, although cationic (positively charged) albumin (BSA) molecules readily extravasate through endothelial junctions (10) , cationic liposomes (larger in comparison) associate directly with the endothelium of tumor vessels (9 , 11) . Cationic liposomes are being used as gene vectors to successfully transfect a variety of mammalian cell types in vitro and in vivo; the ability to form desirable electrostatic interactions with DNA (12) makes them effective gene transfer vehicles and sets them apart from their anionic (negatively charged) counterparts. Additionally, cationic liposomes have been used to incorporate small hydrophobic drugs (13) , difficult to accomplish by other means, and hence are being considered for more broad clinical purposes (14) .

The vascular network is a highly accessible target for tumor therapy (15) , but to be successful, an antivascular strategy should target as many tumor vessels as possible. We have taken the first step toward optimizing liposome-based delivery to improve the next generation of targeting strategies. Although cationic lipid content influenced the extent of liposome uptake by organs such as the lungs and spleen, net tumor uptake was not affected significantly. Additional analysis of the tumor revealed that cationic charge did, however, determine the partitioning between the interstitial and vascular compartments. Highly charged cationic liposomes associated avidly with tumor vessels compared with low-charge content, suggesting a change in the relative distribution of liposomes in tumors. Although net liposome (vascular plus interstitial) localization in the tumor was not dependent on liposome formulation, studies with cationized ferritin molecules showed that patchy and irregular anionic domains exist along the capillary endothelium in a brain tumor model (16) . This is consistent with our studies using relatively large cationic molecules to target tumor vessels in the window chambers. We also found that changing the tumor type (LS174T versus MCa IV) and the host microenvironment (s.c. versus cranial) did not influence the affinity of cationic liposomes for tumor vessels significantly.

Overall tumor uptake was not affected by liposome charge in the biodistribution studies, and the intravital microscopy results supported this. Total (vascular plus interstitial) localization in the tumor was not dependent on liposome formulation (P > 0.05). However, considering the vascular and interstitial compartments individually, charge affected vascular (P < 0.05) but not interstitial (P > 0.05) uptake. This apparent discrepancy in the mass balance is likely caused by the heterogeneity in accumulation and the corresponding large SE in the measurements in the interstitial compartment. The vascular compartment comprises a small fractional area of the tissue and exhibits relatively less heterogeneity.

The presence of positive charge on liposomes is required to optimally interact with the glycoprotein layer of the endothelium. The coating of the liposome surface with PEGylation confers additional advantages. Cationic lipid-based delivery systems are susceptible to attack by human blood components and are prone to opsonization and phagocytosis (17) and hence premature drug release. PEG has been shown to limit interactions of electroneutral liposomes with biological molecules in vivo (11) and to extend the circulation time of cationic liposomes in blood as well (18) . Although PEGylation of cationic liposomes produced carriers with significantly longer blood half-life times (compared with their non-PEGylated counterparts; Ref. 19 ), these liposomes still retained the ability to associate with the endothelium. Furthermore, the electrophoretic mobilities [-1.56 to 2.38 (µ/s)/(V/cm)] and {zeta} potentials (20–30 mv) for PEGylated liposomes were evaluated, and an increase in cationic lipid content (from 0 to 50 mol %) resulted in a steady increase in both values. Taken together, although PEGylation delayed elimination of cationic liposomes from circulation, the partially unshielded surface charge was indeed sufficient to bind the carrier to tumor vessels; the protective border formed by PEG did not shield the cationic charge from binding to the vascular glycocalyx. Other reports have supported this observation, showing that PEG concentrations of >=6 mol % shield the electric surface potential of cationic liposomes (18) and that higher concentrations (~15 mol %) completely abolish the effect of charged groups on the liposome surface (20) . We chose a concentration of 5 mol % PEG, which retains significant cationic charge (and therefore affinity for anionic sites along the vessel wall) but also provides some protection against clearance. Using this formulation, our liposomes remained in the blood compartment for >=24 h.

Why are cationic liposomes significantly more effective at targeting tumor versus normal vascular networks when each is under the influence of the same organ environment? We speculate that the sluggish and irregular tumor blood flow (1) may encourage more interactions between the cationic liposomes and anionic sites on the angiogenic vasculature compared with the ordered, normal vascular networks that maintain regular blood flow velocities. The quantitative differences observed between the amount of cationic liposomes delivered to normal ({approx}4%) and tumor ({approx}25–28%) vessels in the DSC are consistent with this observation. The specific mechanism(s) involved in selective adherence of cationic liposomes to endothelial cells is not completely known. It is thought that mammalian cells interact with and internalize cationized molecules by endocytosis (9 , 16) and that their associations with vascular cells are mediated in part by proteoglycans (21) . These associations are not limited to endothelial cells but might include cancer cells that come in direct contact with the lumen of mosaic tumor vessels (22) .

For PEGylated cationic liposomes to be effective drug carriers, they must not only arrive at their destination but also kill target cells, either by releasing the contained drug or by being taken up directly by target cells. Preliminary results suggest that the formulation used in our studies might be effective, at least for doxorubicin. Using the intrinsic fluorescence of doxorubicin as a tracer, we analyzed the delivery of this drug to LS174T tumors grown in the dorsal window chamber. We found extensive localization of drug-loaded liposomes associated with the tumor vasculature and little uptake in the interstitial compartment (compared to doxorubicin alone). A diffuse cloud of drug signal surrounded the punctate, suggesting that doxorubicin was being released. More detailed studies are needed to determine the cytotoxic effects of these liposomes and the kinetics of drug release.

One of the primary goals of a successful cancer treatment regimen is to deliver sufficient amounts of drug to tumors while minimizing damage to healthy surrounding tissues. In this study, we observed significant uptake of PEGylated cationic liposomes by the liver compared with tumors; uptake was also observed in kidneys, lungs, and spleen. The accumulation in the liver has been demonstrated elsewhere (14) and studied in detail. Apparently, liposomes associate with several cell types within the liver, including hepatocytes, Kupffer cells, and endothelial cells, as evidenced by isolated and purified liver cell models (23) . Kupffer cells are the primary targets, whereas endothelial cells interact to a lesser extent (23) . Several lines of evidence now suggest that drug-associated cationic liposomal formulations will result in clinical complications, but additional toxicity studies are needed. However, we may be able to use this to our advantage; when cisplatin is encapsulated using a PEGylated cationic liposomal formulation (which results in significant accumulation in the liver), it markedly suppresses liver metastases in tumor (LM8G5)-bearing mice and prolongs animal survival (14) . It is therefore possible that PEGylated cationic liposomes might be effective agents to treat tumors that preferentially metastasize to the liver.

In conclusion, the vehicles used for tumor therapy are as important a consideration as the drug to be delivered. To develop better treatments, we must contend with the fact that intratumor distribution of liposomes can be mediated on the basis of liposome composition. Additional efforts aimed at optimizing macromolecular carrier formulation are warranted, and future studies may reveal the mechanism of the "zip codes" on the tumor vasculature responsible for specific targeting (24) . Regardless of the mechanisms involved in tumor endothelial cell recognition in vivo, these data have implications for vascular-based therapeutic delivery.


   ACKNOWLEDGMENTS
 
We thank Julia Kahn for implantation of the dorsal skin-fold chambers and Sylvie Roberge for her assistance in biodistribution studies. We also thank Drs. William Kaufman and Brian Seed for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant P01-CA80124 and by an NIH fellowship to R. B. C (T32-CA73479). Back

2 Current address: Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115. Back

3 To whom requests for reprints should be addressed, at Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114. Phone: (617) 726-4085; Fax: (617) 726-1962; E-mail: Lance{at}steele.mgh.harvard.edu Back

4 The abbreviations used are: PEG, polyethylene glycol; ROI, regions of interest; FOV, field of view; SCID, severe combined immunodeficiency; PE, phosphatidylethanolamine; DSC, dorsal skin fold chamber; CRW, cranial window; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DOTAP, 1,2-diacyl-3-trimethylammonium propane. Back

Received 8/20/02. Accepted 10/14/02.


   REFERENCES
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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