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Highly selective drug targeting is an important goal in the development of cancer nanotechnologies. In an effort to improve tumor targeting a method was developed to formulate cell membrane lipid-extracted nanoliposomes (CLENs). The main ingredients were extracted directly from the membrane of cancer cells. For this study we used three different breast cancer cell lines (4 T1, BT-20, and SK-BR-3). As controls for the normal breast and cancer tissue environments we employed the normal breast fibroblast (CRL-2089) and ovarian cancer (SK-OV-3) cell lines, respectively. We evaluated physicochemical properties, efficiency of drug loading, cellular uptake, and cytotoxicity. The mean diameter and zeta potential values for the 5 different CLENs were 202 ± 38 nm and − 15 ± 3.8 mv, respectively. Doxorubicin hydrochloride (5 mol%) increased the size of 4 T1-CLENs from 158 ± 2 nm to 212 ± 59 nm, with no significant change in the negatively-charged surface potential. Percent of drug loaded ranged from 40 to 93%, varying according to the ratio of lipid extract to conventional components employed. The additional inclusion of cholesterol and DPPE-PEG5000 increased drug loading in CLENs, similar to Doxil preparations. The most promising cellular uptake and cytotoxicity profiles were observed when the lipid ingredients were derived from the eventual target cell. Given the ability of CLENs to better recognize target cells compared to nanosystems consisting of non-specific lipid extracts or conventional liposome ingredients alone, CLENs has demonstrated early promise as a nano-delivery system for cancer treatment.

One of the longstanding issues limiting the use of chemotherapeutic agents is the lack of tumor specificity, which leads to systemic toxicity. Therefore, various targeted nanocarriers have been developed to concentrate the cytotoxic drug agents at tumor regions to avoid uptake by normal healthy tissues. Previous studies from our laboratory have demonstrated that cell membrane lipid-extracted nanoliposomes (CLENs) are capable of selective targeting compared to nanoliposomes consisting of mainly conventional lipid materials. Also, CLENs were relatively nontoxic when employed at concentrations traditionally used to evaluate nanoparticles in vitro. However, parameters such as lipid composition, size, and surface charge all have a direct impact on liposomal cellular uptake. In this study, we prepared different CLENs different molar ratios of natural and cellular-derived lipids extracted directly from breast cancer cells (4T1), cholesterol, and DPPE-PEG-5000. The formulations were used to investigate the mechanisms of CLENs uptake and determine the underlying mechanisms regulating cellular entry. In the study, CLENs were used to evaluate cytotoxicity and cellular uptake for both target and off-target cell populations. CLENs containing cholesterol and DPPE-PEG-5000 (70/25/5) were able to retain doxorubicin in relevant therapeutic concentrations. CLEN formulations were able to exert selective cytotoxic drug effects against different target breast cancer cells in vitro. 4T1 CLENs containing cholesterol and DPPE-PEG-5000 (70/25/5) demonstrated greater binding to 4T1 (target) cells compared to CLENs prepared using 4T1 lipid extracts alone. The binding was not only temperature- and time-dependent, but also composition- and cell type-dependent. Off-target effect studies showed that 4T1 CLENs were taken up minimally by off-target cells (including normal breast fibroblasts and normal myocytes), compared to controls. Our studies collectively support the use of lipid extracts derived from target cells for selective drug targeting and enhanced cytotoxicity. Future studies will explore underlying mechanisms of cellular entry and cardioprotective function. Citation Format: Hanan M. Alharbi, Robert B. Campbell. An evaluation of breast cancer cellular membrane lipid-extracted nanoliposomes (CLENs) in relation to formulation design, stability, mechanism of cellular entry and cardioprotective function in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4666.

Background: Every year more deaths are attributed to lung cancer compared to colon, prostate, and breast cancers combined. Lung cancer is typically diagnosed at late stages, and has demonstrated poor treatment outcomes due to acquired drug resistance. Unfortunately, as one method is introduced to overcome drug resistance a new mechanism can arise. In an effort to overcome drug resistance lipid extracts (LE) were derived from target cells, and the ingredients were used to develop nanoliposomes that would selectively target a desired cell population compared to controls. The early studies were designed to demonstrate the use of CLENs (cellular membrane lipid-extracted nanoliposomes) for the targeting of lung cancer. Experimental Procedures: CLENs were initially prepared using various ratios of LE/DOPC/Chol/PEG by thin film evaporation. Particle size and zeta potential values were determined for all preparations of CLENs (with and without drug). CLENs were evaluated for selectivity for target compared to control cells. Cytotoxicity studies were performed on various doxorubicin hydrochloride-loaded CLENs using a cell model of lung cancer (ChagGo-K-1). For cellular uptake studies ChaGo-K-1 cells were seeded at 10,000 cells/mL in a 48-well plate and allowed to incubate for 24 hours prior to cell studies. A fluorescence microplate reader was used to determine fluorescence intensity values for the two studies. Results: ChaGo-K-1 CLENs demonstrated an average particle size of 206+/-2 nm and zeta-potential of -21+/-3 mV, illustrating favorable characteristics for drug delivery and interstitial drug targeting. The cellular uptake studies suggested that ChaGo-K-1 CLENs were selective for ChaGo-K-1 cells in vitro, when compared to all other control varieties of CLENs. Drug-loaded ChaGo-K-1 CLENs displayed significant cytotoxicity against ChaGo-K-1 cells compared to control cells. Fluorescence microscopy revealed association of ChagGo-K-1 CLENs with target cells. Conclusions: Based on promising early finding CLENs may represent a relatively tumor specific drug delivery system that can be used to overcome drug resistance, improve efficacy, and to limit off-target drug effects. Preclinical studies will investigate whether the relative selectivity and cytotoxicity observed against lung cancer cells in vitro translates to favorable tumor uptake and efficacy in vivo. Citation Format: Jasmine King, Aarohi Bhatt, Drew Goodrich, Thomas Preteroti, Hanan Alharbi, Robert Campbell. Development and in vitro evaluation of cell membrane lipid-extracted nanoliposomes for lung cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3876.

Background: Liposome-encapsulated doxorubicin has proven to be an effective drug delivery method to cancer cells. Lipid extracts derived from human prostate cancer cells along with conventional components were employed to develop cell membrane lipid-extracted nanoliposomes (CLENs) for prostate cancer treatment. In an effort to balance efficiency of drug loading with cellular targeting efficiency we report on physicochemical characteristics (i.e., particle size, zeta potential, drug loading efficiency) and cellular uptake studies. Methods: Five preparations of CLENS were prepared consisting of variable concentrations of DOPC and CRL-1740 lipid extract material varying as a function of optimized fixed ratios of conventional components (Cholesterol and DSPE-PEG5000). The controls used to investigate the effect of the different components on drug incorporation efficiency were preparations of DOPC (100%) and CRL-1740 (100%). Doxorubicin hydrochloride (5 mol%) was employed during the preparation of all formulations. Samples were readily prepared by thin film evaporation. For cellular uptake studies CRL-1740 cells were seeded at 1 x 104 mL in 48-well plate and incubated for 24 hours prior to use. The cells were next exposed to various amounts of rhodamine labeled CLENS for 24 hrs. Cellular uptake was determined using fluorescence intensity values determined by a fluorescence microplate reader. Fluorescence microscopy supplement cellular uptake studies. Results: The DOPC drug-loaded liposome preparation consistently achieved the lowest percent of drug incorporation. Likewise, 100% CRL-1740 CLENs demonstrated a significant degree of formulation instability, resulting in a relatively low percent of drug incorporation compared to preparations that consisted of lipid extract and conventional components. The finding were supported by values determined for zeta potential and particle size. Cellular uptake studies also favored the preparations that consisted of lipid extract material and conventional components compared to each alone. The results collectively support the importance of balancing lipid extract material derived from prostate cancer target cells with cholesterol and DSPE-PEG5000 for formulation development and cellular uptake. Conclusion: We formulated seven preparations of CLENS as part of the drug platform. The most promising candidates were determined following completion of the study. Future studies should evaluate the cytotoxicity and safety profile of the most promising formulations vivo. Citation Format: Abubker Omaer, Ali Alqarni, Carlos Randulfe, Hanan Alharbi, Robert Campbell. Formulating a cell membrane lipid-extracted nanoliposome (CLENS) drug platform for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3881.

Breast cancer is a major cause of cancer death in women worldwide. Breast cancer is a heterogeneous disease characterized by variant pathological features, disparate responses to therapeutics, and substantial differences in patient's long-term survival. The use of drug delivery systems such as liposomes can favorably alter the pharmacokinetic and biodistribution profile of commonly used drugs used to treat breast cancer disease. An important feature of a drug delivery system is its ability to recognize and selectively target tumor cells with relatively high affinity when compared to healthy tissues. For this reason, the goal of this study was to develop a novel nanoliposome system that would target tumor cells to a significantly greater extent when compared to more conventional liposome systems. In this study, natural lipids extracted directly from breast cancer cells were used to prepare cellular lipid-extracted nanoliposomes (CLENs). The rationale is that an array of different lipids with a unique composition profile would represent a more relevant source of lipid material when compared to more traditional methods of liposome preparation. We therefore investigated whether the inclusion of extracted lipids derived from breast cancer cells would improve the selective uptake of liposomes by breast cancer cells, and related formulation features. To achieve these goals, three breast cancer cell lines were used (4T1, BT-20, and SK-BR-3), a normal breast fibroblast (CRL-2089), and an ovarian cancer cell line (SK-OV-3- used as a negative control). The size of CLENs fell within the size range of 120- 203 nm and possessed a negatively-charged liposome surface charge potential (ranging between -10.97 and -20.97 mV). The nanoliposomes were also non-toxic to cells within the concentration range evaluated (up to 1μmol). Furthermore, without exception, the cellular uptake of CLENs was greatest when the extracted material used to prepare CLENs was the same as the target cell from which the lipids were extracted. In addition, when the breast cancer cell lines (4T1, SK-BR-3 and BT-20) were used to prepare CLENs, SK-OV-3 and CRL-2089 cell lines took up CLENs to a lesser extent, suggesting a more selective and differential cellular uptake. In conclusion, the novel nanoliposome platform represents a promising drug delivery alternative to more conventional nano drug delivery systems. Additional formulation studies are currently underway. Citation Format: Hanan M. Alharbi, Robert B. Campbell. Employing a unique panel of breast cancer cellular lipids extracts in the development of a novel nanoliposome drug platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2015-5513