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    Research Grants Awarded

    Multiresponsive Liposomes For The Targeted Therapy Of Advanced Breast Cancer

    Grant Mechanism:
    Career Catalyst Research

    Scientific Abstract:
    BACKGROUND Breast cancer is the most common cancer among women. About 1 in 8 women may have invasive breast cancer at some time during their lives and a chance 1 in 33 of dying from the disease. Breast cancer death rates are decreasing probably due to early detection, but cases of advanced disease still have no cure. Although excellent chemotherapeutic agents are available, chemotherapy is third to surgery and radiation. This is largely due to the low accumulation of chemotherapeutics at the tumors relative to the drug accumulation at normal organs (toxicities), and due to low drug bioavailability in vivo within the cancer cells that constitute the tumors (the inability of chemotherapeutics to reach their molecular site of action). A new treatment modality is needed for advanced breast carcinoma. In this work, we propose to engineer a new generation of ?multi-responsive? liposomes as drug carriers that selectively change their surface morphology in response to cancer-specific stimuli to address the issue of tumor accumulation and drug bioavailability in metastatic breast cancer in vivo, and particularly for tumors with grown vasculature. The proposed liposomes will be administered intravenously to largely accumulate into the interstitium of breast cancer tumors and then specifically and effectively to release their chemotherapeutic contents into the cytoplasm of breast cancer cells. HYPOTHESIS For targeted drug delivery to metastatic tumors with developed vasculature, preferential accumulation and retention of liposomes in the tumor interstitium depends primarily on liposome?s size, and is distinct from the cancer-targeting ligands and cellular internalization mechanisms. Only after liposomes are localized in the tumor interstitium, are cancer-targeting ligands necessary to enhance adhesion of liposomes to cancer cells, and to mediate their cellular internalization. At all other times, during circulation in the blood, ?decoration? of the drug carrier surface with tumor-seeking ligands will activate non-desirable interactions with the immune and reticuloendothelial systems resulting in accumulation of drug carriers in healthy organs. At these sites, liposomes will release their therapeutic contents killing healthy cells and increasing toxicity. With the above rationale in mind we propose ?multi-responsive? liposomes with targeting antibodies on their surface and membrane-tunable properties, that: (a) 'hide' the targeting-ligands from their surface during circulation in the blood, and only 'expose' and 'cluster' the targeting ligands on their surface after localization of the liposomes within the tumor acidic interstitium (pH=6.7) with the aim to increase specific targeting and reduce normal organ uptake, and (b) after antibody-mediated endocytosis, at the acidic pH of the endosomal pathway (5.5-5.0), the liposomes are tuned to effectively release the encapsulated contents in vivo by a mechanism not used before with the aim to increase the bioavailability of delivered therapeutics. Both 'tunable' properties are based on the same simple molecular mechanism: liposomes are comprised of titratable charged ?domain-forming? rigid lipids that are tuned to form phase-separated lipid domains as a response to pH. These liposomes are composed of rigid-bilayer membranes and are covered with PEG-chains to retain long circulation times in the blood that would enhance their accumulation within the tumor interstitium. These liposomes are triggered to form lipid-phase separated domains resembling ?lipid rafts?: clustering of targeting antibodies within the ?rafts? increases multivalency and targeting to cancer cells; formation of ?rafts? creates pronounced grain boundaries across the membrane bilayer followed by fast and extensive release of therapeutic agents intracellularly. SPECIFIC AIMS AIM 1: Investigation of rigid lipid compositions with targeting Trastuzumab-linked lipids for formation of ?multi-responsive? liposomes that result in maximum and sharper difference in binding of liposomes to breast cancer cells between the physiological pH and the tumor interstitial pH in vitro. We will evaluate the effect of the liposomal membrane composition and surface modification by grafted polymers and targeting ligands on the formation of lipid separated domains that ?screen? the targeting ligands from the liposome surface at physiological pH and effectively ?expose? and ?cluster? the targeting ligands at the liposome surface at the acidic pH of the tumor interstitium to maximize binding and uptake by breast cancer cells. We will develop targeted liposome compositions that exhibit minimal binding to breast cancer cells at pH 7.4 and maximum binding at pH 6.7. AIM 2: Investigation of rigid lipid compositions for stable retention of contents at physiological pH, and extended and fast release of contents in the endosomal pH in vitro. We will investigate the effect of rigid-lipid composition on the formation of lipid separated domains in liposome membranes with the objective to develop liposomes that stably retain their contents at neutral pH (during circulation) and extensively and rapidly release their contents at the endosomal pH (5.5-5.0) of breast cancer cells. We will develop liposomes that will exhibit superior killing in vitro compared to Doxil liposomes. AIM 3: Evaluation in vivo. We will choose the best compositions from the previous aims, and also combine both features in a single liposome and will demonstrate that the proposed liposomes result in lower accumulation in normal organs, in higher accumulation in tumors, and in higher ?bioavailability? in vivo compared to targeted Doxil.

    Lay Abstract:
    Breast cancer is the most common cancer among women, and cases of advanced disease still have no cure. Although excellent chemotherapeutic agents are available, chemotherapy is third to surgery and radiation, largely due to two main reasons. First, due to the low accumulation of chemotherapeutics at the tumors relative to their accumulation at normal (healthy) organs which translates into increased toxicities. And second, due to the low drug bioavailabilities within the cancer cells that constitute the tumors owing to the inability of drugs to reach their molecular sites of action within the malignant cells. In this grant application we propose to address these two issues of paramount importance in drug delivery of chemotherapeutics for the therapy of advanced breast cancer. We propose to study and develop a novel class of drug delivery carriers - ?multiresponsive? liposomes- that will be administered intravenously. The proposed liposomes will largely accumulate into the breast cancer tumors, and will also specifically and effectively release their chemotherapeutic contents into the cytoplasm of breast cancer cells. These liposomes, if successful, will enable administration of chemotherapeutic doses that can reach the lethal levels of absorbed doses at the tumors, resulting in tumor regression and killing, without reaching the normal organ limiting toxicities that harm the patient. These novel liposomes, will have great potential to lead to significant reductions in breast cancer mortality. The main design feature if these liposomes, is that they are designed to respond to their environment and to form separated lipid-phases on their membranes that resemble ?lipid rafts? (?patches? made of lipids). Nature uses lipid-membranes as a universal surface ?wrap? around cells to control critical functions by reorganizing lipid-membranes into ?rafts?. Lipid-rafts are nanometer-sized lipid-domains of laterally phase separated lipids whose occurrence coincides with locally confined and clustered membrane proteins and other macromolecules on the surface of cells. These lipid heterogeneities cause changes in membrane continuity and in local multivalency that seem to control biological activities ranging from signal transduction, to membrane trafficking, to viral infection mechanisms. The liposomes we propose to design and develop contain such components that depending on their immediate environment they can be tuned to form ?model lipid rafts? to ?reveal? targeting-ligands, to alter their ?multivalency?, and/or to regulate the release of their encapsulated therapeutic contents. This is a project that uses existing materials (lipids, PEG-linked lipids, and targeting ligand-linked lipids) in entirely novel ways. Our innovation is to use molecular and supramolecular ?multiresponsive switches? on liposomes to address divergent obstacles presented in vivo for successful delivery of drugs. The proposed technology for ?multiresponsive liposomes? is general and can be used for various tumor-targeting ligands (antibodies, aptamers, etc). These liposomes can be used as carriers for delivery of chemotherapeutics, and also of radionuclides for internal radiotherapy of cancer, as well as for delivery of vaccines or targeted gene therapy of diseases beyond cancer. Even if the combination of both properties (enhanced specific targeting and enhanced content release) in a single liposome does not give the desirable additive result, each property separately can stand alone as a very significant improvement in liposomal drug carriers for cancer therapy. We have strong evidence from our preliminary data that the proposed liposomes do result in the proposed desirable behavior in vitro. This is a high reward project. Successful completion of the proposed work will have a transformational effect on the treatment strategies for breast cancer.