Nanotechnology offers an alternative to conventional treatment options by enabling different drug delivery and controlled-release delivery strategies. Liposomes being especially biodegradable and in most cases essentially nontoxic offer a versatile platform for several different delivery approaches that can potentially enhance the delivery and targeting of therapies to tumors. Liposomes penetrate tumors spontaneously as a result of fenestrated blood vessels within tumors, leading to known enhanced permeability and subsequent drug retention effects. In addition, liposomes can be used to carry radioactive moieties, such as radiotracers, which can be bound at multiple locations within liposomes, making them attractive carriers for molecular imaging applications. Phage display is a technique that can deliver various high-affinity and selectivity peptides to different targets. In this study, gelatinase-binding peptides, found by phage display, were attached to liposomes by covalent peptide-PEG-PE anchor creating a targeted drug delivery vehicle. Gelatinases as extracellular targets for tumor targeting offer a viable alternative for tumor targeting. Our findings show that targeted drug delivery is more efficient than non-targeted drug delivery.
Liposomal nanotechnology provides a versatile platform for exploring several approaches that can potentially enhance the delivery and targeting of therapies to tumors. As a biodegradable and essentially nontoxic platform, liposomes can be used to encapsulate both hydrophilic and hydrophobic materials and be utilized as drug carriers in drug delivery systems (DDSs). In addition, liposomes can be used to carry radioactive moieties, such as radiotracers, which can be bound at multiple locations within liposomes, making them attractive carriers for molecular imaging applications. In this study, gelatinase-binding peptides were attached to liposomes for synthesizing a targeted drug delivery vehicle.
For active targeting or drug delivery applications or both, intraliposomal encapsylation of multiple targeting agents or therapies can be (i) to the lipid bilayer, which can bind hydrophobic conjugates; (ii) to hydrated compartments for water-soluble components; (iii) by covalent binding directly or by utilizing spacer to the outer lipid leaflet [
The most commonly used nanoformulated drug is Caelyx/Doxil, a liposomal doxorubicin product. It has nearly supplanted doxorubicin in the therapy of ovarian cancer, breast cancer, and Kaposi’s sarcoma. It differs from the former generation liposomal delivery systems, as the outer surface of Caelyx/Doxil is coated with PEG chains that protect the liposomes from being opsonized by components of the immune system in the circulation. These stealth-type liposomes have longer circulation half-times than those for uncoated liposomes. In addition, they are safer than the native drugs themselves (e.g., Caelyx/Doxil is not cardiotoxic, a major concern for native doxorubicin delivery).
For cancer-based applications, peptides that can selectively detect and target metastatic disease and tumor invasive potential may offer critical prognostic information. Metastatic invasion is promoted by the attachment of tumor cells to the extracellular matrix, the degradation of matrix components by tumor-associated proteases, and the cellular movement into the area modified by protease activity. Matrix metalloproteases (MMPs) represent a family of enzymes capable of degrading the basement membrane and extracellular matrix (ECM), thus contributing to tissue remodeling and cell migration [
MMPs may be divided into subgroups, one comprised of type IV collagenases (gelatinases) such as MMP-2 and MMP-9, which play major roles in tumor growth, angiogenesis, and metastatic disease. These gelatinases degrade type IV collagen (and its breakdown product, gelatin) and comprise the primary structural component of the ECM, enabling tumor cells to gain access to the rest of the body. Overexpression and/or prognostic significance of gelatinases have been examined in a range of cancer types, including ovarian cancer [
In this study, binding peptides (BPs) extracted from MMP-9 were attached to liposomes for synthesizing a targeted drug delivery vehicle. Downregulation of MMP-9 is known to exert inhibitory effects on endothelial cell migration and tube formation [
One of the first known specific gelatinase inhibitors, a cyclic MMP-9-binding peptide identified by random phage display libraries (i.e., CTTHWGFTLC peptide later CTT1), has previously been shown to have high affinity not only to MMP-9, but also to MMP-2 [
Chemical structure of 125I-CTT2-peptide. CTT2-peptide is a 17-amino acid peptide with a disulphide bridge between the two cysteines. The amino terminal end of the peptide is amidated to increase its stability. Upon iodination, peptide labeling occurs on the aromatic ring of the tyrosine amino acid.
We initially present the synthesis of PEG-PE-CTT2 peptide-bound micelles and liposomes. The feasibility of utilizing micellar and liposomal nanoformulations as therapeutic delivery vehicles to achieve efficacy in ovarian carcinoma models was explored by attaching the radioiodinated CTT peptide tracer, 125I-CTT2 peptide, to these platforms and loading them with doxorubicin, an inherently fluorescent chemotherapeutic agent. Biodistribution studies of both targeted nanoformulations were performed in normal and immunosuppressed subcutaneous human xenograft models using the CTT2-peptide.
All reagents, unless stated otherwise, were obtained from Sigma-Aldrich (St Louis, Missouri, USA) and culture media from Gibco Life Technologies (Paisley, Scotland). PEG-PE-NHS was from Avanti Polar Lipids Inc. (Alabama, USA) as all the other lipids used in this study.
Peptides were synthesized on an Applied Biosystems 433A (Foster City, CA, USA) automatic synthesizer using Fmoc-chemistry. For disulfide generation, peptides were dissolved at 1 mg/ml in 0.05 M ammonium acetate (pH 8) and mixed with H2O2 for 40 min at room temperature so that 0.5 ml of 3% H2O2 was added per 100 mg peptide. The peptides were purified by reversed phase HPLC, and the molecular weight was identified by mass spectrometry analysis.
Coupling bioactive peptides to PEGylated lipids can alter the pharmacokinetics and dynamics of these peptides. For pharmaceutical formulation purposes, CTT2-peptide (Figure
Chemical structure of CTT2-PEG3400-DSPE. CTT2-PEG3400-DSPE was synthesized by coupling CTT2-peptide to PEG3400-DSPE, followed by purification of the reaction product from the initial mixture.
Schematic illustration of CTT2-PEG-3400-DSPE liposome [
In this procedure, the peptide called CTT2 (cyclo-GRENYHGCTTHWGFTLC-NH2) was covalently attached to PEG phospholipids through the chemical reaction between the terminal amine of the peptide and the functional NHS (hydroxysuccinimidyl) group at the end of the PEG phospholipid polymer chain. The reaction between the terminal amine and the active succinimidyl ester of the PEG carboxylic acid produced a stable amide linkage. Different molar ratios of the peptide and the PEG phospholipid, as well as the reaction times, were varied to optimize the coupling reaction. Up to several hundred CTT2-PEG-lipid molecules can be attached to the surface of each liposome.
CTT2 peptide (8.8 mg) and DSPE-PEG3400-NHS (100 mg) were dissolved in 2 milliliters (ml) dimethylformamide. CTT2 peptide solution (500
For all studies, samples were reconstituted by adding 100
Monomers or CTT2-PEG3400-DSPE (i.e., CTT2-PEG-lipid) spontaneously formed micelles
CTT-2-peptide-targeted liposomes were synthesized either by incorporating CTT2-PEG-lipid onto the surface of commercially available liposomes or by combining CTT2-micelles with liposomal formulations. Prior studies have shown that incubation of certain lipids with liposomes can result in intraliposomal inclusion of these lipids as a consequence of micellar-liposomal fusion [
CTT2-SL liposomes were made by pipetting the above-mentioned lipid mixture except the CTT2-PEG-lipid, to a round bottomed flask, dried under nitrogen and lyophilized for 2 h to remove trace amounts of chloroform. Doxorubicin liposomes were prepared by using standard pH gradient technique [
To synthesize CTT2-PEG-3400-DSPE Caelyx/doxil-liposomes, CTT2-PEG-DSPE (1 mg) was suspended in 400
The incorporation efficiency, the percentage of total activity contained in the liposome fractions, was measured by using radioisotope-labeled peptide and gel filtration to separate the unreacted micelle from the liposome; optimal reaction conditions were found to be 60°C at 30 min (nearly 100% efficient).
The doxorubicin leakage from the liposomes after the incorporation experiments was determined by comparing the amount of free doxorubicin versus liposome-bound doxorubicin before and after the experiment. The leakage was found to be minimal (the leakage before the incorporation was in average 4.5% and after the reaction in average 4.2%).
Radiolabeling of peptides and all liposomal formulations with iodine-125 (125I) was performed using the IODOGEN (Pierce, Rockford, IL). The CTT2-PEG3400-DSPE peptide was labeled with 125I using iodogen as a catalyst. 5 MBq of Na125I (Amersham, Buckinghamshire, England) in 0.5 ml PBS was added to a tube containing 10
The mice were cared for according to the instructions of the animal facility, and the experiments were approved by an ethical committee of Helsinki University, Finland. Male athymic nu/nu mice (6–8 weeks old, Harland) were provided with water and maintained on regular diets ad libitum. Subcutaneous human serous ovarian carcinoma (OV-90) xenograft models were generated by coinjecting equal volumes of cells (
Following single i.v. tracer doses of purified 125I-CTT1-peptide (
Additional distribution data was measured in immunosuppressed mice (
Therapeutic efficacy studies were conducted in subcutaneous A2780 xenografts using doxorubicin, administered as either CTT2-SL liposomes or Caelyx. Commercially available nonliposomal (“free”) drug (i.e., doxorubicin) and saline dilution buffer were used as treatment controls. A2780 ovarian cancer cells (5×106 in 100
Aforementioned treatments were used to collect two independent biodistribution data sets in immunosuppressed OV-90 xenograft mice (
The initial reason to create the CTT-2 peptide was to make a peptide that was more easily iodinated and that offered a spacer that was comfortably used for linking purposes without destroying the bioactivity of the peptide.
In nontumor-bearing mice, greater liver accumulation of the CTT1-peptide was observed than with the CTT2-peptide (Figure
Hepatic accumulation of 125I-CTT2-peptides in normal mice. Liver accumulation of peptides per gram of tissue in normal mice (
In OV-90 xenograft models, substantially higher uptake of 125I-CTT2-peptide was measured in all organs/tissues (Figure
Tissue distribution of a single dose of 125I-CTT2-peptide in immunosuppressed OV-90 xenograft mice. Blood and major organs/tissues were collected at 0.5 hr and 3 hrs p.i. 125I-CTT2-peptide (40
For doxorubicin-containing liposomes, doxorubicin leakage after peptide attachment was assessed by comparing free and liposomal doxorubicin on the basis of fluorometric analysis. Leakage was found to be minimal, with leakage before and after incorporation averaging 4.5% and 4.2%, respectively.
Both OV-90 and mucinous ovarian carcinomas (A2780) were thus selected as xenograft models for subsequent nanoformulation studies. In OV-90 tumor mice, clear targeting of CTT2-micelles was observed, reaching maximum values of 17.6% of the injected dose per gram (%ID/g) of tumor at 6 hrs p.i. (Figure
Tissue distribution of 125I-CTT2-micelle in OV-90 tumor mice. %ID/g values after i.v. injection of CTT2-micelles (200
Doxorubicin concentrations (
Comparison of doxorubicin concentrations in tumors after a single i.v. injection of CTT2-SL liposome or Caelyx. A2780 xenografts (
CTT2-SL liposomal antitumor efficacy data following i.v. bolus injections of CTT2-SL liposome, Caelyx, doxorubicin, and buffer in A2780 xenografts is shown in Figure
Kaplan-Meier plot of the survival of tumor bearing mice. Mice were treated with doxorubicin (9 mg/kg), administered either as CTT2-SL liposome or Caelyx. Controls were injected with doxorubicin (9 mg/kg) or saline dilution buffer. Injections for each treatment group were made at day 0, day 3 and day 6, respectively.
Mouse body weights were monitored throughout the study period (Figure
Mouse body weight changes in each treatment group during the first 32 days of the trial. Mice were treated with 9 mg/kg doxorubicin (calculated doxorubicin equivalents) or saline dilution buffer at day 0, 3 and 6. All values are expressed as mean of 9 mice.
Given the overall improved survival found following treatment of A2780 xenografts with CTT2-SL liposomes, studies were extended to assess treatment response in OV-90 xenograft models. As seen in Figure
Concentrations of doxorubicin in (a) serum and (b) OV-90 xenografts in mice treated with CTT2-SL liposome and Caelyx at 0.5 and 6 hours. Data are represented as a mean of 5 mice ± SD.
Additional serum and tumor uptake measurements conducted with CTT2-SL DSPE-PEG3400 liposomes are shown in Figure
Serum doxorubicin levels. Concentration of doxorubicin in (a) serum and (b) OV-90 xenograft mice (
Gelatinases, as extracellular targets, offer a viable alternative for tumor targeting. In gelatinase-expressing tumors, such as OV-90, targeted liposomal constructs, 125I-CTT2-SL and doxorubicin-containing CTT2, were found to be promising nanotherapeutic delivery vehicles for achieving therapeutic efficacy. Table
Tumor uptake of various liposomal constructs.
Caelyx | CTT2-SL liposome | |
---|---|---|
Targeted formulation | No | Yes |
Concentration-targeted | — | 0.2% |
Analyte | Doxorubicin | Doxorubicin |
Time point (hours) | 6 | 6 |
Tumor uptake (% ID/gram) | 8.1% | 19.0% |
Lipid percent (%) | |||
---|---|---|---|
CTT2-SL liposome | |||
DSPE-mPEG2000 | 3.2 mg/ml | 1.2 mM | 5.5% |
HSPC | 9.6 mg/ml | 12.2 mM | 56.2% |
Cholesterol | 3.2 mg/ml | 8.2 mM | 38.1% |
CTT2-PEG-lipid | 0.2 mg/ml | 0.04 mM | 0.2% |
Total lipids | 16.2 mg/ml | 21.6 mM | 100.00% |
Caelyx | |||
DSPE-mPEG2000 | 3.2 mg/ml | 1.2 mM | 5.5% |
HSPC | 9.6 mg/ml | 12.2 mM | 56.3% |
Cholesterol | 3.2 mg/ml | 8.2 mM | 38.2% |
Total lipids | 16.0 mg/ml | 21.6 mM | 100.0% |