Peptide-modified liposomes for selective targeting of bombesin receptors overexpressed by cancer cells: a potential theranostic agent

Abstract

Objectives: Drug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors.

Methods: A new amphiphilic peptide derivative (MonY-BN) containing the BN(7-14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C(18) alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized.

Results: Liposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with peptide-free DSPC liposomes (1.4% ± 0.2% at 37°C). Incubation of cells with DSPC/ MonY-BN/Dox showed significantly lower cell survival compared with DSPC/Dox-treated cells, in the presence of 100 ng/mL and 300 ng/mL drug amounts, in cytotoxicity experiments. Intravenous treatment of PC-3 xenograft-bearing mice with DSPC/MonY-BN/Dox at 10 mg/kg Dox dose produced higher tumour growth inhibition (60%) compared with nonspecific DSPC/ Dox liposomes (36%) relative to control animals.

Conclusion: The structural and loading properties of DSPC/MonY-BN liposomes along with the observed in-vitro and in-vivo activity are encouraging for further development of this approach for target-specific cancer chemotherapy.

Keywords: PC-3 cells; bombesin peptide; doxorubicin delivery; gastrin-releasing peptide receptors; theranostic applications.

Figure 1

Schematic representation of MonY-Peg27(DTPA)-BN (MonY-BN). Notes: MonY-BN molecule contains five components: a hydrophobic moiety with two C₁₈ alkyl chains, a long polyoxoethilene spacer (Peg27), the DTPA chelating agent, a short linker (Ahoh), and the BN(7–14) peptide. The peptide sequence is reported using the three-letter amino acid code. Abbreviations: BN, bombesin; DTPA, diethylenetriaminepentaacetate; PEG, polyethylene glycol.

Figure 2

Synthetic procedure for obtaining MonY-BN by solid-phase methods. Notes: The empty circle, ○, represents the solid support (Rink amide resin). The first step of the scheme reports the Fmoc removal by using DMF/Pip (70/30) mixture and the amino acid (Fmoc-aa-OH) coupling under standard SPPS conditions. This step is repeated eightfold to obtain the BN(7–14) sequence (reported according the single-letter amino acid code). Successively, solid-phase synthesis continues to bind on the peptide N-terminus to the other organic components. In the last step, cleavage (TFA/TIS/H₂O) from the resin is performed to give a fully unprotected carboxamide product. Abbreviations: BN, bombesin; DMF, N,N-dimethylformamide; DTPA, diethylenetriaminepentaacetate; PEG, polyethylene glycol; SPPS, solid-phase-peptide-synthesis; TFA, trifluoroacetic acid; TIS, tri-isopropylsylane.

Figure 3

Binding assays of DSPC/MonY-BN and DSPC/(C₁₈)₂DTPA¹¹¹In-radiolabeled liposomes on PC-3 cell line overexpressing the GRP at 37°C and 4°C at 1 hour. Abbreviations: BN, bombesin; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DTPA, diethylenetriaminepentaacetate; GRP, gastrin-releasing peptide.

Figure 4

Cytotoxicity of liposomal Dox against human prostatic cancer cells. Notes: PC-3 cells (8000 cells/well) were incubated with DSPC/MonY-BN/Dox, DSPC/Dox, and free Dox at 37°C (liposome formulations containing 500 or 1500 ng of lipids and 100 or 300 ng of Dox, respectively). Control experiments were performed using free Dox, and empty liposomes of DSPC/MonY or DSPC at the same experimental conditions. After 8 hours, the medium was removed, and after an additional 72 hours an MTS assay was performed. Data are expressed as percentage of control. Each value is the mean ± SEM of three experiments performed in triplicate. Abbreviations: BN, bombesin; Dox, doxorubicin; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; SEM, standard error of the mean.

Figure 5

Comparative efficacy study in BALB/c nude mice bearing xenografts (PC-3) on tumor growth. Notes: Treatment started approximately 5 weeks after cell implantation. Mice (n = 5) were administered intravenously with injection of 100 μL of liposome suspensions of DSPC/MonY-BN/Dox, or DSPC/Dox (1.0 × 10⁻² M lipid concentration, at a dose of 10 mg of Dox/kg) or 100 μL of HBS buffer (control). Therapeutic efficacy was assessed by measuring tumor volume (mean ± SD) over time. Results were analyzed using two-way analysis of variance, followed by Bonferroni’s test. * denotes P < 0.05 and ** denotes P < 0.01 versus saline. DSPC/MonY-BN/Dox and DSPC/Dox produce, with respect to the saline solution, a TGI of 60% and 36%, respectively. Abbreviations: BN, bombesin; Dox, doxorubicin; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBS, HEPES buffered saline; SD, standard deviation; TGI, tumor growth inhibition.