Targeted delivery of nano-PTX to the brain tumor-associated macrophages

Abstract

Nanoparticles containing mixed lipid monolayer shell, biodegradable polymer core and rabies virus glycoprotein (RVG) peptide as brain targeting ligand, were developed for brain targeted delivery of paclitaxel (PTX) to treat malignant glioma. RVG conjugated PTX loaded NPs (RVG-PTX-NPs) had the desirable size (~140 nm), narrow size distribution and spherical shape. RVG-PTX-NPs showed poor uptake by neurons and selective targeting to the brain tumor associated macrophages (TAMs) with controlled release and tumor specific toxicity. In vivo studies revealed that RVG-PTX-NPs were significant to cross the blood-brain barrier (BBB) and had specific targeting to the brain. Most importantly, RVG-PTX-NPs showed effectiveness for anti-glioma therapy on human glioma of mice model. We concluded that RVG-PTX-NPs provided an effective approach for brain-TAMs targeted delivery for the treatment of glioma.

Keywords: brain targeted delivery; glioma; nanoparticles; tumor associated macrophages.

Figure 1. Chemical structure of HSPC

A. DSPE-PEG-Mal B. and the preparation of RVG-PTX-NPs C. were schematically illustrated.

Figure 2. AFM was used to examine the morphology of NPs

The images showed that RVG-PTX-NPs had smooth surface and spherical shape. A. The size and size distribution of RVG-PTX NPs were analyzed by DLS B. The released PTX from PTX-NPs and RVG-PTX-NPs was detected by HPLC C. exhibiting slow release profiles.

Figure 3. Toxicity of RVG-PTX-NPs

RVG-PTX-NPs, PTX-NPs, PTX and blank NPs were treated to U87 at PTX concentration of 0.001 to 1 μg/ml for 72 hours. Dose dependent toxicities of RVG-PTX-NPs, PTX-NPs and PTX were analyzed by MTT assay A. Fluorescence microscopy analysis showed the cytopathological changes of U87 cells (B, green) after 24 hours exposure to Rhodamine 6G labeled RVG-PTX-NPs and blank NPs (B, red). The untreated U87 cells served as control.

Figure 4. To test RVG specific binding to macrophages, we made red fluorescence labeled HS-RVG through directly reaction with TRM (HS-RVG+TRM), HS-RVG reduced by βME before reaction with TRM (HS-RVG+βME+TRM), and βME reaction with TRM without HS-RVG (βME+TRM)

BMM were treated to HS-RVG+TRM, HS-RVG+βME+TRM and βME+TRM for 2 hours. The specific binding showed in BMM treatment with (HS-RVG-βME-TRM (red). No specific binding signals seen in cultures treated to HS-RVG-TRM or βME+TRM A. RVG-PTX-NPs were incubated with BMM and neurons for 1, 2 and 4 hours. Flow cytometry was used to determine fluorescence signals when RVG-PTX-NPs uptake by BMM and neurons B. The fluorescence microscope was used to visualize intracellular distribution of RVG-PTX-NPs in BMM and neurons C. The significant increases of red signal were observed in BMM. No red fluorescence was detected in neurons at any time points.

Figure 5. Maximum uptake of RVG-PTX-NPs and PTX-NPs by BMM was counted as experimental time “0” (A, hrs 0) for the release study under fluorescence microscope

Images show that BMM release PTX/PTX-NPs led to loss of red signals following each medium change every 8 hours. HPLC assay was used to detect extracellular PTX in medium (released PTX) and intracellular PTX in cell lysates. The percentage of PTX content released from BMM to the medium showed in B. MTT assay was used to detect that PTX loaded NPs did not cause BMM death C. TAMs treated with RVG-PTX-NPs and PTX-NPs caused alterations in cytokine profiles of IL1α D., IL6 E. and TNFα F. levels (p<0.05). All data were acquired from triplicate experiments.

Figure 6. Cytotoxicity was analyzed in RVG-PTX-NPs pre-loaded TAMs (RVG-PTX-NPs-TAMs) co-cultivation with U87

In the co-cultivation system, RVG-PTX-NPs-TAMs (red, arrow head) were co-cultured with GFP-U87 (green,) for 24 hours (upper panel). Imaging showed RVG-PTX-NPs (red) within TAMs were transported into U87 cells (green, arrow), identifying as yellow-red (merge, arrow). U87 directly treated with RVG-PTX-NPs served as control (lower panel). In contrast, RVG-PTX-NPs-TAMs provided a significant transportation and greater toxicity to U87.

Figure 7. In vivo images were taken at 24

A. and 48 B. hours in live animals after administration of DiSC₃(5) labelled RVG-PTX-NPs to mice via tail vein. Significant amount of fluorescence signals appeared in the head after 24 and 48 hours. The brain sections from human glioma of SCID mouse model were labeled with IBa-1 and GFP to identify TAMs/microglia (C, red) and human glioma U87 cells (C, green). The frozen sections was mounted with DAPI (blue) to examine the tissue distribution of DiSC₃(5) labelled RVG-PTX-NPs (D, red).

Figure 8. Mice were sacrificed at 4 weeks to evaluate the brain size and weight, and histopathological changes

In contrast to normal mouse (A, upper panel), untreated glioma mice (A, lower panel) exhibited tumor growth, largely increasing the brain size. Tumor growth was prevented by RVG-PTX-NPs (A, middle panel), leading to comparable brain size to the normal control. The brain sections were histopathologically evaluated by antibody to GFP to identify the GFP-U87 cells (B and C, brown). Representative images revealed significant prevention of U87 growth in RVG-PTX-NPs treated glioma mice (B) compared to untreated group (C). The body D. and tumor E. weight were measured to determine the body loss and tumor growth for evaluation of the anti-glioma efficacy.