Development of a Screening System for Targeting Carriers Using Peptide-Modified Liposomes and Tissue Sections

Abstract

Liposomes have been used as targeting carriers for drug delivery systems (DDSs), and the carriers are able to be modified with targeting ligands, such as antibodies and peptides. To evaluate the targetability of DDS carriers modified with a targeting ligand, culture cells expressing the targeting molecules as well as small animals are used. Furthermore, in vitro and in vivo screening analyses must be repeatedly performed. Therefore, it is important to establish an easy and high-precision screening system for targeting carriers. With this aim, we focused that whether this ex vivo system could easily support assessment of interaction between targeting ligand and its receptor under physiological environment and further screen the DDS carrier-modified with targeting moiety. We examined targeting ability via in vitro, ex vivo, and in vivo analyses using integrin α_vβ₃-targeting C16Y-L. For the in vitro analysis, the cellular uptake of C16Y-L was higher than that of control liposomes in colon26 cells. For the ex vivo analysis, we performed an immunohistochemical analysis using colon26 tumor sections. C16Y-L was specifically attached to the tumor sections, as found in the in vitro analysis. Moreover, to evaluate the ex vivo–in vivo correlation, we examined the intratumoral localization of C16Y-L. This result showed that C16Y-L was accumulated not only in the tumor tissue but also in the tumor vasculature after the intravenous injection of C16Y-L, suggesting that the ex vivo peptide-modified liposomal analysis was correlated with the in vivo analysis. Thus, the ex vivo peptide-modified liposomal analysis may be an easy and rapid screening system with high-precision and for consideration in in vivo conditions.

Recently, remarkable progress in drug discovery technology has been achieved, allowing chemical compounds with useful bioactivity, such as anticancer agents, to be synthesized in a short span of time. However, in the clinical setting, the use of anticancer agents is limited by their side effects. To overcome these side effects, there has been a recent focus on drug formulations incorporated into drug delivery systems (DDSs). DDSs have a wide variety functions (e.g., PEGylation^1–5) or controlled release using pH^6,7)). One DDS technology is the targeting system. Targeting systems aid in reducing drug side effects while enhancing the therapeutic effect that is targeted to a specific site. In this regard, nano-sized carriers, such as liposomes and micelles, have been developed for cancer treatment targeting systems, and these carriers are often modified with targeting ligands, such as antibodies,^8–10) folate,¹¹⁾ transferrin,¹²⁾ sugars,¹³⁾ and peptides,^14–17) to enhance their selectivity to tumor tissues. To develop useful carriers, it is important to evaluate their targeting function via in vitro and in vivo assays. Biological cells are affected by external forces such as shear stress and signal transduction in in vivo, and cell lines that are cultured in vitro under a given set of circumstances often lose their functions, such as their differentiation ability. Therefore, data acquired from in vivo analyses often do not correspond to that of in vitro analyses. These experiments may be time-consuming and a waste of effort, among other issues. Therefore, the establishment of an easy, high-precision screening system for targeting carriers is required.

Previously, we developed integrin α_vβ₃-targeting liposomes (C16Y liposomes: C16Y-L).¹⁷⁾ The C16Y peptide is a 12-amino acid synthetic peptide, which is a modified C16 peptide, derived from the globular domain of the laminin γ1 chain that binds to endothelial cell integrins, α_vβ₃ and α₅β₁.^18,19) C16Y-L can selectively target mouse melanoma B16 cells that highly express integrin α_vβ₃. Recently, it was reported that integrin α_vβ₃ is expressed in colon26 cells,²⁰⁾ and colon26 cells were used to screen for integrin α_vβ₃-targeting carriers.²¹⁾

In this study, to establish an easy, high-precision screening system for targeting carriers, we focused that whether this ex vivo system could easily support assessment of interaction between C16Y-L and tumor section (Fig. 1). We hypothesized screening system which was considered in vivo condition lead to more precision analysis and therefore developed ex vivo screening system using peptide-modified liposomes and tissue sections. First, we examined whether C16Y-L interacts with colon26 cells and colon26 tumor tissue sections. Next, we examined the intratumoral localization of C16Y-L to evaluate the ex vivo–in vivo correlation.

Fig. 1. Schematic of the Screening System for Targeting Carriers Using Peptide-Modified Liposomes and Tissue Sections

Cell lines that are cultured in vitro under a given set of circumstances lose some of their function, such as their ability to differentiate. In screening analyses for targeting carriers, in vivo analyses do not necessary correspond to in vitro analyses. For the in vivo screening of targeting carriers, we used peptide-modified liposomes and tissue sections.

MATERIALS AND METHODS

Materials

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoylphosphatidylethanolamine-methoxy-polyethyleneglycol (DSPE-PEG₂₀₀₀-OMe), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-polyethyleneglycol-maleimide (DSPE-PEG₂₀₀₀-Mal) were purchased from NOF Corporation (Tokyo, Japan). Tris(2-carboxyethyl)-phosphine hydrochloride (TCEP) was purchased from Thermo Fisher Scientific Inc. (San Jose, CA, U.S.A.). For cell culture, Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Kohjin Bio Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Equitech Bio Inc. (Kerrville, TX, U.S.A.). All other materials were used without further purification.

Preparation of Liposomes and C16Y Peptide-Modified Liposomes

Liposomes composed of DSPC and DSPE-PEG₂₀₀₀-OMe at a molar ratio of 94 : 6 were prepared via a reverse-phase evaporation method, as described previously.^16,17) In brief, all reagents were dissolved in 1 : 1 (v/v) chloroform/diisopropylether. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (10 mM, pH 6.0) was then added to the lipid solution, and the mixture was sonicated and evaporated at 65°C. The organic solvent was completely removed, and the size of the liposomes was adjusted to approximately 100–200 nm using extruding equipment and sizing filters (pore size: 200 nm, Nuclepore Track-Etch Membrane, Whatman plc, U.K.). After the sizing, the liposomes were passed through a 0.45-µm pore size filter (Syringe filter, ASAHI TECHNOGLASS Co., Chiba, Japan) for sterilization.

The C16Y peptide (DFKLFAVYIKYR-GGC) and a control scramble peptide (IKDYLYFARVKF-GGC) were manually synthesized using a 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase strategy, which was prepared in the COOH terminal amide form and purified via reverse-phase HPLC. The purity and identity of the peptides were confirmed via analytical HPLC and electrospray ionization mass spectrometry at the Central Analysis Center, Tokyo University of Pharmacy and Life Sciences. For the preparation of peptide-modified liposomes, peptides were added to the liposomes and gently mixed, as described previously.¹⁷⁾ In brief, for coupling, the C16Y-Cys peptide, at a molar ratio of 1.5-fold DSPE-PEG₂₀₀₀-Mal, was added to the liposomes in the presence of TCEP (final concentration: 20 mM). The mixture was incubated for 6 h at room temperature to conjugate the cysteines of the C16Y-Cys peptides with the maleimides of the liposomes via a thioether bond. The resulting C16Y-Cys peptide-conjugated liposomes (C16Y-L) and the scramble peptide-conjugated liposomes (Scramble-L) were passed through a Sephadex G-50 spin column to remove any excess peptides. The C16Y-L and Scramble-L were modified with 6 mol% PEG and 2 mol% peptides. The particle size and the ζ potential of the liposomes were measured using a NICOMP 380 ZLS (Particle Sizing Systems, Santa Barbara, CA, U.S.A.). The mean diameter of the C16Y-L particles was estimated as 199.2±9.5 nm, and the ζ potential of the C16Y-L particles was estimated as −6.45±3.12 mV. The mean diameter of the Scramble-L particles was estimated as 160.3±1.9 nm, and the ζ potential of the Scramble-L particles was also estimated as −3.43±4.20 mV. The mean diameter of the non-modified liposomes (PEG-L) was estimated as 165.5±9.5 nm, and the ζ potential of the PEG-L was also estimated as −6.15±1.54 mV.

Cell Lines

A mouse melanoma cell line (B16) and mouse colorectal carcinoma cells (colon26) were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37°C in a humidified 5% CO₂ atmosphere.

Animals and Tumor Models

Male BALB/c mice (6 weeks old) and C57BL/6 (7–8 weeks old) were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan). All animal use and relevant experimental procedures were approved by the Tokyo University School of Pharmacy and the Life Science Committee on the Care and Use of Laboratory Animals. Colon26 cells (1×10⁶ cells/mouse/100 µL) or B16 cells (5×10⁶ cells/mouse/100 µL) in hank’s balanced salt solution were inoculated subcutaneously in the flanks of mice. Ex vivo histological studies were performed when the tumors were approximately 250 mm³. In vivo intratumoral localization distribution test was performed when the tumors were approximately 100–250 mm³.

Flow Cytometry Analysis

The intracellular uptake of the liposomes was determined by flow cytometry analysis. Colon26 (5×10⁴ cells/well) were seeded in a 24-well plate and incubated for 24 h at 37°C in 5% CO₂. The medium was then replaced with DiI-labeled non-modified liposomes (PEG-L), C16Y-L, or Scramble-L diluted with culture medium to a final lipid concentration of 200 nmol/mL. The plates were incubated for 1 h at 37°C. The medium was removed, and subsequently, each cell line was washed with phosphate-buffered saline (PBS) and the cell samples were examined via flow cytometry using a FACScan (Becton Dickinson, San Jose, CA, U.S.A.). Data were collected for 10000 gated events and analyzed using the CELL Quest software program.

Ex Vivo Peptide-Modified Liposomal Analysis

Colon26 cells (1×10⁶ cells/mouse) were inoculated subcutaneously in the right flanks of BALB/c mice. Ten days after tumor inoculation (when the tumor volume reached approximately 250 mm³), the mice were sacrificed, and the tumors and organ tissues (heart, spleen, liver, and kidney) were dissected (n=3). These tissues were fixed in 4% paraformaldehyde, substituted with 20% sucrose, and then embedded in optimal cutting temperature compound (Sakura Finetech, Co., Ltd., Tokyo, Japan) and frozen at −80°C. Each tumor and organ tissue section was prepared with a thickness of 6 µm and mounted on a poly-l-lysine-coated slide. For immunohistochemistry, the tumor sections were blocked with 10% rabbit serum in Tris-buffered saline (TBS) for 30 min at 4°C and incubated with anti-mouse CD31 antibody (1 : 50; BD Pharmingen) overnight at 4°C. Subsequently, the sections were incubated with Alexa Fluor 488 rabbit anti-rat immunoglobulin G (IgG) (1 : 200; Molecular Probes) and DiI-labeled liposomes (800 nmol/mL) for 1 h at room temperature.

For the inhibition assay, recombinant mouse integrin α_vβ₃ (20 µg/mL; R&D Systems, Inc.) was added to the liposomes (400 nmol/mL). The mixture was incubated for 30 min at room temperature. Subsequently, the mixture was added to the tumor sections and incubated for 1 h at room temperature. Each tumor and organ section was then mounted with VECTASHIELD HardSet Mounting Medium with 4′-6-diamidino-2-phenylindole (DAPI) (VECTOR LABORATORIES, INC., U.S.A.) and fluorescently observed using a BZ-9000 microscope (KEYENCE, Japan).

In Vivo Intratumoral Localization of the Liposomes

The B16 cells (5×10⁶ cells/mouse) were inoculated subcutaneously in the right flanks of C57BL/6 mice. Ten days after tumor inoculation (when the tumor volume reached approximately 100–250 mm³), DiI-labeled liposomes (lipid concentration: 10 µmol/kg) were administered via the tail veins of the mice (n=3). At 6 h after injection of the liposomes, the mice were sacrificed, and the tumors were dissected. For immunohistochemistry, we followed the above methods described in the ex vivo histological analysis section.

RESULTS AND DISCUSSION

First, to examine the targeting ability of the C16Y-L to colon26 cells, we performed a flow cytometry analysis and an immunohistochemical analysis. In the flow cytometry analysis, the cells treated with C16Y-L showed enhanced fluorescence intensities compared with those treated with PEG-L and Scramble-L (Fig. 2(A)). Compared to the fluorescence intensity of PEG-L group, that of Scramble-L was slightly higher. It may be due to the electrostatic interaction of Scramble-L and cell line. However, compared to Scramble-L and C16Y-L, the fluorescence intensity of C16Y-L is highest. This means that the association with C16Y-L to cell line results from recognition of receptor, not electrostatic interaction. In addition, to elucidate the specific attachment of C16Y-L to the tumors, an immunohistochemical analysis was performed by directly adding C16Y-L labeled with DiI to the sectioned tumor tissue. This result showed that only C16Y-L was attached to the tumor sections, whereas the control Scramble-L was not (Fig. 2(B)). These results suggested that C16Y-L was strongly associated with the colon26 cells, and the specific association of C16Y-L was sustained in the sectioned tumor tissue. Simultaneously, their specimens were further counterstained with an antibody against CD31 as a characteristic of endothelial-cell-specific markers (Fig. 2(B)). The result showed that C16Y-L attached not only tumor sections but also to part of the tumor vasculature. This result is consistent with our previous in vitro report.¹⁷⁾ Therefore, the ex vivo peptide-modified liposomal analysis may be correlated with the in vitro analysis.

Fig. 2. Specific Affinity of C16Y-L for Tumor Cells (A) and for Tumor Tissue (B)

Cells were treated with DiI-labeled liposomes for 1 h at 37°C. After incubation, the cells were washed, and their fluorescence intensities were measured via flow cytometry. The tumor-bearing mice (n=3) were sacrificed, and each tumor was dissected. The tumor sections were then prepared as slices with widths of 6 µm each. DiI-labeled liposomes were added to each section, and the sections were stained with anti-CD31 antibody to label the endothelial cells. Gray: fluorescence of DiI. White: CD31. Scale bars represented 100 µm.

Next, to determine whether C16Y-L exhibited a specific affinity to tumor tissue sections, we assessed the specific affinity of C16Y-L in normal tissue sections. As shown in Fig. 3, the attachment level of C16Y-L was high in the sectioned tumor tissue but was poor in normal tissue (heart, liver, spleen, and kidney). In addition, almost no attachment of the control liposomes (non-modified liposome or Scramble-L) to any tissue sections was observed. Moreover, to examine the specific binding mechanism of C16Y-L on the sectioned tumor tissue, we attempted to inhibit the attachment of C16Y-L to the tumor tissue by adding recombinant α_vβ₃ protein. As shown in Fig. 4, the attachment of C16Y-L to the sectioned tumor tissue was inhibited by treatment with recombinant α_vβ₃ protein. These results suggested that the C16Y peptide attached to the liposomes via their targeting receptor, integrin α_vβ₃, in ex vivo conditions. Because integrin α_vβ₃-targeting liposomes, termed C16Y-L, recognized the targeting site in ex vivo assays, this ex vivo peptide-modified liposomal analysis may be a useful screening system for targeting carriers. Therefore, we examined whether this screening system was correlated with the in vivo analysis. DiI-labeled liposomes (C16Y-L, non-modified liposome, or Scramble-L) were administered to the mouse tumor model via a tail vein. As shown in Fig. 5, C16Y-L was highly accumulated in the tumor tissue compared with non-modified liposomes and Scramble-L. In addition, C16Y-L was accumulated not only in the tumor tissue but also in the tumor vasculature. This result showed that the ex vivo peptide-modified liposomal analysis was correlated with the in vivo analysis. In this study, we used the mouse colon26-tumor model (ex vivo assay) and the mouse B16 melanoma model (in vivo assay). The expression level of integrin α_vβ₃ is almost same in both tumors,²⁰⁾ and C16Y-L attached to both types of tumor tissues and to the blood vessels. These results suggested that C16Y-L is able to attach to target sites (i.e., integrin α_vβ₃ high-expression sites) regardless of the tumor type and the experimental conditions (ex vivo and in vivo). Therefore, the ex vivo peptide-modified liposomal analysis using sectioned tissues could be a very useful screening array system for targeting carriers. This ex vivo screening method may not reflect in vivo study completely. However, this method may provide useful information regarding the interaction between targeting ligand and its receptor, leading to narrow down the candidate compound (e.g., peptide) and to reduce time consuming and the number of experimental animals used in vivo study. In future, if established the evaluation systems which are combined with immunostaining, it may become more useful screening system. Moreover, the combination of this array system and a disease-specific carrier (e.g., tumor targeting carrier) could lead to disease diagnosis using pathological tissue sections.

Fig. 3. Attachment of C16Y-L to Normal Tissue and to Tumor Tissue

The tumor-bearing mice (n=3) were sacrificed, and the tumors and organ tissues were dissected. Each tumor and organ tissue section was prepared with a thickness of 6 µm. DiI-labeled liposomes were added to each section, and the sections were incubated for 1 h at room temperature. Gray: fluorescence of DiI. Scale bars represented 100 µm.

Fig. 4. Inhibition of the Attachment of C16Y-L by the Recombinant α_vβ₃ Protein on Tumor Tissue

C16Y-L was first incubated with the recombinant α_vβ₃ protein for 30 min at 37°C. Subsequently, the tumor sections were treated with the mixture for 1 h at room temperature. Gray: DiI. Scale bars represented 100 µm.

Fig. 5. Intravenous Injection and Localization of C16Y-L in Tumors

The tumor-bearing mice (n=3) were intravenously injected with DiI-labeled liposomes when the tumor volume reached 100–250 mm³ (10 d after tumor inoculation). The liposomes were injected as lipids at a dose of 10 µmol/kg. At 6 h after injection, each tumor was dissected, and the tumor sections were sliced at widths of 6 µm. Each section was stained with an anti-CD31 antibody to label endothelial cells. White: CD31. Gray: fluorescence of DiI. Scale bars represent 20 µm.

CONCLUSION

In this study, we developed a screening system for targeting carriers. We examined and compared its targeting ability via in vitro, ex vivo, and in vivo analyses using integrin α_vβ₃-targeting liposomes. In the in vitro analysis, the cellular uptake of C16Y-L was higher than the uptake of PEG-L in colon26 cells. In the ex vivo analysis, we examined the interactions of C16Y-L with colon26 tumor sections. In this result, C16Y-L-specific attachment was observed, as determined by an in vitro analysis. Moreover, to evaluate the ex vivo–in vivo correlation, we examined the intratumoral localization of C16Y-L. C16Y-L was accumulated in regions of high integrin α_vβ₃ expression, as identified in the in vitro and ex vivo peptide-modified liposomal analyses. Thus, the ex vivo peptide-modified liposomal analysis may be a useful screening system with an easy, high-precision and for consideration in in vivo conditions.

Acknowledgment

This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities.

Conflict of Interest

The authors declare no conflict of interest.

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