Use of Targeted Liposome-based Chemotherapeutics to Treat Breast Cancer.

The use of nanocarriers such as liposomes to deliver anticancer drugs to tumors can significantly enhance the therapeutic index of otherwise unencapsulated cytotoxic agents. This is in part because of the fact that the phospholipid bilayer can protect healthy sensitive tissue from the damaging effects of these types of drugs. Furthermore, the ease with which the phospholipid bilayer surface can be modified to allow for polyethylene glycol incorporation resulting in pegylated liposomes allow for increased circulation times in vivo, and thus an overall increase in the concentration of the drug delivered to the tumor site. This explains the clinical success of the liposomal-based drug Doxil, which has proven to be quite efficacious in the treatment of breast cancer. However, significant challenges remain involving poor drug transfer between the liposome and tumor cells with this type of nontargeted drug delivery system. Thus, future work involves the development of "smart" drugs, or targeted drug delivery intended for improved colocalization between the drug and cancerous cells. While it is not possible to entirely discuss such a rapidly growing field of study involving many different types of chemotherapeutics here, in this review, we discuss some of the recent advancements involving the development of targeted liposome-based chemotherapeutics to treat breast cancer.

The treatment of breast cancer using liposome technology.

Liposome-based chemotherapeutics used in the treatment of breast cancer can in principle enhance the therapeutic index of otherwise unencapsulated anticancer drugs. This is partially attributed to the fact that encapsulation of cytotoxic agents within liposomes allows for increased concentrations of the drug to be delivered to the tumor site. In addition, the presence of the phospholipid bilayer prevents the encapsulated active form of the drug from being broken down in the body prior to reaching tumor tissue and also serves to minimize exposure of the drug to healthy sensitive tissue. While clinically approved liposome-based chemotherapeutics such as Doxil have proven to be quite effective in the treatment of breast cancer, significant challenges remain involving poor drug transfer between the liposome and cancerous cells. In this review, we discuss the recent advancements made in the development of liposome-based chemotherapeutics with respect to improved drug transfer for use in breast cancer therapy.

The benefits and challenges associated with the use of drug delivery systems in cancer therapy.

The use of drug delivery systems as nanocarriers for chemotherapeutic agents can improve the pharmacological properties of drugs by altering drug pharmacokinetics and biodistribution. Among the many drug delivery systems available, both micelles and liposomes have gained the most attention in recent years due to their clinical success. There are several formulations of these nanocarrier systems in various stages of clinical trials, as well as currently clinically approved liposomal-based drugs. In this review, we discuss these drug carrier systems, as well as current efforts that are being made in order to further improve their delivery efficacy through the incorporation of targeting ligands. In addition, this review discusses aspects of drug resistance attributed to the remodeling of the extracellular matrix that occurs during tumor development and progression, as well as to the acidic, hypoxic, and glucose-deprived tumor microenvironment. Finally, we address future prospective approaches to overcoming drug resistance by further modifications made to these drug delivery systems, as well as the possibility of coencapsulation/coadministration of various drugs aimed to surmount some of these microenvironmental-influenced obstacles for efficacious drug delivery in chemotherapy.

Staged stromal extracellular 3D matrices differentially regulate breast cancer cell responses through PI3K and beta1-integrins.

BACKGROUND: Interactions between cancer cells and stroma are critical for growth and invasiveness of epithelial tumors. The biochemical mechanisms behind tumor-stromal interactions leading to increased invasiveness and metastasis are mostly unknown. The goal of this study was to analyze the direct effects of staged stroma-derived extracellular matrices on breast cancer cell behavior. METHODS: Early and late three-dimensional matrices were produced by NIH-3T3 and tumor-associated murine fibroblasts, respectively. After removing fibroblasts, extracted matrices were re-cultured with breast epithelial cells of assorted characteristics: MCF-10A (non-tumorigenic), MCF-7 (tumorigenic, non-invasive), and MDA-MB-231 (tumorigenic, invasive). Effects prompted by staged matrices on epithelial cell's growth, morphology and invasion were determined. Also, matrix-induced velocity, directionality and relative track orientation of invasive cells were assessed in the presence or absence of inhibitors of phosphoinositide-3 kinase (PI3K) and/or beta-1 integrin. RESULTS: We observed that assorted breast epithelial cells reacted differently to two-dimensional vs. staged, control (early) and tumor-associated (late), three-dimensional matrices. MCF-10A had a proliferative advantage on two-dimensional substrates while MCF-7 and MDA-MB-231 showed no difference. MCF-10A and MCF-7 formed morphologically distinguishable aggregates within three-dimensional matrices, while MDA-MB-231 exhibited increased spindle-shape morphologies and directional movements within three-dimensional matrices. Furthermore, MDA-MB-231 acquired a pattern of parallel oriented organization within tumor-associated, but not control matrices. Moreover, tumor-associated matrices induced PI3K and beta1-integrin dependent Akt/PKB activity in MDA-MB-231 cells. Interestingly, beta1-integrin (but not PI3K) regulated tumor-associated matrix-induced mesenchymal invasion which, when inhibited, resulted in a change of invasive strategy rather than impeding invasion altogether. CONCLUSION: We propose that both cells and matrices are important to promote effective breast cancer cell invasion through three-dimensional matrices and that beta1-integrin inhibition is not necessarily sufficient to block tumor-matrix induced breast cancer cell invasion. Additionally, we believe that characterizing stroma staging (e.g., early vs. late or tumor-associated) might be beneficial for predicting matrix-induced cancer cell responses in order to facilitate the selection of therapies.

Effects of drug hydrophobicity on liposomal stability.

A major obstacle in drug delivery is the inability to effectively deliver drugs to their intended biological target without deleterious side-effects. Delivery vehicles such as liposomes can minimize toxic side-effects by shielding the drug from reaction with unintended targets while in systemic circulation. Liposomes have the ability to accommodate both hydrophilic and hydrophobic drugs, either in the internal aqueous core or the lipid bilayer, respectively. In the present study, fluorescein and rhodamine have been used to model hydrophilic and hydrophobic drugs, respectively. We have compared the stabilities of liposomes encapsulating these fluorophores as a function of lipid content, time, and temperature. At 25 and 37 degrees C, liposomes containing distearoyl phosphatidylcholine as the major phospholipid component were found to be more stable over time than those containing dipalmitoyl phosphatidylcholine, regardless of the fluorophore encapsulated. Liposomes loaded with fluorescein were found to be more stable than those with rhodamine. Dipalmitoyl phosphatidylcholine liposomes that encapsulated rhodamine were the least stable. The results indicate that the physical properties of the drug cargo play a role in the stability, and hence drug delivery kinetics, of liposomal delivery systems, and desired drug release times can be achieved by adjusting/fine-tuning the lipid compositions.

Targeted drug delivery utilizing protein-like molecular architecture.

Nanotechnology-based drug delivery systems (nanoDDSs) have seen recent popularity due to their favorable physical, chemical, and biological properties, and great efforts have been made to target nanoDDSs to specific cellular receptors. CD44/chondroitin sulfate proteoglycan (CSPG) is among the receptors overexpressed in metastatic melanoma, and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical "peptide-amphiphile" (alpha1(IV)1263-1277 PA), which binds CD44/CSPG, has been constructed and incorporated into liposomes of differing lipid compositions. Liposomes containing distearoyl phosphatidylcholine (DSPC) as the major bilayer component, in combination with distearoyl phosphatidylglycerol (DSPG) and cholesterol, were more stable than analogous liposomes containing dipalmitoyl phosphatidylcholine (DPPC) instead of DSPC. When dilauroyl phosphatidylcholine (DLPC):DSPG:cholesterol liposomes were prepared, monotectic behavior was observed. The presence of the alpha1(IV)1263-1277 PA conferred greater stability to the DPPC liposomal systems and did not affect the stability of the DSPC liposomes. A positive correlation was observed for cellular fluorophore delivery by the alpha1(IV)1263-1277 PA liposomes and CD44/CSPG receptor content in metastatic melanoma and fibroblast cell lines. Conversely, nontargeted liposomes delivered minimal fluorophore to these cells regardless of the CD44/CSPG receptor content. When metastatic melanoma cells and fibroblasts were treated with exogeneous alpha1(IV)1263-1277, prior to incubation with alpha1(IV)1263-1277 PA liposomes, to potentially disrupt receptor/liposome interactions, a dose-dependent decrease in the amount of fluorophore delivered was observed. Overall, our results suggest that PA-targeted liposomes can be constructed and rationally fine-tuned for drug delivery applications based on lipid composition. The selectivity of alpha1(IV)1263-1277 PA liposomes for CD44/CSPG-containing cells represents a targeted-nanoDDS with potential for further development and application.

Peptide-mediated targeting of liposomes to tumor cells.

One of the biggest obstacles for efficient drug delivery is specific cellular targeting. Liposomes have long been used for drug delivery, but do not possess targeting capabilities. This limitation may be circumvented by surface coating of colloidal delivery systems with peptides, proteins, carbohydrates, vitamins, or antibodies that target cell surface receptors or other biomolecules. Each of these coatings has significant drawbacks. One idealized system for drug delivery combines stabilized "protein module" ligands with a colloidal delivery vehicle. Prior studies have shown that peptide-amphiphiles, whereby both a peptide "head group" and a lipid-like "tail" are present in the same molecule, can be used to engineer collagen-like triple-helical or alpha-helical miniproteins. The tails serve to stabilize the head group structural elements. These peptide-amphiphiles can be designed to bind to specific cell surface receptors with high affinity. Structural stabilization of the integrated targeting ligand in the peptide-amphiphile system equates to prolonged in vivo stability through resistance to proteolytic degradation. Liposomes have been prepared incorporating a melanoma targeting peptide-amphiphile ligand, and shown to be stable with retention of peptide-amphiphile triple-helical structure. Encapsulated fluorescent dyes are selectively delivered to cells. In this chapter we describe the methods and techniques employed in the preparation and characterization of peptide-amphiphiles and peptide-amphiphile-targeted large and small unilamellar vesicles (LUVs and SUVs). Fluorescence microscopy is subsequently utilized to examine the targeting capabilities of peptide-amphiphile LUVs, which should allow for improved drug selectivity towards melanoma vs normal cells based on differences in the relative abundance of the targeted cell surface receptors.