3.4.1 Cancer Chemotherapy
The pH-sensitive liposomes have been tested for numerous applications in drug delivery.27,29 Engineered liposomes capable of spontaneous accumulation in tumors and ischemic areas via EPR effect and further penetration and drug delivery inside tumor or ischemic cellsviathe action of cell-penetrating peptides (CPP) have been reported. These liposomes were simultaneously bearing on their surface CPP (TAT peptide, TATp) moieties and protective PEG chains.
Estrogen
Estrogen receptors
Figure 3.4 Estrogen-anchored pH-sensitive liposomes as nanomodule designed for site-specific delivery of doxorubicin in breast cancer therapy.
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After accumulation in the target site, the PEGylated liposomes lose the PEG coating due to the lowered pH-induced hydrolysis, and penetrate inside cellsvia the now-exposed TATp moieties. These liposomes rendered promising results for chemotherapy in cell cultures as well as in ischemic cardiac tissues using the Langendorff perfused rat heart model and in mice bearing tumors.30
Korenet al.31prepared pH-sensitive PEGylated long-circulating liposomes modified with TATp moieties and cancer-specific monoclonal antibodies (mAb). PE was PEGylated using a degradable pH-sensitive hydrazone bond between PE and long shielding PEG chains (PEG(2k)-Hz-PE). TATp was conjugated to a short PEG1000-PE spacer and mAb to a long PEG chain (PEG3400-PE). At pH 7.4, surface TATp moieties are ‘‘hidden’’ by the long PEG chains. Upon exposure to lower pH, the hydrazone bond is broken, the long PEG chains are removed and the TATp moieties become exposed.
Enhanced cellular uptake of the TATp-containing immunoliposomes was observedin vitroafter pre-treatment at lower pH. The presence of mAb 2C5 on the liposome surface further enhanced the interaction between the carrier and tumor cells but not normal cells. Furthermore, this multi-functional immuno- Doxilspreparation showed increased cellular cytotoxicity of B16-F10, HeLa and MCF-7 cells when pre-incubated at lower pH, indicating TATp exposure and activity. This multi-functional immunoliposomal nanocarrier is claimed for intra-cellular drug delivery after exposure to the lowered pH environment typical of solid tumors.31 More recently, nanoengineered estrogen receptor (ER) targeted pH-sensitive liposomes for the site-specific intra-cellular delivery of doxorubicin have been developed for breast cancer therapy. Estrone, a bioligand, was anchored on the surface of pH-sensitive liposomes (ES-pH-sensitive-SL) for drug targeting to ERs overexpressed by breast cancer cells. The ES-pH-sensitive-SL showed fusogenic potential at acidic pH (5.5) andin vitro cytotoxicity studies (MCF-7 cells) proved this formulation to be more cytotoxic than non-pH-sensitive targeted liposomes. Intra-cellular delivery and nuclear localization of doxorubicin was confirmed by fluorescence microscopy. Further, in vivo biodistribution and antitumor activity of the formulations were evaluated on tumor-bearing female Balb/c mice after intravenous administration. The ES-pH-sensitive-SL suppressed the breast tumor growth more efficiently than non-pH-sensitive targeted liposomes and free doxorubicin. The ES-pH-sensitive-SL increased the therapeutic efficacy, being promising nanocarriers for the targeted intra-cellular delivery of anti- cancer agents to breast tumors with reduced systemic side effects.32
PEG-coated pH-sensitive and PEG-folate-coated pH-sensitive liposomes containing (159)Gd-DTPA-BMA and radiolabeled through neutron activation technique were developed forin vivotests of antitumoral activity and toxicity on mice bearing solid Ehrlich tumor. The results showed that after 31 days of treatment, animals treated with radioactive formulations had a lower increase in tumor volume and a significantly higher percentage of necrosis.
Furthermore, mice treated with radioactive formulations exhibited lower weight gain without significant hematological or biochemical changes, except for toxicity to hepatocytes.33
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Cisplatin is one of the most active cytotoxic agents and is widely appliedvia intra-peritoneal route in the treatment of peritoneal carcinomatosis. However, cisplatin, a low-molecular-weight compound, is rapidly absorbed by the capillaries in the intra-peritoneal serosa and transferred to the bloodstream, inducing the appearance of systemic side effects, such as nephrotoxicity.
Furthermore, the intra-peritoneal cisplatin chemotherapy is limited to patients whose residual tumor nodules are less than 0.5 cm in diameter after surgical debulking. A tissue distribution study in solid Ehrlich tumor-bearing mice revealed that, after administering a 6 mg/kg single intravenous bolus injection of either free radiolabeled cisplatin or stealth pH-sensitive liposomes containing radiolabeled cisplatin, the area under the plasma concentration-time curve (AUC) for stealth pH-sensitive liposomes was 2.1-fold larger than that obtained for free cisplatin.34 Similar improved biodistribution and targeted delivery of cisplatin to tumor-bearing mice was observed in another study.35 Long-circulating and pH-sensitive liposomes containing cisplatin successfully avoid severe side effects as well as drug resistance. However, physical (i.e.
aggregation/fusion) and chemical instability during storage may limit the use of these drug carriers as medicines. Freeze-drying may be a success strategy to improve the stability before use.36Immunoliposomes directed by monoclonal antibodies may efficiently target the drug to tumor tissues.37Long-circulating pH-sensitive liposomes with epidermal growth factor receptor (EGFR) antibody tested on A549 cells and Balb/c-nu/nu mouse tumor model demon- strated efficient and targeted delivery of gemcitabine for tumors that over- express the EGFR.38 Drug targeting is expected to be optimized in the near future as further knowledge about internalization pathways becomes available.39
3.4.2 Gene Delivery
The number of biotechnological products such as nucleic acids, proteins and peptides that enter in the therapeutic arsenal is notably increasing. However their in vivoefficacy can be severely comprised by the unfavorable physico- chemical characteristics. The major obstacle for cell penetration is the large size and hydrophilic nature of the biotechnological molecules, which demand the use of a carrier able to overcome cellular barriers and facilitate cytosolic delivery.40Therefore, many different drug-delivery systems including liposomes have been investigated for this purpose. Liposomes are able to provide protection and targeting of the encapsulated macromolecule and may promote cellular internalization.39–41
Gene therapy requires the development of multi-functional vectors that could overcome the barrier effects of the membranes of the cell, endosome and nucleus. A pH-sensitive multi-functional gene vector has been developed to attain long circulation without using PEG but showing tumor cellular uptake of the gene carrier.42DNA was firstly condensed with protamine into a cationic core that was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes and o-carboxymethyl-chitosan (CMCS) were
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alternately adsorbed onto the templateviaa layer-by-layer technique, to render CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD).
In vitro test with isolated tumor (HepG2) cells and in vivo evaluation into tumor-bearing mice confirmed the transfection efficiency. Other reports also confirmed the relevance of the fusogenic properties of the pH-sensitive liposomal membranes for intra-cellular gene transfection.43–45
The pH-sensitive stealth liposomes have been reported as suitable vectors for targeting therapeutic peptides to the nucleous.46 Cellular uptake of peptide- loaded liposomes has been investigated in Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. Two different formulations were tested:
long circulating classical liposomes [soybean phosphatidylcholine : CHOL : PEG-750-DSPE (47 : 47 : 6 molar% ratio)] and pH-sensitive stealth liposomes [DOPE : CHEMS : CHOL : PEG750-DSPE (43 : 21 : 30 : 6 molar% ratio)]. The difference between both formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was observed as a function of time. Using pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non-tumorigenic breast cancer cells.46In summary, the available information demonstrates the utility of pH-sensitive liposomes as intra-cellular carrier for bioactives.
3.4.3 Tumor Diagnosis
Liposomes are excellent candidates for the development of theranostic agents and multi-modal imaging probes, since they can release the entrapped imaging probe/radioactive agent/drug in response to a change of physico-chemical variables like pH, redox potential or concentration of specific enzymes that usually occur in the early asymptomatic stage of several diseases such as cancer.47 The pH-sensitive liposomes trapping 99mTc have been used for biodistribution studies and scintigraphic imaging in Ehrlich tumor-bearing mice. They accumulate in tumor tissue with high tumor-to-muscle ratio and can be useful for diagnosis of tumors.48
Paramagnetic pH-sensitive liposomes have also been developed as imaging tools for visualizing drug-delivery and release processes by means of Magnetic Resonance Imaging (MRI). The proposed formulation allowed the fast and full release of gadoteridol at pH 5.5. The leakage of the imaging reporter from the vesicles was associated with a relaxivity enhancement that allowed its visual- ization by MRI. It was observed that the release mechanism implies the protonation of the basic sites that leads to vesicle aggregation, thus enabling the expression of the fusogenic property.49