Monoclonal antibody 2C5-modified doxorubicin-loaded liposomes with significantly enhanced therapeutic activity against intracranial human brain U-87 MG tumor xenografts in nude mice

Abstract

Liposomes, modified with monoclonal antibodies, are suitable carriers for targeted delivery of chemotherapeutic drugs into brain tumors. Here, we investigate the therapeutic efficacy of monoclonal anticancer antibody 2C5-modified long-circulating liposomes (LCL) loaded with doxorubicin (2C5-DoxLCL) for the treatment of U-87 MG human brain tumors in an intracranial model in nude mice. In vitro, 2C5-DoxLCL is significantly more effective in killing the U-87 MG tumor cells than Doxil^® (commercial doxorubicin-loaded PEGylated LCL) or DoxLCL modified with a non-specific IgG. 2C5-immunoliposomes also demonstrate a significantly higher accumulation in U-87 MG tumors compared to all controls in a subcutaneous model. The treatment of intracranial U-87 MG brain tumors in nude mice with 2C5-DoxLCL provides a significant therapeutic benefit over control formulations, substantially reducing the tumor size and almost doubling the survival time. Thus, monoclonal antibody 2C5-modified LCL can specifically target the anticancer drugs to brain tumors, leading to improved therapeutic treatment of brain tumor in an intracranial model, in vivo.

Introduction

Despite the advances in therapeutic modalities, the median survival time of patients with brain tumor, ∼12 months after the initial diagnosis and treatment, has not changed over the past decades [10]. The reason for such an insufficient outcome is the non-responsiveness of brain tumors to chemotherapeutic treatment, which occurs due to the existence of tumor cell multidrug resistance (MDR) [4, 5] and insufficient drug accumulation in tumors in the process of traditional therapy [2, 34]. To overcome these obstacles, various alternative therapeutic strategies have been explored to improve the drug delivery within brain tumors [33]. Long-circulating liposomes (LCL) offer several advantages that can be exploited to deliver anticancer drugs into brain tumors. Liposome accumulation in tumors proceeds passively and is mediated by the enhanced permeability and retention (EPR) phenomenon [25]. Since the blood–brain barrier is disrupted in high-grade gliomas with all the components of the tumor blood vessels, i.e., endothelial cells, pericytes and the basement membrane, showing significant abnormalities (leaky vasculature) compared to normal cerebral vessels [8, 39], LCL can extravasate in such areas and accumulate in the interstitial space of tumors delivering there their payload [15, 40, 41]. In addition, the liposome-mediated enhanced drug accumulation in the tumor interstitium through the EPR effect can partially overcome the MDR by creating local high drug concentrations around and within the tumor [20, 27].

The therapeutic efficacy of the treatment can be improved further by modifying the liposomes with tumor-specific targeting ligands, such as monoclonal antibodies. The resulting immunoliposomes target the tumor “actively” via specific recognition and binding to tumor cell surface antigens [31]. Previous studies in our laboratory have identified a novel class of anticancer antibodies that show high affinity to the surface of a variety of tumor cells, but not normal cells [16, 17]. A representative of this class, the monoclonal antibody 2C5 (mAb 2C5), possesses the nucleosome-restricted specificity and targets the tumor cells by recognizing the nucleosomes that are present on the surface of tumor cells being originated from apoptotically dying neighboring tumor cells [17, 18]. This antibody being attached to drug-loaded pharmaceutical nanocarriers, such as liposomes or polymeric micelles, improves their association with cancer cells both in vitro and in vivo and increases their antitumor activity [24, 38].

In an attempt to expand the potential applicability of this approach onto brain tumors, we have shown in our preliminary studies that liposomes modified with this mAb 2C5, 2C5-immunoliposomes, specifically and efficiently recognize and bind different types of human brain tumor cells in vitro and also display significantly enhanced accumulation in subcutaneously implanted brain tumors in mice [11]. The specificity of 2C5-immunoliposome binding to cancer cells, including U-87 MG cells, was confirmed in this study by the fact that the excess of free mAb 2C5 significantly inhibited the binding of immunoliposomes with the cells.

Here, we demonstrate a significantly enhanced therapeutic efficacy of long-circulating 2C5-immunoliposomes loaded with doxorubicin (2C5-DoxLCL) in the treatment of human brain tumors in an intracranial model in nude mice.

Materials and methods

Cell culture

U-87 MG human astrocytoma cells (American Type Culture Collection, Manassas, VA, USA) were grown in DMEM medium (Cellgro, Herndon, VA, USA) with fetal bovine serum to 10%, sodium pyruvate to 1 mM, and penicillin and streptomycin to 50 U/ml and 50 μg/ml, respectively, at 37°C and 5% CO₂.

Modification of Doxil with antibodies

Doxil, a commercially available preparation of doxorubicin in PEGylated liposomes (ALZA Corp.), was purchased from Ortho Biotech Products LP (Raritan, NJ, USA). The mAb 2C5 was produced and purified by Harlan Bioproducts for Science Inc. (Indianapolis, IN, USA) from the cell line provided by our laboratory. Control bovine antibody IgG was obtained from Serologicals Proteins Inc. (Kankakee, IL, USA). For our purposes, Doxil was modified with antibodies (mAb 2C5 or non-specific IgG) by the “micelle transfer method” at the initial ratio of 169 μg Ab per μmol of phospholipid (PL), as described in [11] with slight modification. Briefly, the antibody was initially modified with p-nitrophenyl carbonyl-PEG-PE (pNP-PEG-PE) groups. For this purpose, the excess of pNP-PEG-PE (obtained as a film by evaporation from the chloroform solution) was incubated with the antibody solution in 50 mM Tris-buffered saline, pH 8.7 for 24 h at 4°C to allow for antibody modification via the pNP-activated PEG termini with the simultaneous hydrolysis of non-reacted pNP groups. As a result, Ab-PEG-PE was formed, which associates into the loose micelles. The antibody micelles were then incubated with Doxil at 169 μg Ab/μmol PL for 24 h at 4°C at final PL concentration of 2.96 mM, followed by the dialysis (300,000 Da membrane cut-off size) against HEPES-buffered saline, pH 7.4, for 2 days at 4°C.

The size of Doxil and antibody-modified DoxLCL liposomes was determined by the Coulter N4 MD Submicron Particle Size Analyzer (Coulter Electronics, Arlington, TX, USA). The concentration of doxorubicin after Doxil modification was determined by lysing liposomes with acidified isopropanol (90% isopropanol with 0.075 M hydrochloric acid) and measuring the absorbance at 480 nm [1] using Hitachi U-1500 spectrophotometer. The ratio of free versus total doxorubicin present after antibody modification of Doxil was determined by changing the pH of liposomes from 7.4 to 10.5 and measuring the absorbance at 600 nm with and without lysis of liposomes with 0.5% Triton X-100 [28].

Cytotoxicity of various doxorubicin preparations toward U-87 MG astrocytoma cells

U-87 MG astrocytoma cells were seeded in 96-well plate at the density 10,000 cells per well and were allowed to adhere to the plate for 48 h. The medium was removed, the cells were washed in complete serum-free media (SFM) thrice and treated with 2C5-DoxLCL, control Doxil and control non-specific IgG-DoxLCL at 200 μg/ml doxorubicin concentration. Dilutions of liposomes were made in SFM, and the plate was incubated for 48 h at 37°C and 5% CO₂. The medium was removed and the cells were washed again as before. The cells were incubated with CellTiter 96^® AQueous One Solution Cell Proliferation reagent (Promega Corporation, Madison, WI, USA) for 1 h at 37°C and 5% CO₂. The absorbance of viable cells was measured at 490 nm using a Labsystems Multiscan MCC/340 microplate reader.

Subcutaneous brain tumor model in nude mice

The experiments were performed under the protocol No.011022R, approved by the Institutional Animal Care and Use Committee in accordance with the Principles of Laboratory Animal Care (NIH publication No.85–23, revised in 1985). The female nude mice (Charles River Laboratories, Wilmington, MA, USA), 6–8 weeks old, were inoculated with 15 × 10⁶ U-87 MG cells in 200 μl of media (without FBS) subcutaneously into the rear left flanks. The mice were examined every day for tumor formation. The tumor volume was measured with vernier calipers using the formula:

Histology of U-87 MG tumors grown subcutaneously in nude mice

The subcutaneous tumors were removed by killing the mice by cervical dislocation. The tumors were fixed in 10% buffered formalin, pH 7.4, overnight, followed by 20% sucrose exchange overnight at 4°C. The tumors were then frozen in Tissue-freezing media (TBS Inc., NC, USA) under isopentane and stored at −80°C until sectioning. The tumor sections were taken at 8 μm thickness using a Microtome cryostat (TBS Inc., NC, USA) and stained with hematoxylin (Fisher Scientific, Pittsburgh, PA, USA) and eosin (Sigma, St. Louis, MO, USA) to check the morphology of the tumors.

Accumulation of various liposomal preparations in subcutaneously grown U-87 MG tumors in nude mice

Hydrogenated soy phosphatidylcholine (HPC), cholesterol (Chol), and methoxy PEG₂₀₀₀-DSPE (mPEG-PE) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Diethylene triamine pentaacetic acid-phosphatidyl ethanolamine (DTPA-PE) was synthesized in our laboratory as described in [23].

U-87 MG astrocytoma cells were injected subcutaneously as described above. For in vivo accumulation studies, plain liposomes carrying 0.5 mol% DTPA-PE were prepared by the lipid film hydration method [22] with the composition and size mimicking Doxil (HPC:Chol:mPEG-PE = 1.5:1:0.12 m/m). The liposomes were modified with antibodies (mAb 2C5 and non-specific IgG) at 169 μg Ab/μmol PL initial ratio. Plain liposomes and immunoliposomes were radiolabeled with Indium (¹¹¹In) (Perkin-Elmer, Boston, MA, USA) by the transchelation onto the liposomal DTPA-PE from the ¹¹¹In-citrate complex [35]. On day 9 post-tumor cell injections, the tumor-bearing mice were randomly divided into three groups of five mice each. Each mouse was injected with 100 μl of radiolabeled liposomes (2.24 mg/ml PL, 5–6 μCi per mouse) via the tail vein. The mice were killed by cervical dislocation at different time points, and the tumor and the adjacent normal tissues were removed. ¹¹¹In radioactivity was quantified using a Beckman 5500B γ-counter. Tumor-to-muscle accumulation ratio and tumor-to-blood ratio were calculated from the percentage-injected dose/g of tissue parameter.

Intracranial model of U-87 MG astrocytoma in nude mice

The surgical procedures were performed under sterile conditions in accordance with the protocol No.050614R, approved by the Institutional Animal Care and Use Committee in accordance with the Principles of Laboratory Animal Care (NIH publication No.85-23, revised in 1985). For intracranial tumor establishment, U-87 MG astrocytoma cells were harvested by trypsinization, washed two times in Hank’s balanced salt solution (HBSS) and resuspended in HBSS. The female nude mice, 6–8 weeks old, were anesthetized by intra-peritoneal injection of 80 mg/kg ketamine (Ketaset^®, Fort Dodge Animal Health) and 10 mg/kg xylazine (X-Ject SA, Vetus Animal Health). The dorsal head surface of nude mice was disinfected with betadine and wiped with 70% alcohol. The skin was incised at the midline and retracted. The area around the site of injection was blotted dry. A small hole was drilled 2 mm to the right of the midline using a 25 gauze needle that was fitted with a sleeve to create a hole to a depth of 3 mm into the right cerebral hemisphere. Each mouse was injected slowly (∼30 s) with 2 × 10⁵ cancer cells in 3 μl HBSS, through a Hamilton syringe fitted with a sleeve to restrict brain penetration to 3 mm depth. The skin edges were closed using Krazy glue. The animals were injected with 0.05 mg/kg of buprenorphine hydrochloride (Buprenex^® Injectable, Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA, USA) subcutaneously and returned to the cages. The animals were monitored for the next hour after the surgery and then daily.

The body weights of mice were recorded prior to the surgery and followed regularly. The mice were observed for body weight reduction post-tumor cell injections. They were killed by cervical dislocation when their body weight reduction was more than 20% of their original weight, the time to this moment being referred to as “survival time.”

Intracranial tumor area characterization

For tumor growth estimation, the mice were killed after days 7 and 14 post-tumor cells injection and at the time of weight reduction of more than 20% of their original weight. The brains were removed and fixed in 10% buffered formalin overnight at 4°C. For analysis, the brains were sliced into serial 1 mm thick coronal sections using a mouse brain slicer (Harvard Apparatus Inc., Boston, MA, USA), and stained with H-E. The coronal section with the maximum tumorous area was photographed with a digital camera for analyzing the tumor growth. The tumor area was estimated by Adobe^® Photoshop software (Version 8.0).

Therapeutic efficacy studies on intracranial tumor-bearing mice

To investigate the efficacy of the therapeutic treatment of brain tumors, the tumor-bearing mice were randomly divided into four groups of 6–8 mice each. Therapeutic treatment was provided with 2C5-DoxLCL, and control Doxil and non-specific IgG-DoxLCL at the dose of 2.7 mg/kg doxorubicin via the tail vein injections thrice a week for two consecutive weeks (days 11, 13, 15, 18, 20 and 22 post-tumor inoculation). Mice injected with the buffer were taken as a control untreated group. The survival times (see above) of mice in all groups were recorded. The percent increase in the life span was calculated as {(T − C)/C} × 100, where T and C are the survival times of the treated and control animals [14].

To correlate the therapeutic efficacy of the formulations with the changes in the actual tumor area, some randomly selected therapeutically treated and untreated mice were killed 2 days after the last treatment (day 24 post-tumor injections), and their brain tumor areas were measured as above.

Statistics

The data were presented as mean ± SEM, unless specified otherwise. Student’s t-test was applied for the test of significance. P < 0.05 was considered statistically significant. The survival data was analyzed by log rank Kaplan–Meier method using GraphPad Prism Version 4.03 (Trial) for Windows, GraphPad Software, San Diego, CA, USA, http://www.graphpad.com.

Results

Modification of Doxil with antibody

Doxil (liposome size of 87.0 ± 10.87 nm) was successfully modified with the antibody (mAb 2C5 or non-specific bovine IgG) at the initial ratio of 169 μg of the protein per 1 μmol of the PL initial ratio. The coupling efficiency was above 70%, and the used ratio resulted in the binding of ∼80 antibodies per single 100 nm liposome (as calculated using the number of PL molecules per liposome determined in [9]). The size of mAb 2C5-DoxLCL was 92.4 ± 11.33 nm with quite narrow size distribution (Fig. 1), i.e., practically the same as for the initial Doxil (size 87.0 ± 10.87 nm). The final doxorubicin concentration in immunoliposomes was 0.5 mg/ml at 2.25 mg/ml PL concentration. Doxil contained ∼2% free doxorubicin of the total drug, which was completely removed by dialysis after the modification of Doxil with antibodies. Although we did not specifically investigate a long-term stability of Ab-Doxil conjugates, it was noticed that over a two-week period at 4°C, there was no free drug release or antibody activity drop.

Fig. 1.

Size distribution of a Doxil^® and b 2C5-DoxLCL; values are mean ± standard deviation

Cytotoxicity of 2C5-DoxLCL toward U-87 MG astrocytoma cells in vitro

The cytotoxic effect of 2C5-DoxLCL on U-87 MG astrocytoma cells was estimated in vitro following the IC₅₀-value. The cells were incubated with control Doxil, non-specific IgG-DoxLCL or 2C5-DoxLCL for 24–48 h. After the incubation for 24 h, the IC₅₀ for 2C5-DoxLCL was 175 μg/ml, while Doxil and IgG-DoxLCL did not cause cell death up to 200 μg/ml. Upon increasing the incubation time to 48 h, the IC₅₀ of 2C5-DoxLCL was 6 μg/ml, and IC₅₀ for Doxil and IgG-DoxLCL were 85 and 55 μg/ml, respectively (Fig. 2). Thus, 2C5-DoxLCL is significantly more cytotoxic for U-87 tumor cells than Doxil and non-specific IgG-DoxLCL, causing 50% cell death at 9–14-fold lower dose of doxorubicin. One has to note here that drug-free and plain or antibody-modified liposomes, as well as the antibody itself, were not toxic to the cells at the concentrations used.

Fig. 2.

Cytotoxicity profile of 2C5-DoxLCL toward U-87 MG astrocytoma cells after 48 h incubation; n = 3, *P < 0.01 and **P < 0.05 versus Doxil^® and IgG-DoxLCL

Subcutaneous brain tumor model in nude mice

To check the specific recognition of U-87 MG tumor by 2C5-DoxLCL in vivo, we first performed the experiments with subcutaneously inoculated brain tumor. U-87 MG tumor is usually initiated for different experimental purposes by inoculating (5–8) ×10⁶ cells per mouse [19, 21, 30]. We, however, used 15 × 10⁶ U-87 MG cells per mouse to inoculate the tumor, since mice injected with 5 × 10⁶ cells developed tumors after a lag period of around 30 days at a take rate of 80%, with tumors reaching a volume of 300–1,000 mm³ after as long as 55 days post-inoculation. At the same time, tumors initiated by the inoculation of 15 × 10⁶ cells grew much faster with no lag phase and with 100% take rate. The tumors grew to a size within the range of 100–500 mm³ in 10 days post-inoculation, after which they began to slowly regress.

To ensure that the tumor cells retain their morphological characteristics when injected at such high cell density, we histologically examined and compared tumors grown after the inoculation of 15 × 10⁶ cells and after the inoculation of 5 × 10⁶ cells. In both cases, the cells were spindle-shaped and were organized in fascicles or clusters, creating a typical pattern of U-87 MG astrocytoma growth (pictures not shown).

Accumulation of 2C5-immunoliposomes in subcutaneous U-87 MG tumors in nude mice

2C5-Immunoliposomes (with liposome composition mimicking that of Doxil) showed enhanced accumulation in subcutaneously grown brain tumors compared to the neighboring normal muscle, with significantly higher tumor-to-muscle and tumor-to-blood ratios than control plain liposomes and non-specific IgG-liposomes at all the time points tested, with the difference reaching about two-fold at 24 h post-liposome injection (Fig. 3). The tumor-to-muscle accumulation ratios, 24 h after the liposome administration, was 14.65 ± 0.75, 5.74 ± 0.79, and 7.60 ± 1.55 for 2C5-immunoliposomes, IgG-liposomes and plain liposomes, respectively. The tumor-to-blood accumulation ratios, 24 h post-liposome injection, were 3.19 ± 0.36, 2.05 ± 0.43, and 1.39 ± 0.28 for 2C5-immunoliposomes, IgG-liposomes and plain liposomes, respectively. These results confirm that 2C5-immunoliposomes can specifically recognize and target selected U-87 MG human brain tumors in vivo.

Fig. 3.

Tumor-to-muscle accumulation of 2C5-immunoliposomes within subcutaneous U-87 MG astrocytoma grown in nude mice; n = 5 mice per group, the P-values displayed are for 2C5-immunoliposomes versus plain liposomes and IgG-liposomes, 9 days after tumor inoculation

Characterization of the intracranial U-87 MG astrocytoma in nude mice

For therapeutic activity studies, the intracranial brain tumor model was established with U-87 MG tumor cells in nude mice. Different investigators have proposed inoculating different cell number for establishing intracranial xenografts in nude mice [12, 19, 21, 29]. In our studies, the mice were injected intracranially with 2 × 10⁵ cells in 3 μl HBSS to establish a tumor. After the tumor progresses, the mice begin to lose body weight; so the reduction in animal body weight was considered as an indicator of the brain tumor growth [3].

To assess the tumor growth, the coronal sections of the brain were analyzed by hematoxylin-and-eosin (H-E) staining over a period of time. The section with the maximum tumor area was captured by digital camera. The pixels of the tumor area were counted relative to the total number of pixels within the hand-drawn square of 100 mm². After day 7, the tumor area was 0.55 ± 0.08 mm² and increased to 4.03 ± 0.5 mm² by day 14 of tumor injection. The average tumor area at the median survival time of the mice was 4.58 ± 0.6 mm² (Fig. 4a). Thus, after 14 days of tumor inoculation, the tumor grew to a substantial size covering approximately 9% of the coronal section area. Figure 4a shows the representative coronal sections of the brain stained by H-E for tumor growth characterization.

Fig. 4.

a Quantitative analysis of the H−E stained coronal sections for U-87 MG tumor area estimation; n = 5 mice per group for days 7 and 14; n = 6 mice for days ≥ 22 (median survival = 24.5 days). b Representative pictures of the H–E stained coronal sections of U-87 MG astrocytoma-carrying brains (1 mm thickness). Pictures are modified with Adobe^® Photoshop software for contrast enhancement. Arrows represent the areas of tumor growth

Therapeutic efficacy studies

The therapeutic efficacies of 2C5-DoxLCL, control Doxil and non-specific control IgG-DoxLCL were evaluated by finding the time, after treatment, when the mice were killed because of losing more than 20% of their original body weight. This period was termed conditionally as “survival time.” The average survival time of the untreated mice was 25.5 days; after treatment with different doxorubicin preparations, the survival was significantly prolonged to: 44.5 days with Doxil, 41 days with IgG-DoxLCL and 74 days with 2C5-DoxLCL (Fig. 5). In other words, the survival of the treated mice was increased by 19 days with Doxil, 15.5 days with IgG-DoxLCL and by as much as 48.5 days with 2C5-DoxLCL treatment. This translates to an increase in the life span of 74% with Doxil (P = 0.0003 versus untreated), 61% with IgG-DoxLCL (P = 0.0003 vs. untreated and 0.8173 vs. Doxil) and 190% with 2C5-DoxLCL (P = 0.0127 vs. Doxil and 0.0236 vs. IgG-DoxLCL) treatment. Clearly, the treatment with 2C5-DoxLCL significantly improved the survival time of the mice (by 115–130%) compared to the survival time increase achieved with Doxil and IgG-DoxLCL.

Fig. 5.

Survival curves for different groups of mice; n = 8 in untreated group; n = 6 per group in treated groups. Arrows indicate the time of therapeutic injections

Tumor area of therapeutically treated mice

To correlate the therapeutic effect on the actual tumor area, the brains of some randomly selected therapeutically treated and control mice were evaluated for residual tumor via the H-E staining. Figure 6a shows the quantitative analysis of H-E stained sections. Figure 6b shows the representative H-E stained coronal sections of the therapeutically treated and control mice. Thus, although the tumor area was significantly reduced using different preparations of Doxil, still tumor regression was significantly enhanced by the treatment with 2C5-DoxLCL.

Fig. 6.

a Quantitative analysis of the actual tumor area after the treatment (day 24 post-tumor injections). b Representative pictures of the coronal sections of U-87 MG astrocytoma-carrying brains (1 mm thickness) for actual tumor area estimation via the H–E staining. Pictures are modified with Adobe^® Photoshop software for contrast enhancement. Arrows represent tumorous areas

Discussion

Chemotherapeutic treatment is widely used for brain tumor treatment; however, the outcome continues to be unsatisfactory. The increase in the local anticancer drug concentration within the tumor may improve the outcome of the drug therapy. Liposomes are suitable carriers to effectively deliver the drugs to brain tumors. Studies have shown that liposomes can overcome the obstacles imposed on brain tumor delivery and that they can accumulate and deliver the therapeutic drug in higher amounts to the brain tumors in intracranial models [36, 37]. Further improvements in the liposome-mediated therapy of brain tumors have been demonstrated by utilizing monoclonal antibodies as a targeting ligand [26, 32] for an enhanced delivery of drug-loaded liposomes into brain tumors. Earlier, we had used cancer-specific mAb 2C5 to modify liposomes and make them capable of specific and efficient targeting of various human brain tumor cells in vitro and in subcutaneous brain tumor model in vivo [11].

Here, we have modified Doxil, a commercial preparation of doxorubicin in long-circulating PEGylated liposomes, with mAb 2C5 and confirmed the ability of such 2C5-DoxLCL to accumulate selectively in a subcutaneous model of the human U-87 MG astrocytoma in nude mice. Further, we have investigated the efficacy of 2C5-DoxLCL in the therapeutic treatment of U-87 MG astrocytoma in an intracranial model in nude mice. The choice of this very tumor for our studies was based on the fact that U-87 MG astrocytoma is a widely used model for subcutaneous and intracranial xenografts [19, 21, 29, 30].

In vitro cytotoxicity studies using 2C5-DoxLCL and U-87 MG astrocytoma cells were performed for 24–48 h. After 24 h incubation of U-87 MG cells with various liposomal preparations, control Doxil and non-specific IgG-DoxLCL did not provide any cell killing up to 200 μg/ml doxorubicin concentration, while 2C5-DoxLCL caused the death of approximately 50% of cells even at 175 μg/ml of doxorubicin. Increasing the incubation time to 48 h still further reveals high cytotoxicity of 2C5-DoxLCL, demonstrating the IC₅₀-value for this preparation 9–14 times lower than for controls. The cytotoxicity of non-targeted doxorubicin-liposomes is determined by the release of free doxorubicin from the liposomes into the extracellular medium [13]. In the case of control Doxil and non-specific IgG-DoxLCL accumulating inside the tumor interstitium because of the EPR effect, the release of the active drug, doxorubicin, takes place in the extracellular environment, and the cell death depends on the subsequent cellular uptake of the released doxorubicin on prolonged incubations [7, 13]. Actively targeted 2C5-DoxLCL can be taken up by cells as a result of the endocytosis after binding to the tumor cell surface-bound nucleosomes and release all doxorubicin, being already inside cancer cells (the mechanism to be published elsewhere), which translates into significantly increased cytotoxicity of this preparation.

For conducting therapeutic efficacy studies, intracranial U-87 MG xenograft was established in nude mice and the treatment schedule included the administration of various preparations at the dose of 2.7 mg/kg doxorubicin thrice a week for two consecutive weeks. This resulted in a weekly dose of 8.1 mg/kg and a total cumulative dose of 16.2 mg/kg doxorubicin. The selected dosing complied with the recommended dosing of 2.5–10 mg/kg mouse per week doxorubicin used with human xenografts in nude mice [32]. The treatment with 2C5-DoxLCL provided a significant therapeutic benefit over controls with a pronounced reduction in the tumor size.

In conclusion, brain tumors can be effectively targeted with 2C5-immunoliposomes delivering its payload in therapeutic amounts directly into the tumor cells and hence increasing the efficiency of the treatment. The mAb 2C5-immunoliposomes loaded with anticancer drugs can be used to significantly enhance the efficacy of the therapy of human brain tumors in vivo.