Article pubs.acs.org/molecularpharmaceutics
Tumor-Penetrating Peptide Mediation: An Effective Strategy for Improving the Transport of Liposomes in Tumor Tissue Zhiqiang Yan,*,†,‡ Yiyi Yang,†,§ Xiaoli Wei,∥ Jian Zhong,† Daixu Wei,† Lu Liu,† Cao Xie,∥ Fei Wang,∥ Lin Zhang,∥ Weiyue Lu,*,∥ and Dannong He†,§ †
National Engineering Research Center for Nanotechnology, Shanghai 200241, P.R. China Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai 200062, P.R. China § School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, P.R. China ∥ Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, P.R. China ‡
ABSTRACT: Currently, the inefficient transport of liposomes in tumor tissue hinders their clinical application. Tumorpenetrating peptides (TPP) are a series of targeting peptides with the function of penetrating tumor blood vessels and tumor stroma. This work aimed to improve the penetration of liposomes in tumor tissues by TPP modification, thereby enhancing the antitumor effect. First, RPARPAR, a TPP, was modified to the surface of liposomes loaded with doxorubicin. The RPARPAR-modified liposomes (RPA-LP) and unmodified liposomes (LP) showed spherical morphology with average sizes about 90 nm. RPA-LP exhibited remarkably increased cellular accumulation by PC-3 tumor cells than LP as evidenced by the cellular uptake test. The in vivo imaging study confirmed that RPARPAR modification significantly increased the liposome accumulation in subcutaneous tumor tissues. RPA-LP could penetrate through tumor blood vessels and tumor stroma and into the deep tumor tissues as evidence by the immunofluorescence staining analysis. The cytotoxicity of RPARPAR-modified doxorubicin liposomes (RPA-LP-DXR) is considerably increased compared with that of doxorubicin liposomes (LP-DXR). The RPA-LP-DXR also showed significantly (p < 0.005) stronger growthinhibiting effect on tumor than LP-DXR, possibly due to the tumor-penetrating ability of RPA-LP and targeted killing of tumor cells. This study proved that TPP mediation may be an effective strategy for improving the transport of liposomes in tumor tissue. KEYWORDS: tumor-penetrating peptide, transport in tumor tissue, targeting, liposomes, doxorubicin
1. INTRODUCTION Nano drug delivery systems have been widely recognized as a paradigm-changing opportunity with the potential to revolutionize the areas of tumor therapy.1 Liposomes are the most extensively used nano drug delivery systems due to their controllable size, ready modifiability, and good biocompatibility.2 Ligand-modified liposomes have especially attracted interest for their improved tumor-targeting ability and antitumor effect due to the mediation of ligand−receptor interaction.3 However, they have not consistently delivered successful outcomes.1 One major factor that contributes to the observed inconsistencies is that the current liposomes cannot effectively reach cancer cells deep inside the tumor.4 The incomplete penetration of liposomes in the tumor tissues hinders the activity of anticancer drugs, and can lead to drug resistance and recurrence of the tumor.4 Therefore, there is an urgent need for an effective way to improve the transport of liposomes in tumor tissues. To achieve homogeneous accumulation in tumor, nanomedicines need to move deeply into the whole tumor tissue.5 However, the delivery of nanomedicines from blood to its © 2013 American Chemical Society
cellular/subcellular target is hindered by the two barriers: tumor vessel barrier and tumor stroma barrier. The former refers to the barrier encountered in the extravasation from blood vessels into the tumor, which is caused by the presence of the vessel wall,6 heterogeneity of vascular permeability,7 and high interstitial fluid pressure in the tumor.8 The latter refers to the barrier encountered in the delivery process from the tumor vessel vicinity deep into tumor tissue, which is caused by the high cellular density, dense extracellular matrix (ECM), and rapidly increased interstitial fluid pressure in the periphery of tumor tissue.9 The two barriers make the nanomedicines penetrate only a few cell diameters into the extravascular tumor tissue.10−12 In addition, the extravasated nanomedicines can stay near blood vessels for a long period of time, further hampering the penetration of the subsequent nanomedicines.13 Received: Revised: Accepted: Published: 218
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Figure 1. Transport of tumor cell-targeted liposomes (A) and TPP-modified liposomes in tumor tissues (B). The tumor cell-targeted liposomes can only enter tumor tissue by the EPR effect and remain near blood vessels, whereas the TPP-modified liposomes can penetrate through tumor blood vessels and tumor stroma, and into the deep tumor tissue.
(FAM) were obtained from Sigma, United States. DMEM medium and fetal bovine serum (FBS) were supplied by Gibco, United States. DAPI was supplied by Roche, Switzerland. Rat anti-mouse CD31 was obtained from Abcam, United States. Goat anti-rat IgG-R was supplied by SantaCruz Biotechnology, United States. Chloroform, sodium chloride, and cholesterol (Chol) were obtained from Sinopharm Chemical Reagent, China. PC-3 prostate cancer cells were supplied by Shanghai Institute of Cell Biology, and MDA-MB-435 melanoma cells were obtained from Shanghai Biosis Biotechnology Co., Ltd. The BALB/c nude mice (male, 20 ± 2 g) were supplied by Shanghai SLAC Laboratory Animal Ltd. (China) and maintained at SPF conditions. The protocol of all animal experiments was approved by the ethics committee of Shanghai Jiao Tong University. 2.2. Synthesis and Characterization of DSPE-PEGRPARPAR. The thiolated RPARPAR (C-RPARPAR) was synthesized by using the BOC solid-phase peptide synthesis technique, then reacted with DSPE-PEG-Mal to obtain DSPEPEG-RPARPAR. The structure of the product was confirmed by 1H NMR and FTIR according to the procedure described previously.21,22 2.3. Preparation and Characterization of RPARPARModified Liposomes (RPA-LP). DXR, FAM, or DiR encapsulated liposomes (including LP-DXR, RPA-LP-DXR, LP-FAM, RPA-LP-FAM, LP-DiR and RPA-LP-DiR) were prepared by the thin-film hydration and extrusion method. HSPC, Chol, DSPE-mPEG2000, and DSPE-PEG-RPARPAR were mixed and dissolved in CHCl3 with a mole ratio of 55:40:5:1 or 0 (1 for RPA-LP, 0 for unmodified liposomes). The solution was subjected to rotary evaporation to remove the CHCl3 and produce a thin film. DXR encapsulated liposomes were prepared by using an ammonium sulfate gradient technique. Briefly, the thin film was hydrated with (NH4)2SO4 solution and extruded with polycarbonate membranes (pore size: 50 nm) by using a Mini-Extruder. The liposomes were passed through a gel filtration column using saline, mixed with DXR solution, and left to stand at 60 °C for 20 min. Next, the liposomes were passed through a gel filtration column again to remove the unencapsulated DXR. For FAM encapsulated liposomes, the film was hydrated with FAM solution, extruded, and passed over a gel filtration column. For DiR encapsulated liposomes, the DiR was dissolved in CHCl3 together with lipids
These problems result in the incomplete inhibition to tumor growth, arousing extensive concerns from researchers.5,12 Recently, a series of targeting peptides with the function of penetrating tumor vessels and tumor stroma were identified by in vivo phage display technology, which are called “tumorpenetrating peptides”(TPP).14,15 These peptides usually contain a key sequence motif R/KXXR/K at the C terminal, which allows specific binding to an overexpressed cell surface receptor, neuropilin-1 on tumor cells and tumor endothelial cells.14 Neuropilin-1 is the coreceptor of VEGD165, playing an essential role in tumor metastasis, angiogenesis, and regulation of vascular permeability.16 Neuropilin-1 is overexpressed on a wide range of tumors, including prostate cancer, breast cancer, melanoma cancer, pancreas cancer, and glioblastoma, but not on endothelial cells in normal tissues.17,18 Several reports have proved that coadministration or conjugation of TPPs can increase the penetration of drugs or nanoparticles into the tumor tissues.19,20 For instance, coadministration of iRGD, a TPP, significantly improved the therapeutic index of doxorubicin and doxorubicin liposomes.19 Peptide RPARPAR is a recently identified TPP, able to cause vascular leakage and increase the extravasation of coadministered firefly luciferase and T7 phage into tissues.14 However, there is no report on the use of RPARPAR-modified drug delivery systems in tumortargeted therapy. In this work, we studied the use of RPARPAR modification to improve the transport of liposomes in tumor tissues. We aimed to verify the hypothesis that, following intravenous administration, the prepared liposomes penetrate through tumor blood vessels and tumor stroma and deep into the extravascular tumor tissue (as illustrated in Figure 1). After that, being mediated by NRP-1 on tumor cells, the liposomes enter tumor cells, and the loaded doxorubicin kills the tumor cells. This study may provide an effective solution for the current dilemma of active targeted therapy of tumors.
2. EXPERIMENTAL SECTION 2.1. Materials. DSPE-PEG-Mal was supplied by Laysan Bio Co., United States. DSPE-mPEG2000 was supplied by Shanghai Advanced Vehicle Technology Pharmaceutical L.T.D., China. Hydrogenated soybean phosphatidylcholine (HSPC) was supplied by Lipoid, Germany. DiR (1,1′-dioctadecyl3,3,3′,3′-tetramethyl indotricarbocyanine iodide) was supplied by Invitrogen, United States. Sephadex G50 and 5-carboxyfluorescein 219
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Figure 3. As examined by the DLS method, all prepared liposomes (LP-DXR, RPA-LP-DXR, LP-FAM, RPA-LP-FAM, LP-DiR, and RPALP-DiR) showed average sizes of ∼90 nm (A). As displayed in the TEM images, these liposomes exhibited nearly spherical shapes with uniform size (B, scale bar = 100 nm).
Figure 2. Molecular structure of DSPE-PEG-RPARPAR (A), and the NMR (B), and FTIR (C) spectra of DSPE-PEG-Mal and DSPE-PEGRPARPAR. The NMR spectrum of DSPE-PEG-Mal showed the maleimide peak at 6.7 ppm, whereas DSPE-PEG-RPARPAR did not. The FTIR spectrum of DSPE-PEG-RPARPAR showed a remarkably enhanced N−H stretch band at 3400 cm−1 and CO stretch band at 1666.8 cm−1 compared with that of DSPE-PEG-Mal.
were examined by UV−vis spectroscopy (for DXR) and fluorescence spectrophotometry (for FAM and DiR). The dynamic light-scattering (DLS) method was used to determine the vesicle size and size distribution of the liposomes using a Zetasizer Nano (Malvern, UK). The morphology of lipsomes was examined by using a transmission electron microscopy (TEM) (JEM-2010FEF, JEOL, Japan). The liposomes were negatively stained by 4% phosphotungstic acid and dried on carbon-coated grids for examination. 2.4. In Vitro Cell Uptake of RPA-LP. The cell uptake study was performed according to previous reports with minor modification.24,25 PC-3 cells were maintained in F-12 medium, and MDA-MB-435 cells were in DMEM medium, both supplemented with 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO2. The in vitro cell uptake of RPA-LP was performed as the following procedures. PC-3 and MDA-MB-435 cells were seeded into 35-mm glass bottom culture dishes and
Table 1. Encapsulated Efficiency and Drug Concentration of FAM-, DiR-, or DXR-Loaded Liposomes with or without RPARPAR Modificationa formulation LP-FAM RPA-LP-FAM LP-DiR RPA-LP-DiR LP-DXR RPA-LP-DXR a
encapsulated efficiency (%) 1.78 1.89 96.1 96.5 94.8 95.2
± ± ± ± ± ±
drug concentration (mg/mL)
0.29 0.28 2.3 1.6 3.1 3.7
0.199 0.203 0.025 0.023 2.33 2.37
± ± ± ± ± ±
0.009 0.010 0.001 0.001 0.09 0.10
Data are represented with mean ± SD (n = 3).
to form the thin film, which was hydrated with saline, extruded, and passed over a gel filtration column. The encapsulation efficiency of these liposomes was determined by the gel filtration method.23 The drug concentrations 220
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Figure 4. In vitro cellular uptake of FAM (A), LP-FAM (B), and RPA-LP-FAM (C) by PC-3 and MDA-MB-435 (inset in C) cells was shown as fluorescent images and flow cytometry data. The accumulation of RPA-LP-FAM in PC-3 cells was significantly enhanced compared with that of LPFAM. The accumulation of RPA-LP-FAM in MDA-MB-435 cells was significantly lower than that in PC-3 cells The red numbers in the rightmost column represent % fluorescence positive cells and mean fluorescence intensity, respectively.
cultured for 24 h. Then the cells were incubated with RPA-LPFAM in cell-culture medium supplemented with 10% FBS for 2 h, washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and examined with a laser scanning confocal microscope (DMI4000 B, LEICA, Germany). LP-FAM was used as control. The quantitative analysis of cell uptake was performed by flow cytometry. After the incubation with RPA-LP-FAM, the cells were washed, trypsinized, centrifuged, resuspended in PBS, and detected with a flow cytometer (FACSAria, BD, United States). 2.5. In Vivo and Ex Vivo Fluorescence Imaging of RPA-LP. The nude mice model of PC-3 tumor was established by subcutaneously injecting tumor cells (5 × 106 cells in 200 μL F-12 medium) into the right shoulder blade. The mice were used for the in vivo imaging test when the diameter of the tumor tissue reached 4−6 mm. RPA-LP-DiR was i.v. injected into tumor-bearing nude mice via the tail vein to examine their targetability, and LP-DiR was used as control. The dose of DiR was 0.1 mg/kg. At predetermined time points, the mice were anesthetized and imaged using a live animal imaging system (CRi, MA, United States, excitation/emission, 730/790 nm). At 48 h postinjection, the mice were sacrificed, and tumor tissues and normal organs (heart, liver, spleen, lung, and kidney) were excised to be examined using the imaging system. 2.6. Immunofluorescence Staining Analysis. Immunofluorescence staining analysis was used to study the tumorpenetrating ability of RPA-LP. RPA-LP-FAM was i.v. injected into the tumor-bearing nude mice via the tail vein, and LP-FAM were as the control. The dose of FAM was 0.94 mg/kg. At 6 h postinjection, the subcutaneous tumor tissues were excised following the sacrifice of the mice, fixed in 4% paraformaldehyde, dehydrated in 30% sucrose solution, embedded in OCT
(Tissue-Tek), and frozen sectioned. The slides were incubated with rat anti-mouse CD31 and then rhodamine-conjugated goat anti-rat IgG-R to label the blood endothelial cells. After incubation with DAPI, the slides were imaged under a laser scanning confocal microscope. 2.7. Growth Inhibitory Effect of RPA-LP-DXR on Tumor Cells. The growth inhibitory effect of RPA-LP-DXR on PC-3 tumor cells was evaluated by MTT assay according to the previously reported procedure.26 The cells were seeded in a 96-well plate (2000 cells in 200 μL per well), and cultured for 24 h. The medium was then substituted with a series of concentrations of DXR, LP-DXR, and RPA-LP-DXR. Three days later, the formulations were removed, and MTT was added and incubated for 4 h. Next, the MTT solution was removed, and DMSO was added to the wells. The absorption representing cell viability was detected using a microplate reader (PowerWave XS, Bio-TEK, United States) at the wavelength of 490 nm. 2.8. Growth Inhibition of RPA-LP-DXR on Tumors in Vivo. The nude mice model of PC-3 tumor was established according to the above-mentioned procedure. Four groups of mice (n = 6) were i.v. injected with N.S., DXR, LP-DXR and RPA-LP-DXR at 29, 35, 41 days postinoculation, respectively. The total DXR dose for each group was 22.5 mg/kg. The growth inhibitory effect on tumors was evaluated by measuring the tumor size every 2 days according to the following formula: tumor volume = length × (width)2/2. The mice were sacrificed on the 51st day postinoculation, and the volume and weight of excised tumor tissue were measured. 2.9. Statistical Analysis. Data were presented as mean ± SD unless otherwise indicated. Unpaired Student’s t test was used to assess the statistical significance of differences 221
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Figure 5. In vivo (A, B) and ex vivo (C) fluorescent images of nude mice bearing subcutaneous tumor after i.v. injection of RPA-LP-DiR (a) and LPDiR (b) via the tail vein. The accumulation of RPA-LP-DiR in tumor tissue was significantly higher than that of LP-DiR (A). The enlarged images of tumor regions showed that RPA-LP-DiR was mainly distributed in the vicinity of tumor blood vessels from 0 to 4 h postinjection (marked by the black arrows) and penetrated into the whole tumor tissue after 4 h (B). RPARPPR modification increased the accumulation of liposomes in livers (C). The color bar at the bottom represents the fluorescence efficiency.
(* for p < 0.05; ** for p < 0.01; *** for p < 0.001.). GraphPad Prism v5.0 was used to measure the IC50 values in the MTT assay.
RPARPAR, the maleimide peak completely disappeared. This indicated that the Michael-type addition between the maleimide group of DSPE-PEG-Mal and thiol moieties of C-RPARPAR was complete. As shown in Figure 2C, in the FTIR spectrum of DSPEPEG-RPARPAR, the bands at 1666.8 cm−1 and 3400 cm−1 were remarkably enhanced compared with that of DSPE-PEGMal. The two bands should be attributed to CO stretching and N−H stretching from the amide groups, respectively. Thus, the enhancement was due to the amide groups from the peptide structure of DSPE-PEG-RPARPAR. This suggested that the peptide RPARPAR had been conjugated to DSPEPEG-Mal.
3. RESULTS 3.1. Characterization of DSPE-PEG-RPARPAR. The molecular structure of DSPE-PEG-RPARPAR is displayed in Figure 2A. Figure 2B shows the NMR spectra of DSPE-PEGMal and DSPE-PEG-RPARPAR. In the spectrum of DSPEPEG-Mal, the methylene protons of DSPE, the repeat units of PEG, and the maleimide group exhibited peaks at 1.26 ppm, 3.7−3.8 ppm, and 6.7 ppm, respectively. In contrast, while the two former peaks remained in the spectrum of DSPE-PEG222
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Figure 6. Immunofluorescence images of tumor tissues of nude mice i.v. injected with LP-FAM and RPA-LP-FAM, respectively. Most of LP-FAM (green) is located adjacent to tumor blood vessels (shown for CD31, red), whereas RPA-LP-FAM (green) is distributed into the whole tumor tissue, demonstrating the tumor-penetrating ability of RPA-LP.
3.2. Characterization of liposomes. The encapsulated efficiency and drug concentration of all liposomes (LP-DXR, RPA-LP-DXR, LP-FAM, RPA-LP-FAM, LP-DiR, and RPA-LPDiR) are shown in Table 1. As examined by the DLS method, all liposomes (LP-DXR, RPA-LP-DXR, LP-FAM, RPA-LP-FAM, LP-DiR, and RPA-LP-DiR) exhibited narrowly distributed vesicle sizes around 90 nm (Figure 3A). These liposomes exhibited nearly spherical shapes with uniform size as displayed in the TEM images (Figure 3B). No obvious differences in encapsulated efficiencies and vesicle sizes were found between RPARPARmodified liposomes and unmodified liposomes. 3.3. In Vitro Cellular Uptake of RPA-LP. The fluorescent images (Figure 4) of cellular uptake showed that the accumulation of RPA-LP-FAM in PC-3 tumor cells was remarkably higher than that of LP-FAM. As shown in the flow cytometry data, the percentage of fluorescence-positive cells and the mean fluorescent intensity were 19.7% and 24.5 for LP-FAM, and 90.9% and 180 for RPA-LP-FAM, respectively. In order to testify to the role of NRP-1 in the cellular uptake of RPA-LP-FAM, an NRP-1-deficient cell line, MDA-MB-435, was used as the control. As a result, the accumulation of RPA-LP-FAM in MDAMB-435 cells was significantly lower than that in PC-3 cells. The results indicated that the mediation of NRP-1 enabled the active internalization of liposomes by PC-3 tumor cells. 3.4. In Vivo Tumor-Targeting Ability of RPA-LP. The in vivo fluorescent images showed that the fluorescent intensity of tumor regions for RPA-LP-DiR was significantly higher than that for LP-DiR at different time points (Figure 5A). From the enlarged images of the tumor regions (Figure 5B), we can see that from 0 to 4 h postinjection, the RPA-LP-DiR was mainly distributed at the vicinity of the tumor blood vessels (marked by the black arrows), which was possibly due to the specific binding of RPA-LP-DiR to NPR-1 on the tumor blood vessels. From 4 to 48 h postinjection, RPA-LP-DiR spread into the whole tumor tissue, possibly due to the tumor-penetrating ability of RPA-LP. As shown in the ex vivo fluorescent image (Figure 5C), liposomes displayed the highest accumulation in tumor tissue, next was liver and spleen, and then other organs. RPARPPR modification increased the accumulation of liposomes in livers, which should be attributed to the increased
phagocytosis of liposomes through mononuclear phagocyte system caused by RPARPPR on the surface of liposomes. 3.5. Tumor-Penetrating Function of RPA-LP. Immunofluorescence analysis of tumor tissue was performed to investigate the tumor-penetrating function of RPA-LP. The fluorescence images (Figure 6) showed that at 6 h postinjection most of LP-FAM stayed in the vicinity of tumor blood vessels. In contrast, most of RPA-LP-FAM spread into the deep tumor tissue, demonstrating the good tumor-penetrating function of RPA-LP. The results suggested that the modification of the tumorpenetrating peptide RPARPAR enabled liposomes to penetrate through tumor blood vessels and tumor stroma, thereby possibly enhancing their antitumor effect. 3.6. Cytotoxicity of RPA-LP-DXR. The results of cytotoxicity of DXR, LP-DXR, and RPA-LP-DXR (Figure 7)
Figure 7. Inhibitory effect of DXR, LP-DXR, and RPA-LP-DXR on PC-3 tumor cells as examined by MTT assay. RPA-LP-DXR exhibited much lower IC50 values than did LP-DXR, indicating that RPARPAR modification increased the in vitro growth inhibitory effect of DXR liposomes.
showed that the IC50 values were 43.65 μM for LP-DXR and 10.47 μM for RPA-LP-DXR, indicating that RPARPAR modification increased the in vitro growth inhibitory effect of liposomal DXR. 3.7. In Vivo Antitumor Effect of RPA-LP-DXR. The results of the antitumor effect were displayed as the relative tumor volume profiles (Figure 8 A) and the weight and volume of ex vivo tumor tissues (Figure 8B). Compared with the control N.S., RPA-LP-DXR (p < 0.001), LP-DXR (p < 0.001), 223
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tumor vessels, depending on the tumor type and status.27 For instance, it is usually very weak in prostate cancer, pancreatic cancer, metastatic liver cancer, and glioblastoma.8,28 Second, the barriers of tumor blood vessels and tumor stroma hinder the transport of nano drug delivery systems into deep tumor tissue, rendering the uniform delivery of nano drug delivery systems extremely difficult.5 In order to enhance the drug transport in tumor tissue, a wide variety of methods have been investigated, including lowering tumor interstitial fluid pressure29 or modulating tumor ECM structure.30 However, these techniques seem premature now because of the complex interaction involving various physiological parameters.13 Our results proved that TPP-modified liposomes could penetrate through tumor blood vessels and deep into the tumor tissues, and inhibit the growth of tumor cells in the whole tumor. This study suggests that the TPP mediation is an effective strategy for improving the transport of nanomedicines in the tumor tissues. According to previous studies, the mechanism of the tumorpenetrating ability of free TPPs is that the interaction of TPPs with NRP-1 can cause vascular-induced leakage. The interaction may induce the vesiculo−vacuolar organelles transport pathway that allows the TPPs to escape from the vasculature and spread into the deep tumor tissue.4,5 This study proved that, when TPPs are coupled on the surface of liposomes, their interactions with NRP-1 are still able to cause vascular leakage and tissue penetration in vivo. Several reports evidenced that the coadministration of unconjugated TPP could improve the penetration of nanomedicines into the deep tumor tissues, which was probably due to the activation of the bulk transport pathway.19,31 In this study, the TPP was conjugated to the surface of liposomes to enable them to penetrate the tumor blood vessels and tumor tissues. This means that both conjugated and unconjugated TPP can lead to tumor penetration of nanomedicines. In addition TPP-modified liposomes can be called “multi-functional nanoparticles”, since they not only possess tumor-penetrating property but also have targetability to tumor cells and tumor blood vessels. The simultaneous killing effect to tumor cells and tumor endothelial blood cells in the whole tumor tissue produces a synergistic antitumor effect.
Figure 8. Antitumor effect of DXR, LP-DXR, and RPA-LP-DXR shown as the relative tumor volume profiles (A) and the weight and volume of ex vivo tumor tissues (B). DXR, LP-DXR, and RPA-LPDXR all exhibited considerable growth inhibiting effect compared with N.S. (p < 0.001). RPA-LP-DXR showed significantly stronger antitumor effect than LP-DXR (p < 0.005). The relative tumor volume was calculated as the ratio of tumor volume to initial tumor volume. Arrows indicate the administration day.
and DXR (p < 0.001) all showed considerable growth inhibitory effect on tumors based on the tumor weight. Furthermore, RPA-LP-DXR showed the strongest efficacy among all the experimental groups, significantly stronger than LP-DXR (p < 0.005). These results indicated that RPARPAR modification increased the antitumor efficacy of DXR liposomes, which should be attributed not only to the specific binding with NRP-1 overexpressed on tumor cells and tumor blood vessels, but also to the tumor-penetrating ability of RPA-LP.
5. CONCLUSION In summary, the modification of RPARPAR enabled the penetration of liposomes through tumor blood vessels and into the deep tumor tissues and significantly enhanced the antitumor effect of DXR liposomes. This study may present an effective strategy for improving the transport of liposomes into tumor tissue.
4. DISCUSSION In this study, the tumor-penetrating peptide RPARPAR was used to modify the DXR liposomes to improve the transport efficiency in tumor tissues. The regular peptide-mediated nano drug delivery systems can only be extravasated from blood vessels to tumor tissue by EPR effect, and bind with the tumor cells near the blood vessels. By contrast, our results suggested that RPARPAR modification enabled the liposomes to penetrate through tumor blood vessels and tumor stroma and into the deep tumor tissues, and bind with tumor cells in the whole tumor tissues. Eventually, the RPARPAR modification significantly enhanced the antitumor effect of liposomal DXR. Currently, the inconsistent clinical outcomes of nano drug delivery systems could be attributed to several factors. First, although the EPR effect is an important factor that favors tumor accumulation, it is not applicable for all tumors due to the variation of the degree of tumor vascularization and porosity of
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Corresponding Authors
*Telephone: +86 21 3429 1286. Fax: +86 21 3429 1286. Email:
[email protected]. *Telephone: +86 21 5198 0094. Fax: +86 21 5288 0090. Email:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2013CB932500), National Natural Science Foundation of China (81202471, 51203024), International Cooperation Project from Science and Technology Commission 224
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tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 2010, 328 (5981), 1031−1035. (20) Ruoslahti, E. Peptides as targeting elements and tissue penetration devices for nanoparticles. Adv. Mater. 2012, 24 (28), 3747−3456. (21) Yan, Z.; Wang, F.; Wen, Z.; Zhan, C.; Feng, L.; Liu, Y.; Wei, X.; Xie, C.; Lu, W. LyP-1-conjugated PEGylated liposomes: A carrier system for targeted therapy of lymphatic metastatic tumor. J. Controlled Release 2012, 157 (1), 118−125. (22) Yan, Z.; Zhan, C.; Wen, Z.; Feng, L.; Wang, F.; Liu, Y.; Yang, X.; Dong, Q.; Liu, M.; Lu, W. LyP-1-conjugated doxorubicin-loaded liposomes suppress lymphatic metastasis by inhibiting lymph node metastases and destroying tumor lymphatics. Nanotechnology 2011, 22 (41), 415103. (23) Arien, A.; Dupuy, B. Encapsulation of calcitonin in liposomes depends on the vesicle preparation method. J Microencapsulation 1997, 14 (6), 753−760. (24) Yu, Z.; Schmaltz, R. M.; Bozeman, T. C.; Paul, R.; Rishel, M. J.; Tsosie, K. S.; Hecht, S. M. Selective tumor cell targeting by the disaccharide moiety of bleomycin. J. Am. Chem. Soc. 2013, 135 (8), 2883−2886. (25) Li, C.; Wang, Y.; Zhang, X.; Deng, L.; Zhang, Y.; Chen, Z. Tumor-targeted liposomal drug delivery mediated by a diseleno bondstabilized cyclic peptide. Int. J. Nanomed. 2013, 8, 1051−1062. (26) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65 (1−2), 55−63. (27) Bae, Y. H. Drug targeting and tumor heterogeneity. J. Controlled Release 2009, 133 (1), 2−3. (28) Zhan, C.; Wei, X.; Qian, J.; Feng, L.; Zhu, J.; Lu, W. Co-delivery of TRAIL gene enhances the anti-glioblastoma effect of paclitaxel in vitro and in vivo. J. Controlled Release 2012, 160 (3), 630−636. (29) Fan, Y.; Du, W.; He, B.; Fu, F.; Yuan, L.; Wu, H.; Dai, W.; Zhang, H.; Wang, X.; Wang, J.; Zhang, X.; Zhang, Q. The reduction of tumor interstitial fluid pressure by liposomal imatinib and its effect on combination therapy with liposomal doxorubicin. Biomaterials 2013, 34 (9), 2277−2288. (30) Beyer, I.; Li, Z.; Persson, J.; Liu, Y.; van Rensburg, R.; Yumul, R.; Zhang, X. B.; Hung, M. C.; Lieber, A. Controlled extracellular matrix degradation in breast cancer tumors improves therapy by trastuzumab. Mol. Ther. 2011, 19 (3), 479−489. (31) Gu, G.; Gao, X.; Hu, Q.; Kang, T.; Liu, Z.; Jiang, M.; Miao, D.; Song, Q.; Yao, L.; Tu, Y.; Pang, Z.; Chen, H.; Jiang, X.; Chen, J. The influence of the penetrating peptide iRGD on the effect of paclitaxelloaded MT1-AF7p-conjugated nanoparticles on glioma cells. Biomaterials 2013, 34 (21), 5138−5148.
of Shanghai Municipality (12520708000), Shanghai Rising-Star Program (B-type) (12QB1402800), Traditional Chinese medicine scientific research fund project in Zhejiang province (2013ZB137), and Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China (SDD2011-06).
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