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Nano-formulations composed of cell membrane-specific cellular lipid extracts derived from target cells: physicochemical characterization and in vitro evaluation using cellular models of breast carcinoma

Nano-formulations composed of cell membrane-specific cellular lipid extracts derived from target... 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 (5mol%)increased thesizeof4T1-CLENs from 158 ±2nmto212 ± 59nm, with no significantchangeinthenegatively- chargedsurfacepotential. Percentofdrugloadedrangedfrom40to93%,varying accordingtothe ratiooflipid extract to conventional components employed. The additional inclusion of cholesterol and DPPE-PEG increaseddrugloading 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. Keywords: Breast cancer, Cell lines, Cholesterol, Liposomes, Drug delivery systems Background A current area of research investigation involves the An important goal of chemotherapy is to eradicate the development of nanomedicines capable of recognizing, tumor mass while causing minimal side effects to the and selectively targeting, exploitable tumor features for patient. Liposomes have been employed to help achieve cancer therapy. (Campbell et al., 2002; Torchilin, 2005; this goal. The vesicle type has improved tumor targeting Sharma et al., 2006; Campbell, 2006; Sadzuka et al., 2003) and when optimised can favorably alter the pharmacoki- A few advances in the field of nanoliposome development netic and biodistribution profile of the drug. (Campbell include the inclusion of poly-ethylene glycol (PEG)-PE in et al., 2002; Deshpande et al., 2013; Torchilin, 2005) nano-size drug delivery systems (i.e., liposomes, micelles). Although effective, issues involving limited interstitial (Dan, 2002) The liposome formulation product Doxil drug transport, off-target drug effects and formulation contains PEG, and as a result can successfully exploit the instability still remain. (Deshpande et al., 2013; Torchilin, relatively large tumor vascular pore openings owing to 2005;Sharmaet al., 2006). extended circulation properties afforded by PEG. (Sadzuka et al., 2003; Eloy et al., 2014; Hatakeyama et al., 2013;Jain * Correspondence: robert.campbell@mcphs.edu &Jain, 2008; Ryan et al., 2008;Vemuri&Rhodes, 1995; Department of Pharmaceutical Sciences, MCPHS University, 19 Foster Street, Verma et al., 2008) PEG has versatile functions. For Worcester, MA 01608, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Alharbi and Campbell AAPS Open (2018) 4:5 Page 2 of 9 example, its inclusion in cationic liposomes permits prefer- MO). All chemicals and solvents used in this study were of ential tumor targeting, relatively longer circulation of analytical grade and obtained from Fisher Scientific (Pitts- cationic liposomes while significantly decreasing uptake by burgh, PA). vessels in healthy tissues. (Campbell et al., 2002; Campbell, 2006) Efforts to both gain access to and exploit the differ- Cell culture ential expression of various receptors in tumors for treat- Human breast cancer cell lines BT-20 (HTB-19), ment when compared to normal tissues has improved with SKBR3 (HTB-30), murine mammary cell line 4 T1 advances in liposome technology. (Torchilin, 2005; (CRL-2539), normal mammary fibroblast cell line Sharma et al., 2006;Lu&Low, 2002)Suchexamples CCD-1069SK (CRL-2089), and ovarian cancer cell line include the significant folate receptor-mediated uptake SK-OV-3 (HTB-77) were obtained from ATCC in athymic mice-bearing tumors overexpressing folate (American Type Culture Collection, Manassas, VA). receptor-positive cells, compared to non-folate All cell line cultures were grown in a humidified receptor-labeled systems for which reduced uptake atmosphere of 5% CO at 37 °C. was observed. (Mathias et al., 1998) Regardless of the specific variety of nanomedicine employed most drug delivery systems are prepared from Cellular lipid extraction natural and/or synthetic lipid ingredients. Moreover, the The method used to extract the cellular lipid material ratio of the ingredients employed rarely resemble the was modified from the original Bligh and Dyer composition profile of the target cell membrane. In an method established in 1959. (Dabbas et al., 2008)The effort to achieve more selective targeting we designed, standard gravimetric quantification method involves prepared and evaluated the use of cell membrane the use of three different solvents. In brief, when the lipid-extracted nanoliposomes (CLENs) in vitro. The cells reached approximately 90% confluence, they process involved the extraction of cellular lipid material were trypsinized and collected. The pellet of cells was from intended target cells, and the use of extracted lipid diluted with 1X PBS (phosphate buffer saline). The material with (and without) conventional ingredients for average number of cells in a pellet determined the formulation and in vitro experimentation. Moreover, a proportion of solvents used according to a modified single organ tissue environment was the primary focus chart (Table 1). The three solvents were added of this investigation. The tissue model was additionally step-wise as follows: 1:2 chloroform-methanol mix- selected to represent an organ tissue environment for ture, chloroform, and distilled water, vortexed inter- which the nano drug platform is being developed. For mittently between solvent additions. The final mixture this reason, the cellular lipid material used to prepare was centrifuged at 1000 rpm at 4 °C for 5 min to ob- CLENs was derived from breast tissue, except for when tain a two-phased system; the bottom lipid layer was negative controls were employed. aspirated and transferred into a glass tube and refrig- The composition profile of extracted lipid components erated until a sufficient volume was obtained. of CLENs more closely resemble the target cell mem- brane compared to non-specific preparations in terms of composition. For this reason, we investigated whether Lyophilization method CLENs prepared from lipid extracts derived from target The extracted lipids were dehydrated to form a film cells will associate with the intended target cells to a using a rotary evaporator system, and a heating bath greater extent when compared to CLENs prepared from (Buchi B-491) (Flawil, Switzerland) with temperatures non-specific lipid extracts, and/or conventional nanoli- maintained at 40-50 °C. Sucrose (0.2 M) was added posomal preparations. to serve as a cryoprotectant during the process of lyophilization using a FreeZone Freeze Dry system Methods (Labconco, Kansas City, MO). The lipid powder was Materials weighed and subsequently dissolved in 1 mL of The lipids 1, 2-dioleoyl-Sn-glycero-3-[phospho-rac-(1-gly- chloroform. All lipid chloroform stocks used to pre- cerol)] (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine pare CLENs were stored at − 80 °C. Prior to using (DOPC), Cholesterol (Chol), 1,2-dipalmitoryl-sn-glycero-3- the lipid extracts for preparation of CLENs batches of [phospho-ethanolamine (Polyethylene glycol)]-5000 (DPPE the freeze dried lipid extract were evaluated by -PEG ), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolami LCMS. All lipid components were identified, and the ne-N- (lissamine rhodamine B sulfonyl) (rhodamine ), relative abundance and molecular weights for each -DPPE were purchased from Avanti Polar Lipids (Alabaster, AL). lipid extract type was determined, resulting in average Sulforhodamine B (SRB) and Doxorubicin hydrochloride molecular weights. The final concentrations were sub- (98% HPLC) were purchased from Sigma-Aldrich (St Louis, sequently determined for the lipid extract stocks and Alharbi and Campbell AAPS Open (2018) 4:5 Page 3 of 9 Table 1 Modified chart of the Dyer and Bligh lipid extraction method Cell Number (millions) (5–9.9) (10–24.9) (25–49.9) (50–74.9) (75–99.9) 1:2 CHCl : MeOH 0.750 1.9 3.75 5.7 7.5 CHCl 0.250 0.625 1.25 1.875 2.5 dH O 0.250 0.625 1.25 1.875 2.5 Total Volume 1.45 3.65 7.25 10.95 14.50 later used to prepare CLENs in absence and presence respectively. The percentage of doxorubicin incorpo- of drug agents. rated was calculated using the following equation: Percent of drug Incorporated CLENs and conventional liposome preparations Fluorescence intensityðÞ after dialysis sample The CLENs used in this study were generally derived ¼ Χ 100 Fluorescence intensityðÞ before dialysis sample from breast cancer cell lines. We named the CLENs by the cell line from which the lipid material was de- rived (i.e., 4 T1 CLENs, BT-20 CLENs etc.). The com- Cellular uptake studies position of the nanoliposome formulation employed Cells were seeded at 1 × 10 /mL in a 48-well plate and in- was as follows: Doxil comprised of DSPC/chol/ LP cubated at 37 °C. Following 24 h of incubation, rhodamine PEG- (50/45/5). 4 T1 CLENs were employed with labeled CLENs prepared from specific cell lines were added the inclusion of chol (0, 10, 25, or 50 mol%) and to the respective well at different concentrations. Following DPPE-PEG (0, 2,5,or10mol%). Whennecessary, an additional 24 h of incubation the plates were washed rhodamine- label was included in CLENs at the DPPE with 1X PBS and analyzed using a fluorescence microplate ratio of 1 mol%. CLENs were prepared by thin film reader (BioTek Instruments, Winooski, Vermont). hydration method as previously reported (Campbell et al., 2002; Dandamudi & Campbell, 2007;Kalra & Cytotoxicity of doxorubicin-loaded CLENs Campbell, 2006; DeSantis et al., 2014). Particle size Sulforhodamine B (SRB) assay was used to determine and zeta (ζ) potential were determined following five percent viability of control based on the amount of basic minutes of sonication using a 90 Plus Particle/Zeta proteins in viable cells following exposure to different Potential Analyzer (Brookhaven Instruments, Holts- CLENs. Cells were seeded at 1 × 10 per ml in a 48-well ville, NY). plate. Columns of cells in the 48 well plate were exposed to either doxil, free doxorubicin, SKBR-3, BT-20 or 4 T1 Drug incorporation efficiency CLENs-loaded with doxorubicin (5 mol%). Following a Doxorubicin hydrochloride (5 mol%) was loaded in 24 h incubation period at 37 °C, cells were treated with conventional and 4 T1 CLENs. To determine drug in- variousconcentrationsofdifferent typesofCLENsloaded corporation efficiency, the two fractions (incorporated with doxorubicin Hcl. The following day, SRB assay was and un-incorporated free drug) were separated. First, utilized to determine the percent of cell viability. Briefly, a required volume was removed from each prepar- plates were washed twice with 1X PBS and the cells were ation type and stored at 4 °C. Next, each preparation fixed with 50% wt/vol TCA (Trichloroacetic acid) and was centrifuged using an ultra-centrifugation system stored at 4 °C for 1 h. Next, plates were washed five times at 13,000 rpm for 15 min, and a pre-determined vol- by distilled deionized water, stained by 0.4% w/v of SRB ume was removed from the preparation and stored at dye, and placed in the dark for 30 min. Excess dye was 4 °C. The centrifuged formulation was transferred to washed minimum of four times with 1% v/v acetic acid and a Float-A-lyzer system with 1000 MW semipermeable left to dry completely. Finally, 1 mL of 1X PBS was added membrane (Fisher Scientific, Pittsburgh, PA) and to each well and the plate was analyzed using a fluores- placed in a beaker filled with 1X PBS at 4 °C over- cence microplate reader. The fluorescence intensity was night. The next day, a required volume from the dia- measured at excitation wavelength of 540/20 nm and emis- lyzed formulation was removed and used for analysis. sion wavelength of 590/20 nm using FLX 800 Fluorescence The three samples (before centrifugation, after centri- Microplate Reader (Bio-tech Instruments, Winooski, VT). fugation, and after dialysis) were added to a 96-well plate to determine doxorubicin fluorescence intensity Cellular toxicity of CLENs using a fluorescence microplate reader. The fluores- To determine the level of cellular toxicity of CLENs cence intensity was measured at excitation and emis- compared to controls the Sulforhodamine B assay was sion wavelengths of 540/20 nm and 590/20 nm, performed as reported elsewhere (Dabbas et al., 2008; Alharbi and Campbell AAPS Open (2018) 4:5 Page 4 of 9 Dandamudi & Campbell, 2007;Kalra &Campbell, 2006), for four different cell lines: three mammary epithelial and as briefly described under the section cytotoxicity of (4 T1, BT-20, SK-BR-3), and one mammary fibroblast doxorubicin-loaded CLENs. (CRL-2089). Each cell line variety was exposed to five different types of CLENs. A toxicity profile for each Statistics preparation is shown in Fig. 1. All CLENs demonstrated Statistical analysis was performed using ANOVA (ana- a relatively non-toxic effect against cellular growth lysis of variance) followed by Turkey’s multiple compari- within the concentration range evaluated. In comparison son procedure as post- hoc, and two-tailed Student’s to the untreated control minimal toxicity was observed t-test using Sigma Plot for Windows (Systat Software, for CLENs. CLENs demonstrated similar toxicity profiles San Jose, CA). Results are presented as mean ± SD, n =6. compared to doxil (data not shown). LP P < 0.05 and P < 0.001 were considered statistically sig- nificant and denoted as * and **, respectively. Cellular uptake of CLENs Cellular uptake studies were performed to determine the Results extent to which CLENs were taken up by various breast Characterization of CLENs cancer cell lines. Figure 2 showed significant cellular The physicochemical properties such as particle size and uptake of CLENs when 4 T1 cells were exposed to 4 T1 surface charge potential are important to assess the CLENs, compared to other CLEN varieties. Similar quality of the preparation, including drug incorporation, results were observed for when the BT-20 cell line was stability and drug release characteristics. Particle size exposed to BT-20 CLENs (Fig. 3). Minimal uptake was and zeta (ζ) potential of the different CLENs were deter- observed for when SK-OV-3 (the negative control) cells mined using the PALS zeta potential analyzer. The uni- were exposed to both 4 T1 and BT-20 CLENs (Figs. 2 modal particle size distribution ranged between 150 and and 3). Four different preparations of 4 T1-CLENs all 263 nm, with an increase in size observed for prepara- containing 25 mol% of cholesterol and different ratios of tions including cholesterol and DPPE-PEG- . All DPPE-PEG- were evaluated for cell uptake against 5000 5000 preparations exhibited negative zeta potential values ran- the 4 T1 cell line (Fig. 4). 4 T1-CLENs containing ging between − 11 to − 21 mV (Table 2). 5 mol% of DPPE-PEG- demonstrated significant up- take compared to the other PEG-containing preparations Incorporation efficiency of doxorubicin in 4 T1-CLENs (Fig. 4). In the next study, we used the optimized ratio The percent of drug incorporated in a drug carrier mol- of PEG- and compared the CLENs type to a ecule is an important step in formulation development. nano-system with a lipid composition resembling Doxil For this reason, we evaluated the incorporation efficiency (referred to here as Doxil (Doxil lipid preparation LP for doxorubicin in 4 T1-CLENs (Table 3). The inclusion without doxorubicin)). The inclusion of DPPE-PEG- of 25 mol% cholesterol, and DPPE-PEG- (2 or 5 mol%) (5 mol%) significantly enhanced the cellular uptake of in 4 T1-CLENs demonstrated the best results. Drug in- CLENs when compared to Doxil (Fig. 5). LP corporation was similar to the incorporation of doxorubi- cin in Doxil (Table 3). The incorporation of the drug Cytotoxicity studies of doxorubicin-loaded CLENs increased the size of 4 T1-CLENs, with no observable Enhanced cellular uptake and selective drug targeting are effect on the values for zeta potential. important features of a drug delivery system, but how each translates overall to therapy is critical to success. For this In vitro cellular toxicity profile of CLENs reason, we next evaluated cytotoxicity of three different The SRB assay was performed for different preparations doxorubicin-loaded CLENs for 4 T1, BT-20 and SKBR-3 of CLENs and compared to the relative effects of con- variety, doxil, and free doxorubicin (0.5 μmol/ml) against ventional liposomes. The toxicity profile was determined the growth of 4 T1 cells in vitro. The results showed a significant decrease in the growth of 4 T1 cells following Table 2 Characterization of different CLENs developed from exposure to 4 T1 doxorubicin-loaded CLENs compared to breast cells untreated cells (P < 0.001). The percent of viable cells was # Nanoliposomal Preparations Particle Size (nm) Zeta potential (mV) 50 and 46% for concentrations of 100 and 150 nmol/mL, 1 Doxil 150 ± 2.0 - 23 ± 0.9 LP respectively (Fig. 6). We observed no significant difference 2 4 T1 CLENs 158 ± 1.9 - 17 ± 2.5 in cytotoxicity among 4 T1 doxorubicin-loaded CLENs, doxil, and free doxorubicin. (Fig. 6). Interestingly, the 3 BT-20 CLENs 263 ± 1.4 - 21 ± 3.5 non-specific SKBR-3 CLENs, forwhich similaramounts of 4 SK-BR-3 CLENs 187 ± 2.1 − 13 ± 1.1 drug agent was incorporated, was significantly less effective 5 CRL-2089 CLENs 203 ± 1.9 − 15 ± 1.3 compared to 4 T1 doxorubicin-loaded CLENs, doxil, and 6 SK-OV-3 CLENs 200 ± 2.2 − 11 ± 2.3 free doxorubicin (data not shown). Alharbi and Campbell AAPS Open (2018) 4:5 Page 5 of 9 Table 3 Characterization and evaluation of 4 T1 CLENs loading efficiency # Nanoliposomes mol% Particle Size (nm) Z-Potential (mV) Dox Incorporation Efficiency % 1 4 T1/chol 100/0 131 ± 6.5 − 14 ± 0.4 63 ± 1.3 2 4 T1/ chol 90/10 184 ± 0.4 − 20 ± 1.2 64 ± 1.9 3 4 T1/ chol 75/25 239 ± 4.3 −18 ± 2.50 86 ± 0.9 4 4 T1/ chol 50/50 282 ± 0.3 −20 ± 2.50 44 ± 0.9 5 4 T1/ chol /PEG- 73/25/2 180 ± 5.2 −10 ± 1.2 81 ± 1.5 6 4 T1/ chol /PEG- 70/25/5 179 ± 0.2 −13 ± 1.5 93 ± 0.9 7 4 T1/ chol /PEG- 65/25/10 290 ± 0.4 −10 ± 1.7 41 ± 0.3 8 Doxil 45/50/5 168 ± 0.6 −13 ± 1.5 95 ± 1.1 LP Discussion lipid extracts from various breast cancer cell lines. Breast cancer is a major cause of cancer death in Perhaps the lipid extracts could one day be optimized women worldwide (Hassan et al., 2010; Jemal et al., to target specific stages of tumor maturation and de- 2011; Moulder & Hortobagyi, 2008; Rivenbark et al., velopment. However, the general procedure employed 2013). The disease is characterized by variant patho- for lipid extraction presented here was optimized for logical features, disparate responses to therapeutics, cell lines regardless of staging. Once the procedure and substantial differences in patient’slong-term sur- was established no additional modifications were vival (Verma et al., 2008; Jemal et al., 2011;Rivenbark necessary (Fig. 7). CLENs contain a wider range of et al., 2013; Taylor et al., 1990). Targeted drug deliv- different lipid components with varied acyl chain ery systems (i.e., liposomes) can improve the pharma- lengths and degree of unsaturation compared to more cokinetic and bio-distribution profile of commonly conventional liposomal preparations. In CLENs the used drugs used for the treatment of disease (Camp- lipids are naturally employed in ratios unique to the bell et al., 2002; Torchilin, 2005; Sharma et al., 2006; target cell, and the fractional makeup of the many Campbell, 2006; Kalra & Campbell, 2006; Allen et al., different lipid components appears to make them less 1991). Our studies were performed to evaluate differ- recognizable by non-target cell populations. This is ent nanoliposome formulations derived from natural ideal when selective drug targeting if needed. Fig. 1 In vitro toxicity profile of CLENs. Cells were seeded at 1 × 10 cells/mL in a 48 well plate and incubated at 37 °C. Percent of cell viability was determined following 24 h of exposure to 10 μmol/mL of respective CLENs. CLENs were added to plates seeded with the same cell line used in the general extraction and development of CLENs; a) 4 T1 cells were exposed to 4 T1 CLENs, b) BT-20 cells were exposed to BT-20 CLENs, c) CRL-2089 cells were exposed to CRL-2089 CLENs and d) SKBR-3 cells were exposed to SKBR-3 CLENs. Data are presented as mean ± S.D.; (n =6) Alharbi and Campbell AAPS Open (2018) 4:5 Page 6 of 9 Fig. 2 4 T1 cellular uptake studies. An amount of 1 × 10 4 T1 cells Fig. 4 Effect of cholesterol and PEG inclusion on cellular uptake of were seeded in a 48 well plate. After 24 h, cells were exposed to CLENs. Four different preparations of 4 T1 CLENs containing different concentration of fluorescently labeled CLENs and incubated 25 mol% of cholesterol and different mol% of DPPE-PEG- (0, 2, 5 with the cells for an additional 24 h. The most significant cellular and 10%) were evaluated for cellular uptake against 4 T1 cell line. uptake was observed with 4 T1 cells compared to the other breast Rhodamine-labeled CLENs were used for this study. The cells were cells. The least amount of cellular uptake was observed with the seeded in a 48-well plate followed by the addition of CLENs for ovarian cancer (negative control) cell line, SK-OV-3. Data are 24 h. The fluorescence intensity used here as an indicator of cell presented as mean ± S.D.; (n =6) uptake was measured using a fluorescence microplate reader. CLENs containing 5 mol% of DPPE-PEG- demonstrated the most significant uptake compared to other varieties within the Investigating the physicochemical characteristics of concentration range employed. Data are presented as mean ± S.D.; (n =6) a drug carrier molecule is an important step to un- derstanding formulation quality, stability, drug release rates and intracellular fate. The average particle size for CLENs was between 100 and 200 nm, the ideal In general, the greatest degree of selectivity was size range for I.V. administered formulations (Torchi- observed when the breast cancer (target) cells were ex- lin, 2005;Sharmaetal., 2006;Campbell, 2006; posed to CLENs prepared from lipids derived from the Pasenkiewicz-Gierula et al., 2000). The zeta potential target cell. When the target cell population was exposed values for CLENs were negatively-charged, suggesting to lipid material from a different source the results were the vehicle is likely to accumulate in the tumor inter- less impressive, suggesting the mechanism(s) underlying stitial environment following I.V. administration. cellular uptake is a function of the lipid composition pro- (Campbell et al., 2002;Campbell, 2006; DeSantis file of CLENs, which is cell line-dependent (Figs. 2 and 3). et al., 2014; Pasenkiewicz-Gierula et al., 2000). The incorporation of chemotherapeutic agents in lipo- Regardless of thecelltype fromwhich the CLENs somes has been shown to enhance the therapeutic index were derived the CLENs were relatively non-toxic to of incorporated drug agents, either by increasing the drug target cells. This is not surprising given the similar concentration in tumor cells, or by decreasing exposure to composition profile between the target cell membrane normal healthy tissues (Sharma et al., 2006;Vemuri & and the nano delivery system. Rhodes, 1995; Pasenkiewicz-Gierula et al., 2000). Fig. 3 BT-20 cellular uptake studies. An amount of 1 × 10 BT-20 cells were seeded in a 48 well plate. After 24 h, cells were exposed Fig. 5 Comparing cellular uptake of CLENs and Doxil . An amount LP to different concentrations of fluorescently labeled CLENs for 24 h. of 1 × 10 4 T1 cells were seeded in a 48- well plate. After 24 h, cells The data show cellular uptake of CLENs by all the cells, with the were incubated for an additional 24 h with various concentrations of highest degree of uptake observed with BT-20 cells, and the least rhodamine labeled CLENs (containing 5 mol% of PEG- ) and amount of cellular uptake was observed with the (negative control) Doxil . The data show that CLENs containing 5 mol% of DPPE-PEG- LP ovarian cancer cell line, SK-OV-3. Data are presented as were taken up to a greater extent compared to the mean ± S.D.; (n= 6) conventional Doxil . Error bars indicate mean ± S.D. (n =6) LP Alharbi and Campbell AAPS Open (2018) 4:5 Page 7 of 9 amount of drug incorporated. The most significant in- crease in incorporated drug was observed following the in- clusion of 25 mol% cholesterol (86% drug loaded). The percent cholesterol is greater than that typically used to prepare conventional liposomes (Raffy & Teissié, 1999; Ohvo-Rekila et al., 2002; Cui et al., 2014). Approximately 44% of drug was incorporated in CLENs consisting of 50 mol% cholesterol, the lowest percent of drug incorpo- rated compared to others evaluated (Table 3;25>10>0> 50 mol%). Previously published reports support a co- relationship between relatively high cholesterol content and liposome instability (Tseng et al., 2007; Decker et al., Fig. 6 Cytotoxicity of doxorubicin-loaded CLENs. 4 T1 cells were 4 2012; Pedrosa et al., 2015). The optimal ratio of cholesterol seeded at 1 X 10 per ml in a 48-well plate. Cells were exposed to with respect to conventional components employed is different concentrations of doxorubicin-loaded CLENs (4 T1, BT-20, and SKBR-3). SRB assay was used to determine percent of cell necessary; the balanced optimization will prevent viability 24 h following exposure to doxorubicin (5 mol%). Error bars premature drug release and formulation instability. indicate mean ± S.D. (n =6) The cellular uptake of CLENs by the target cells was greater overall when compared to Doxil . Moreover, 4 T1 LP Cholesterol and DPPE-PEG- are commonly used to doxorubicin loaded-CLENs demonstrated more significant optimize nanoliposome formulations (Sadzuka et al., 2003; growth inhibitory effects. Data also support greater activ- Hatakeyama et al., 2013; Pasenkiewicz-Gierula et al., 2000). ity against intended target cells compared to non-specific Cholesterol is known to increase the packing order and the target (control) cell populations. A greater degree of rigidity of liposomes. The decrease in the permeability of selectivity was observed when both the lipid extracts used the lipid bilayer due to the inclusion of cholesterol has to prepare CLENs and the target cell population were been shown to improve drug retention for various derived from the same organ tissue environment (i.e. lipid-based nano-sized drug delivery systems. (Raffy & breast). This result was highly consistent and reprodu- Teissié, 1999; Tseng et al., 2007; Sadzuka et al., 2006). cible. However, the most desirable results were achieved PEGylation has offered opportunities to formulate drug when CLENs were prepared directly from the target cell, carrier molecules that can evade opsonization and rela- when compared to target cells that shared the organ tissue tively rapid blood clearance (Sadzuka et al., 2003; environment only (Figs. 2 and 3). Pasenkiewicz-Gierula et al., 2000;Drummondetal., 1999). Not yet identified are the key determinants of selective In our study, the inclusion of 5 mol% DPPE-PEG- in targeting, as well the driving forces underlying cellular CLENs improved cellular uptake over other ratios evalu- uptake of CLENs. Preliminary evaluation of the entire ated (5 mol% > 0, 2 and 10 mol% DPPE-PEG- ). The chloroform-soluble (lipid) fraction of 4 T1 cells by LC/ experimental finding is particularly noteworthy, suggesting MS revealed a variety of glycosphingolipids among other a quite different role of PEG in CLENs compared to more lipid classes (manuscript in preparation). The exact conventional stealth liposomes. CLENs varied in the mechanism for why the complex lipid mixture and Fig. 7 Isolation of lipid extracts from target cells in preparation of CLENs. The schematic shows the process for preparing CLENs from isolation of the chloroform-soluble fraction which corresponds to the lipid extract phase mixture. The inclusion of cholesterol and DPPE-PEG in CLENs improved drug incorporation, stability, cellular uptake, and cytotoxicity among other formulation properties Alharbi and Campbell AAPS Open (2018) 4:5 Page 8 of 9 unique lipid ratio profile of CLENs preferentially accu- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in mulate in target cells is not known. Of interest, following published maps and institutional affiliations. evaluation of a different nanosystem composition, Pedrosa and colleagues showed that relatively short-chained glyco- Received: 24 April 2018 Accepted: 8 July 2018 sphingolipids (SC-GSLs) selectively entered target cell membranes when cells were exposed to SC-GSLs-modified References liposomes. 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Cancer Res 62(23):6831–6836 Cui ZK, Edwards K, Orellana AN, Bastiat G, Benoit JP, Lafleur M (2014) Impact of Conclusions interfacial cholesterol-anchored polyethylene glycol on sterol-rich non- CLENs represent a novel nanoliposome drug platform phospholipid liposomes. J Colloid Inter Sci 428:111–120 capable of recognizing target cells with relatively high ef- Dabbas S, Kaushik RR, Dandamudi S, Kuesters GM, Campbell RB (2008) Importance of the liposomal cationic lipid content and type in tumor vascular targeting: ficiency compared to more conventional nano-systems. physicochemical characterization and in vitro studies using human primary and The drug carrier was relatively non-toxic to cells when transformed endothelial cells. Endothelium 15(4):189–201 used at concentrations traditionally used to evaluate Dan N (2002) Effect of liposome charge and PEG polymer layer thickness on cell–liposome electrostatic interactions. Biochim Biophys Acta 1564(2):343–348 nanoparticles in vitro. Our studies collectively support Dandamudi S, Campbell RB (2007) The drug loading, cytotoxicty and tumor the use of cellular membrane lipid extracts in combin- vascular targeting characteristics of magnetite in magnetic drug targeting. ation with more conventional components of drug deliv- Biomaterials 28(31):4673–4683 Decker C, Fahr A, Kuntsche J, May S (2012) Selective partitioning of cholesterol ery systems to achieve more selective drug targeting. and a model drug into liposomes of varying size. Chem Phys Lipids Overall, CLENs were most efficient when applied against 165(5):520–529 intended target cell populations. However, the organ tis- DeSantis C, Ma J, Bryan L, Jemal A (2014) Breast cancer statistics, 2013. CA Cancer J Clin 64(1):52–62 sue environment appears to play a role in mechanism(s) Deshpande PP, Biswas S, Torchilin VP (2013) Current trends in the use of underlying cell uptake. The results could thus vary de- liposomes for tumor targeting. Nanomedicine (Lond) 8(9):1509–1528 pending on the host tissue environment. For this reason, Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid subsequent studies should investigate the relationship tumors. Pharmacol Rev 51(4):691–743 between CLENs and the tissue environment. Eloy JO, Claro de Souza M, Petrilli R, Barcellos JP, Lee RJ et al (2014) Liposomes as carriers of hydrophilic small molecule drugs: strategies to enhance Abbreviations encapsulation and delivery. Colloids Surf B Biointerfaces 123c:345–363 Chol: Cholesterol; CLENs: Cell membrane lipid extracted nanoliposomes; Hassan MS, Ansari J, Spooner D, Hussain SA (2010) Chemotherapy for breast DOPC: 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine; DOPG: 1, 2-dioleoyl-Sn- cancer (review). Onco Rep 24(5):1121–1131 glycero-3-[phospho-rac-(1-glycerol)]; Doxil: Stealth liposomal doxorubicin; Hatakeyama H, Akita H, Harashima H (2013) The polyethyleneglycol dilemma: Doxil : Doxil lipid preparation only (no drug loaded); DPPE-PEG : 1,2- LP − 5000 advantage and disadvantage of PEGylation of liposomes for systemic genes dipalmitoryl-sn-glycero-3- [phospho-ethanolamine (PEG)]-5000; Rhodamine and nucleic acids delivery to tumors. Biol Pharm Bull 36(6):892–899 : 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine DPPE Jain A, Jain SK (2008) PEGylation: an approach for drug delivery. A review. Crit rhodamine B sulfonyl); SC-GSLs: Short-chained glycosphingolipids; Rev Ther Drug Carrier Syst 25(5):403–447 SRB: Sulforhodamine B; TCA: Trichloroacidic acid Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. C A 61(2):69–90 Acknowledgements Kalra AV, Campbell RB (2006) Development of 5-FU and doxorubicin-loaded We thank the Saudi Arabian Culture Mission and Umm Al-Qura University for cationic liposomes against human pancreatic cancer: implications for tumor host institution sponsorship and support for H.A. vascular targeting. Pharm Res 23(12):2809–2817 Lu Y, Low PS (2002) Folate-mediated delivery of macromolecular anticancer Funding therapeutic agents. Adv Drug Deliv Rev 54(5):675–693 The research study was funded in part by MCPHS University, and the Mathias CJ, Wang S, Waters DJ, Turek JJ, Low PS et al (1998) Indium-111-DTPA- National Science Foundation CMMI Grant No. 1232339 for corresponding folate as a potential folate-receptor-targeted radiopharmaceutical. J Nuc Med author R.B.C. 39(9):1579–1585 Moulder S, Hortobagyi GN (2008) Advances in the treatment of breast cancer. Authors’ contributions Clin pharm thera 83(1):26–36 This manuscript represents work submitted in partial fulfillment of the Ohvo-Rekila H, Ramstedt B, Leppimaki P, Slotte JP (2002) Cholesterol interactions Doctoral Degree at MCPHS University for first author (HA), under the with phospholipids in membranes. Prog Lipid Res 41(1):66–97 direction of (Dr. RBC). HA participated in the experimental design, Pasenkiewicz-Gierula M, Rog T, Kitamura K, Kusumi A (2000) Cholesterol effects manuscript draft and performed the experiments. Supervisor for project RBC on the phosphatidylcholine bilayer polar region: a molecular simulation conceived of the study, participated in the design, coordination and drafts of study. Biophys J 78(3):1376–1389 the manuscript. Both authors read and approved the final manuscript. Pedrosa LR, Ten Hagen TL, Suss R, van Hell A, Eggermont AM, Verheij M et al (2015) Short-chain glycoceramides promote intracellular mitoxantrone Competing interests delivery from novel nanoliposomes into breast cancer cells. Pharma res The authors declare that they have no competing interests. 32(4):1354–1367 Alharbi and Campbell AAPS Open (2018) 4:5 Page 9 of 9 Raffy S, Teissié J (1999) Control of lipid membrane stability by cholesterol content. Biophys J 76(4):2072–2080 Rivenbark AG, O'Connor SM, Coleman WB (2013) Molecular and cellular heterogeneity in breast cancer: challenges for personalized medicine. Am J Path 183(4):1113–1124 Ryan SM, Mantovani G, Wang X, Haddleton DM, Brayden DJ (2008) Advances in PEGylation of important biotech molecules: delivery aspects. Expert Opin Drug Deliv 5(4):371–383 Sadzuka Y, Kishi K, Hirota S, Sonobe T (2003) Effect of polyethyleneglycol (PEG) chain on cell uptake of PEG-modified liposomes. J Liposome Res 13(2):157–172 Sadzuka Y, Sugiyama I, Tsuruda T, Sonobe T (2006) Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin. Int J Pharm 312(1–2):83–89 Sharma G, Anabousi S, Ehrhardt C, Ravi Kumar MN (2006) Liposomes as targeted drug delivery systems in the treatment of breast cancer. J Drug Deliv 14(5):301–310 Taylor KMG, Taylor G, Kellaway IW, Stevens J (1990) Drug entrapment and release from multilamellar and reverse-phase evaporation liposomes. Int J Pharm 58(1):49–55 Torchilin VP (2005) Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4(2):145–160 Tseng LP, Liang HJ, Chung TW, Huang YY, Liu DZ (2007) Liposomes incorporated with cholesterol for drug release triggered by magnetic field. J Med Bio Eng 27(1):29–34 Vemuri S, Rhodes CT (1995) Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharm Acta Helv 70(2):95–111 Verma S, Dent S, Chow BJ, Rayson D, Safra T (2008) Metastatic breast cancer: the role of pegylated liposomal doxorubicin after conventional anthracyclines. Can Treat Rev 34(5):391–406 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AAPS Open Springer Journals

Nano-formulations composed of cell membrane-specific cellular lipid extracts derived from target cells: physicochemical characterization and in vitro evaluation using cellular models of breast carcinoma

AAPS Open , Volume 4 (1) – Jul 23, 2018

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Copyright © 2018 by The Author(s)
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Biomedicine; Pharmaceutical Sciences/Technology; Pharmacology/Toxicology
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10.1186/s41120-018-0025-1
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Abstract

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 (5mol%)increased thesizeof4T1-CLENs from 158 ±2nmto212 ± 59nm, with no significantchangeinthenegatively- chargedsurfacepotential. Percentofdrugloadedrangedfrom40to93%,varying accordingtothe ratiooflipid extract to conventional components employed. The additional inclusion of cholesterol and DPPE-PEG increaseddrugloading 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. Keywords: Breast cancer, Cell lines, Cholesterol, Liposomes, Drug delivery systems Background A current area of research investigation involves the An important goal of chemotherapy is to eradicate the development of nanomedicines capable of recognizing, tumor mass while causing minimal side effects to the and selectively targeting, exploitable tumor features for patient. Liposomes have been employed to help achieve cancer therapy. (Campbell et al., 2002; Torchilin, 2005; this goal. The vesicle type has improved tumor targeting Sharma et al., 2006; Campbell, 2006; Sadzuka et al., 2003) and when optimised can favorably alter the pharmacoki- A few advances in the field of nanoliposome development netic and biodistribution profile of the drug. (Campbell include the inclusion of poly-ethylene glycol (PEG)-PE in et al., 2002; Deshpande et al., 2013; Torchilin, 2005) nano-size drug delivery systems (i.e., liposomes, micelles). Although effective, issues involving limited interstitial (Dan, 2002) The liposome formulation product Doxil drug transport, off-target drug effects and formulation contains PEG, and as a result can successfully exploit the instability still remain. (Deshpande et al., 2013; Torchilin, relatively large tumor vascular pore openings owing to 2005;Sharmaet al., 2006). extended circulation properties afforded by PEG. (Sadzuka et al., 2003; Eloy et al., 2014; Hatakeyama et al., 2013;Jain * Correspondence: robert.campbell@mcphs.edu &Jain, 2008; Ryan et al., 2008;Vemuri&Rhodes, 1995; Department of Pharmaceutical Sciences, MCPHS University, 19 Foster Street, Verma et al., 2008) PEG has versatile functions. For Worcester, MA 01608, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Alharbi and Campbell AAPS Open (2018) 4:5 Page 2 of 9 example, its inclusion in cationic liposomes permits prefer- MO). All chemicals and solvents used in this study were of ential tumor targeting, relatively longer circulation of analytical grade and obtained from Fisher Scientific (Pitts- cationic liposomes while significantly decreasing uptake by burgh, PA). vessels in healthy tissues. (Campbell et al., 2002; Campbell, 2006) Efforts to both gain access to and exploit the differ- Cell culture ential expression of various receptors in tumors for treat- Human breast cancer cell lines BT-20 (HTB-19), ment when compared to normal tissues has improved with SKBR3 (HTB-30), murine mammary cell line 4 T1 advances in liposome technology. (Torchilin, 2005; (CRL-2539), normal mammary fibroblast cell line Sharma et al., 2006;Lu&Low, 2002)Suchexamples CCD-1069SK (CRL-2089), and ovarian cancer cell line include the significant folate receptor-mediated uptake SK-OV-3 (HTB-77) were obtained from ATCC in athymic mice-bearing tumors overexpressing folate (American Type Culture Collection, Manassas, VA). receptor-positive cells, compared to non-folate All cell line cultures were grown in a humidified receptor-labeled systems for which reduced uptake atmosphere of 5% CO at 37 °C. was observed. (Mathias et al., 1998) Regardless of the specific variety of nanomedicine employed most drug delivery systems are prepared from Cellular lipid extraction natural and/or synthetic lipid ingredients. Moreover, the The method used to extract the cellular lipid material ratio of the ingredients employed rarely resemble the was modified from the original Bligh and Dyer composition profile of the target cell membrane. In an method established in 1959. (Dabbas et al., 2008)The effort to achieve more selective targeting we designed, standard gravimetric quantification method involves prepared and evaluated the use of cell membrane the use of three different solvents. In brief, when the lipid-extracted nanoliposomes (CLENs) in vitro. The cells reached approximately 90% confluence, they process involved the extraction of cellular lipid material were trypsinized and collected. The pellet of cells was from intended target cells, and the use of extracted lipid diluted with 1X PBS (phosphate buffer saline). The material with (and without) conventional ingredients for average number of cells in a pellet determined the formulation and in vitro experimentation. Moreover, a proportion of solvents used according to a modified single organ tissue environment was the primary focus chart (Table 1). The three solvents were added of this investigation. The tissue model was additionally step-wise as follows: 1:2 chloroform-methanol mix- selected to represent an organ tissue environment for ture, chloroform, and distilled water, vortexed inter- which the nano drug platform is being developed. For mittently between solvent additions. The final mixture this reason, the cellular lipid material used to prepare was centrifuged at 1000 rpm at 4 °C for 5 min to ob- CLENs was derived from breast tissue, except for when tain a two-phased system; the bottom lipid layer was negative controls were employed. aspirated and transferred into a glass tube and refrig- The composition profile of extracted lipid components erated until a sufficient volume was obtained. of CLENs more closely resemble the target cell mem- brane compared to non-specific preparations in terms of composition. For this reason, we investigated whether Lyophilization method CLENs prepared from lipid extracts derived from target The extracted lipids were dehydrated to form a film cells will associate with the intended target cells to a using a rotary evaporator system, and a heating bath greater extent when compared to CLENs prepared from (Buchi B-491) (Flawil, Switzerland) with temperatures non-specific lipid extracts, and/or conventional nanoli- maintained at 40-50 °C. Sucrose (0.2 M) was added posomal preparations. to serve as a cryoprotectant during the process of lyophilization using a FreeZone Freeze Dry system Methods (Labconco, Kansas City, MO). The lipid powder was Materials weighed and subsequently dissolved in 1 mL of The lipids 1, 2-dioleoyl-Sn-glycero-3-[phospho-rac-(1-gly- chloroform. All lipid chloroform stocks used to pre- cerol)] (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine pare CLENs were stored at − 80 °C. Prior to using (DOPC), Cholesterol (Chol), 1,2-dipalmitoryl-sn-glycero-3- the lipid extracts for preparation of CLENs batches of [phospho-ethanolamine (Polyethylene glycol)]-5000 (DPPE the freeze dried lipid extract were evaluated by -PEG ), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolami LCMS. All lipid components were identified, and the ne-N- (lissamine rhodamine B sulfonyl) (rhodamine ), relative abundance and molecular weights for each -DPPE were purchased from Avanti Polar Lipids (Alabaster, AL). lipid extract type was determined, resulting in average Sulforhodamine B (SRB) and Doxorubicin hydrochloride molecular weights. The final concentrations were sub- (98% HPLC) were purchased from Sigma-Aldrich (St Louis, sequently determined for the lipid extract stocks and Alharbi and Campbell AAPS Open (2018) 4:5 Page 3 of 9 Table 1 Modified chart of the Dyer and Bligh lipid extraction method Cell Number (millions) (5–9.9) (10–24.9) (25–49.9) (50–74.9) (75–99.9) 1:2 CHCl : MeOH 0.750 1.9 3.75 5.7 7.5 CHCl 0.250 0.625 1.25 1.875 2.5 dH O 0.250 0.625 1.25 1.875 2.5 Total Volume 1.45 3.65 7.25 10.95 14.50 later used to prepare CLENs in absence and presence respectively. The percentage of doxorubicin incorpo- of drug agents. rated was calculated using the following equation: Percent of drug Incorporated CLENs and conventional liposome preparations Fluorescence intensityðÞ after dialysis sample The CLENs used in this study were generally derived ¼ Χ 100 Fluorescence intensityðÞ before dialysis sample from breast cancer cell lines. We named the CLENs by the cell line from which the lipid material was de- rived (i.e., 4 T1 CLENs, BT-20 CLENs etc.). The com- Cellular uptake studies position of the nanoliposome formulation employed Cells were seeded at 1 × 10 /mL in a 48-well plate and in- was as follows: Doxil comprised of DSPC/chol/ LP cubated at 37 °C. Following 24 h of incubation, rhodamine PEG- (50/45/5). 4 T1 CLENs were employed with labeled CLENs prepared from specific cell lines were added the inclusion of chol (0, 10, 25, or 50 mol%) and to the respective well at different concentrations. Following DPPE-PEG (0, 2,5,or10mol%). Whennecessary, an additional 24 h of incubation the plates were washed rhodamine- label was included in CLENs at the DPPE with 1X PBS and analyzed using a fluorescence microplate ratio of 1 mol%. CLENs were prepared by thin film reader (BioTek Instruments, Winooski, Vermont). hydration method as previously reported (Campbell et al., 2002; Dandamudi & Campbell, 2007;Kalra & Cytotoxicity of doxorubicin-loaded CLENs Campbell, 2006; DeSantis et al., 2014). Particle size Sulforhodamine B (SRB) assay was used to determine and zeta (ζ) potential were determined following five percent viability of control based on the amount of basic minutes of sonication using a 90 Plus Particle/Zeta proteins in viable cells following exposure to different Potential Analyzer (Brookhaven Instruments, Holts- CLENs. Cells were seeded at 1 × 10 per ml in a 48-well ville, NY). plate. Columns of cells in the 48 well plate were exposed to either doxil, free doxorubicin, SKBR-3, BT-20 or 4 T1 Drug incorporation efficiency CLENs-loaded with doxorubicin (5 mol%). Following a Doxorubicin hydrochloride (5 mol%) was loaded in 24 h incubation period at 37 °C, cells were treated with conventional and 4 T1 CLENs. To determine drug in- variousconcentrationsofdifferent typesofCLENsloaded corporation efficiency, the two fractions (incorporated with doxorubicin Hcl. The following day, SRB assay was and un-incorporated free drug) were separated. First, utilized to determine the percent of cell viability. Briefly, a required volume was removed from each prepar- plates were washed twice with 1X PBS and the cells were ation type and stored at 4 °C. Next, each preparation fixed with 50% wt/vol TCA (Trichloroacetic acid) and was centrifuged using an ultra-centrifugation system stored at 4 °C for 1 h. Next, plates were washed five times at 13,000 rpm for 15 min, and a pre-determined vol- by distilled deionized water, stained by 0.4% w/v of SRB ume was removed from the preparation and stored at dye, and placed in the dark for 30 min. Excess dye was 4 °C. The centrifuged formulation was transferred to washed minimum of four times with 1% v/v acetic acid and a Float-A-lyzer system with 1000 MW semipermeable left to dry completely. Finally, 1 mL of 1X PBS was added membrane (Fisher Scientific, Pittsburgh, PA) and to each well and the plate was analyzed using a fluores- placed in a beaker filled with 1X PBS at 4 °C over- cence microplate reader. The fluorescence intensity was night. The next day, a required volume from the dia- measured at excitation wavelength of 540/20 nm and emis- lyzed formulation was removed and used for analysis. sion wavelength of 590/20 nm using FLX 800 Fluorescence The three samples (before centrifugation, after centri- Microplate Reader (Bio-tech Instruments, Winooski, VT). fugation, and after dialysis) were added to a 96-well plate to determine doxorubicin fluorescence intensity Cellular toxicity of CLENs using a fluorescence microplate reader. The fluores- To determine the level of cellular toxicity of CLENs cence intensity was measured at excitation and emis- compared to controls the Sulforhodamine B assay was sion wavelengths of 540/20 nm and 590/20 nm, performed as reported elsewhere (Dabbas et al., 2008; Alharbi and Campbell AAPS Open (2018) 4:5 Page 4 of 9 Dandamudi & Campbell, 2007;Kalra &Campbell, 2006), for four different cell lines: three mammary epithelial and as briefly described under the section cytotoxicity of (4 T1, BT-20, SK-BR-3), and one mammary fibroblast doxorubicin-loaded CLENs. (CRL-2089). Each cell line variety was exposed to five different types of CLENs. A toxicity profile for each Statistics preparation is shown in Fig. 1. All CLENs demonstrated Statistical analysis was performed using ANOVA (ana- a relatively non-toxic effect against cellular growth lysis of variance) followed by Turkey’s multiple compari- within the concentration range evaluated. In comparison son procedure as post- hoc, and two-tailed Student’s to the untreated control minimal toxicity was observed t-test using Sigma Plot for Windows (Systat Software, for CLENs. CLENs demonstrated similar toxicity profiles San Jose, CA). Results are presented as mean ± SD, n =6. compared to doxil (data not shown). LP P < 0.05 and P < 0.001 were considered statistically sig- nificant and denoted as * and **, respectively. Cellular uptake of CLENs Cellular uptake studies were performed to determine the Results extent to which CLENs were taken up by various breast Characterization of CLENs cancer cell lines. Figure 2 showed significant cellular The physicochemical properties such as particle size and uptake of CLENs when 4 T1 cells were exposed to 4 T1 surface charge potential are important to assess the CLENs, compared to other CLEN varieties. Similar quality of the preparation, including drug incorporation, results were observed for when the BT-20 cell line was stability and drug release characteristics. Particle size exposed to BT-20 CLENs (Fig. 3). Minimal uptake was and zeta (ζ) potential of the different CLENs were deter- observed for when SK-OV-3 (the negative control) cells mined using the PALS zeta potential analyzer. The uni- were exposed to both 4 T1 and BT-20 CLENs (Figs. 2 modal particle size distribution ranged between 150 and and 3). Four different preparations of 4 T1-CLENs all 263 nm, with an increase in size observed for prepara- containing 25 mol% of cholesterol and different ratios of tions including cholesterol and DPPE-PEG- . All DPPE-PEG- were evaluated for cell uptake against 5000 5000 preparations exhibited negative zeta potential values ran- the 4 T1 cell line (Fig. 4). 4 T1-CLENs containing ging between − 11 to − 21 mV (Table 2). 5 mol% of DPPE-PEG- demonstrated significant up- take compared to the other PEG-containing preparations Incorporation efficiency of doxorubicin in 4 T1-CLENs (Fig. 4). In the next study, we used the optimized ratio The percent of drug incorporated in a drug carrier mol- of PEG- and compared the CLENs type to a ecule is an important step in formulation development. nano-system with a lipid composition resembling Doxil For this reason, we evaluated the incorporation efficiency (referred to here as Doxil (Doxil lipid preparation LP for doxorubicin in 4 T1-CLENs (Table 3). The inclusion without doxorubicin)). The inclusion of DPPE-PEG- of 25 mol% cholesterol, and DPPE-PEG- (2 or 5 mol%) (5 mol%) significantly enhanced the cellular uptake of in 4 T1-CLENs demonstrated the best results. Drug in- CLENs when compared to Doxil (Fig. 5). LP corporation was similar to the incorporation of doxorubi- cin in Doxil (Table 3). The incorporation of the drug Cytotoxicity studies of doxorubicin-loaded CLENs increased the size of 4 T1-CLENs, with no observable Enhanced cellular uptake and selective drug targeting are effect on the values for zeta potential. important features of a drug delivery system, but how each translates overall to therapy is critical to success. For this In vitro cellular toxicity profile of CLENs reason, we next evaluated cytotoxicity of three different The SRB assay was performed for different preparations doxorubicin-loaded CLENs for 4 T1, BT-20 and SKBR-3 of CLENs and compared to the relative effects of con- variety, doxil, and free doxorubicin (0.5 μmol/ml) against ventional liposomes. The toxicity profile was determined the growth of 4 T1 cells in vitro. The results showed a significant decrease in the growth of 4 T1 cells following Table 2 Characterization of different CLENs developed from exposure to 4 T1 doxorubicin-loaded CLENs compared to breast cells untreated cells (P < 0.001). The percent of viable cells was # Nanoliposomal Preparations Particle Size (nm) Zeta potential (mV) 50 and 46% for concentrations of 100 and 150 nmol/mL, 1 Doxil 150 ± 2.0 - 23 ± 0.9 LP respectively (Fig. 6). We observed no significant difference 2 4 T1 CLENs 158 ± 1.9 - 17 ± 2.5 in cytotoxicity among 4 T1 doxorubicin-loaded CLENs, doxil, and free doxorubicin. (Fig. 6). Interestingly, the 3 BT-20 CLENs 263 ± 1.4 - 21 ± 3.5 non-specific SKBR-3 CLENs, forwhich similaramounts of 4 SK-BR-3 CLENs 187 ± 2.1 − 13 ± 1.1 drug agent was incorporated, was significantly less effective 5 CRL-2089 CLENs 203 ± 1.9 − 15 ± 1.3 compared to 4 T1 doxorubicin-loaded CLENs, doxil, and 6 SK-OV-3 CLENs 200 ± 2.2 − 11 ± 2.3 free doxorubicin (data not shown). Alharbi and Campbell AAPS Open (2018) 4:5 Page 5 of 9 Table 3 Characterization and evaluation of 4 T1 CLENs loading efficiency # Nanoliposomes mol% Particle Size (nm) Z-Potential (mV) Dox Incorporation Efficiency % 1 4 T1/chol 100/0 131 ± 6.5 − 14 ± 0.4 63 ± 1.3 2 4 T1/ chol 90/10 184 ± 0.4 − 20 ± 1.2 64 ± 1.9 3 4 T1/ chol 75/25 239 ± 4.3 −18 ± 2.50 86 ± 0.9 4 4 T1/ chol 50/50 282 ± 0.3 −20 ± 2.50 44 ± 0.9 5 4 T1/ chol /PEG- 73/25/2 180 ± 5.2 −10 ± 1.2 81 ± 1.5 6 4 T1/ chol /PEG- 70/25/5 179 ± 0.2 −13 ± 1.5 93 ± 0.9 7 4 T1/ chol /PEG- 65/25/10 290 ± 0.4 −10 ± 1.7 41 ± 0.3 8 Doxil 45/50/5 168 ± 0.6 −13 ± 1.5 95 ± 1.1 LP Discussion lipid extracts from various breast cancer cell lines. Breast cancer is a major cause of cancer death in Perhaps the lipid extracts could one day be optimized women worldwide (Hassan et al., 2010; Jemal et al., to target specific stages of tumor maturation and de- 2011; Moulder & Hortobagyi, 2008; Rivenbark et al., velopment. However, the general procedure employed 2013). The disease is characterized by variant patho- for lipid extraction presented here was optimized for logical features, disparate responses to therapeutics, cell lines regardless of staging. Once the procedure and substantial differences in patient’slong-term sur- was established no additional modifications were vival (Verma et al., 2008; Jemal et al., 2011;Rivenbark necessary (Fig. 7). CLENs contain a wider range of et al., 2013; Taylor et al., 1990). Targeted drug deliv- different lipid components with varied acyl chain ery systems (i.e., liposomes) can improve the pharma- lengths and degree of unsaturation compared to more cokinetic and bio-distribution profile of commonly conventional liposomal preparations. In CLENs the used drugs used for the treatment of disease (Camp- lipids are naturally employed in ratios unique to the bell et al., 2002; Torchilin, 2005; Sharma et al., 2006; target cell, and the fractional makeup of the many Campbell, 2006; Kalra & Campbell, 2006; Allen et al., different lipid components appears to make them less 1991). Our studies were performed to evaluate differ- recognizable by non-target cell populations. This is ent nanoliposome formulations derived from natural ideal when selective drug targeting if needed. Fig. 1 In vitro toxicity profile of CLENs. Cells were seeded at 1 × 10 cells/mL in a 48 well plate and incubated at 37 °C. Percent of cell viability was determined following 24 h of exposure to 10 μmol/mL of respective CLENs. CLENs were added to plates seeded with the same cell line used in the general extraction and development of CLENs; a) 4 T1 cells were exposed to 4 T1 CLENs, b) BT-20 cells were exposed to BT-20 CLENs, c) CRL-2089 cells were exposed to CRL-2089 CLENs and d) SKBR-3 cells were exposed to SKBR-3 CLENs. Data are presented as mean ± S.D.; (n =6) Alharbi and Campbell AAPS Open (2018) 4:5 Page 6 of 9 Fig. 2 4 T1 cellular uptake studies. An amount of 1 × 10 4 T1 cells Fig. 4 Effect of cholesterol and PEG inclusion on cellular uptake of were seeded in a 48 well plate. After 24 h, cells were exposed to CLENs. Four different preparations of 4 T1 CLENs containing different concentration of fluorescently labeled CLENs and incubated 25 mol% of cholesterol and different mol% of DPPE-PEG- (0, 2, 5 with the cells for an additional 24 h. The most significant cellular and 10%) were evaluated for cellular uptake against 4 T1 cell line. uptake was observed with 4 T1 cells compared to the other breast Rhodamine-labeled CLENs were used for this study. The cells were cells. The least amount of cellular uptake was observed with the seeded in a 48-well plate followed by the addition of CLENs for ovarian cancer (negative control) cell line, SK-OV-3. Data are 24 h. The fluorescence intensity used here as an indicator of cell presented as mean ± S.D.; (n =6) uptake was measured using a fluorescence microplate reader. CLENs containing 5 mol% of DPPE-PEG- demonstrated the most significant uptake compared to other varieties within the Investigating the physicochemical characteristics of concentration range employed. Data are presented as mean ± S.D.; (n =6) a drug carrier molecule is an important step to un- derstanding formulation quality, stability, drug release rates and intracellular fate. The average particle size for CLENs was between 100 and 200 nm, the ideal In general, the greatest degree of selectivity was size range for I.V. administered formulations (Torchi- observed when the breast cancer (target) cells were ex- lin, 2005;Sharmaetal., 2006;Campbell, 2006; posed to CLENs prepared from lipids derived from the Pasenkiewicz-Gierula et al., 2000). The zeta potential target cell. When the target cell population was exposed values for CLENs were negatively-charged, suggesting to lipid material from a different source the results were the vehicle is likely to accumulate in the tumor inter- less impressive, suggesting the mechanism(s) underlying stitial environment following I.V. administration. cellular uptake is a function of the lipid composition pro- (Campbell et al., 2002;Campbell, 2006; DeSantis file of CLENs, which is cell line-dependent (Figs. 2 and 3). et al., 2014; Pasenkiewicz-Gierula et al., 2000). The incorporation of chemotherapeutic agents in lipo- Regardless of thecelltype fromwhich the CLENs somes has been shown to enhance the therapeutic index were derived the CLENs were relatively non-toxic to of incorporated drug agents, either by increasing the drug target cells. This is not surprising given the similar concentration in tumor cells, or by decreasing exposure to composition profile between the target cell membrane normal healthy tissues (Sharma et al., 2006;Vemuri & and the nano delivery system. Rhodes, 1995; Pasenkiewicz-Gierula et al., 2000). Fig. 3 BT-20 cellular uptake studies. An amount of 1 × 10 BT-20 cells were seeded in a 48 well plate. After 24 h, cells were exposed Fig. 5 Comparing cellular uptake of CLENs and Doxil . An amount LP to different concentrations of fluorescently labeled CLENs for 24 h. of 1 × 10 4 T1 cells were seeded in a 48- well plate. After 24 h, cells The data show cellular uptake of CLENs by all the cells, with the were incubated for an additional 24 h with various concentrations of highest degree of uptake observed with BT-20 cells, and the least rhodamine labeled CLENs (containing 5 mol% of PEG- ) and amount of cellular uptake was observed with the (negative control) Doxil . The data show that CLENs containing 5 mol% of DPPE-PEG- LP ovarian cancer cell line, SK-OV-3. Data are presented as were taken up to a greater extent compared to the mean ± S.D.; (n= 6) conventional Doxil . Error bars indicate mean ± S.D. (n =6) LP Alharbi and Campbell AAPS Open (2018) 4:5 Page 7 of 9 amount of drug incorporated. The most significant in- crease in incorporated drug was observed following the in- clusion of 25 mol% cholesterol (86% drug loaded). The percent cholesterol is greater than that typically used to prepare conventional liposomes (Raffy & Teissié, 1999; Ohvo-Rekila et al., 2002; Cui et al., 2014). Approximately 44% of drug was incorporated in CLENs consisting of 50 mol% cholesterol, the lowest percent of drug incorpo- rated compared to others evaluated (Table 3;25>10>0> 50 mol%). Previously published reports support a co- relationship between relatively high cholesterol content and liposome instability (Tseng et al., 2007; Decker et al., Fig. 6 Cytotoxicity of doxorubicin-loaded CLENs. 4 T1 cells were 4 2012; Pedrosa et al., 2015). The optimal ratio of cholesterol seeded at 1 X 10 per ml in a 48-well plate. Cells were exposed to with respect to conventional components employed is different concentrations of doxorubicin-loaded CLENs (4 T1, BT-20, and SKBR-3). SRB assay was used to determine percent of cell necessary; the balanced optimization will prevent viability 24 h following exposure to doxorubicin (5 mol%). Error bars premature drug release and formulation instability. indicate mean ± S.D. (n =6) The cellular uptake of CLENs by the target cells was greater overall when compared to Doxil . Moreover, 4 T1 LP Cholesterol and DPPE-PEG- are commonly used to doxorubicin loaded-CLENs demonstrated more significant optimize nanoliposome formulations (Sadzuka et al., 2003; growth inhibitory effects. Data also support greater activ- Hatakeyama et al., 2013; Pasenkiewicz-Gierula et al., 2000). ity against intended target cells compared to non-specific Cholesterol is known to increase the packing order and the target (control) cell populations. A greater degree of rigidity of liposomes. The decrease in the permeability of selectivity was observed when both the lipid extracts used the lipid bilayer due to the inclusion of cholesterol has to prepare CLENs and the target cell population were been shown to improve drug retention for various derived from the same organ tissue environment (i.e. lipid-based nano-sized drug delivery systems. (Raffy & breast). This result was highly consistent and reprodu- Teissié, 1999; Tseng et al., 2007; Sadzuka et al., 2006). cible. However, the most desirable results were achieved PEGylation has offered opportunities to formulate drug when CLENs were prepared directly from the target cell, carrier molecules that can evade opsonization and rela- when compared to target cells that shared the organ tissue tively rapid blood clearance (Sadzuka et al., 2003; environment only (Figs. 2 and 3). Pasenkiewicz-Gierula et al., 2000;Drummondetal., 1999). Not yet identified are the key determinants of selective In our study, the inclusion of 5 mol% DPPE-PEG- in targeting, as well the driving forces underlying cellular CLENs improved cellular uptake over other ratios evalu- uptake of CLENs. Preliminary evaluation of the entire ated (5 mol% > 0, 2 and 10 mol% DPPE-PEG- ). The chloroform-soluble (lipid) fraction of 4 T1 cells by LC/ experimental finding is particularly noteworthy, suggesting MS revealed a variety of glycosphingolipids among other a quite different role of PEG in CLENs compared to more lipid classes (manuscript in preparation). The exact conventional stealth liposomes. CLENs varied in the mechanism for why the complex lipid mixture and Fig. 7 Isolation of lipid extracts from target cells in preparation of CLENs. The schematic shows the process for preparing CLENs from isolation of the chloroform-soluble fraction which corresponds to the lipid extract phase mixture. The inclusion of cholesterol and DPPE-PEG in CLENs improved drug incorporation, stability, cellular uptake, and cytotoxicity among other formulation properties Alharbi and Campbell AAPS Open (2018) 4:5 Page 8 of 9 unique lipid ratio profile of CLENs preferentially accu- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in mulate in target cells is not known. Of interest, following published maps and institutional affiliations. evaluation of a different nanosystem composition, Pedrosa and colleagues showed that relatively short-chained glyco- Received: 24 April 2018 Accepted: 8 July 2018 sphingolipids (SC-GSLs) selectively entered target cell membranes when cells were exposed to SC-GSLs-modified References liposomes. 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Nanomedicine (Lond) 8(9):1509–1528 pending on the host tissue environment. For this reason, Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid subsequent studies should investigate the relationship tumors. Pharmacol Rev 51(4):691–743 between CLENs and the tissue environment. Eloy JO, Claro de Souza M, Petrilli R, Barcellos JP, Lee RJ et al (2014) Liposomes as carriers of hydrophilic small molecule drugs: strategies to enhance Abbreviations encapsulation and delivery. Colloids Surf B Biointerfaces 123c:345–363 Chol: Cholesterol; CLENs: Cell membrane lipid extracted nanoliposomes; Hassan MS, Ansari J, Spooner D, Hussain SA (2010) Chemotherapy for breast DOPC: 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine; DOPG: 1, 2-dioleoyl-Sn- cancer (review). Onco Rep 24(5):1121–1131 glycero-3-[phospho-rac-(1-glycerol)]; Doxil: Stealth liposomal doxorubicin; Hatakeyama H, Akita H, Harashima H (2013) The polyethyleneglycol dilemma: Doxil : Doxil lipid preparation only (no drug loaded); DPPE-PEG : 1,2- LP − 5000 advantage and disadvantage of PEGylation of liposomes for systemic genes dipalmitoryl-sn-glycero-3- [phospho-ethanolamine (PEG)]-5000; Rhodamine and nucleic acids delivery to tumors. Biol Pharm Bull 36(6):892–899 : 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine DPPE Jain A, Jain SK (2008) PEGylation: an approach for drug delivery. A review. Crit rhodamine B sulfonyl); SC-GSLs: Short-chained glycosphingolipids; Rev Ther Drug Carrier Syst 25(5):403–447 SRB: Sulforhodamine B; TCA: Trichloroacidic acid Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. C A 61(2):69–90 Acknowledgements Kalra AV, Campbell RB (2006) Development of 5-FU and doxorubicin-loaded We thank the Saudi Arabian Culture Mission and Umm Al-Qura University for cationic liposomes against human pancreatic cancer: implications for tumor host institution sponsorship and support for H.A. vascular targeting. Pharm Res 23(12):2809–2817 Lu Y, Low PS (2002) Folate-mediated delivery of macromolecular anticancer Funding therapeutic agents. Adv Drug Deliv Rev 54(5):675–693 The research study was funded in part by MCPHS University, and the Mathias CJ, Wang S, Waters DJ, Turek JJ, Low PS et al (1998) Indium-111-DTPA- National Science Foundation CMMI Grant No. 1232339 for corresponding folate as a potential folate-receptor-targeted radiopharmaceutical. J Nuc Med author R.B.C. 39(9):1579–1585 Moulder S, Hortobagyi GN (2008) Advances in the treatment of breast cancer. Authors’ contributions Clin pharm thera 83(1):26–36 This manuscript represents work submitted in partial fulfillment of the Ohvo-Rekila H, Ramstedt B, Leppimaki P, Slotte JP (2002) Cholesterol interactions Doctoral Degree at MCPHS University for first author (HA), under the with phospholipids in membranes. Prog Lipid Res 41(1):66–97 direction of (Dr. RBC). HA participated in the experimental design, Pasenkiewicz-Gierula M, Rog T, Kitamura K, Kusumi A (2000) Cholesterol effects manuscript draft and performed the experiments. Supervisor for project RBC on the phosphatidylcholine bilayer polar region: a molecular simulation conceived of the study, participated in the design, coordination and drafts of study. Biophys J 78(3):1376–1389 the manuscript. Both authors read and approved the final manuscript. 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J Liposome Res 13(2):157–172 Sadzuka Y, Sugiyama I, Tsuruda T, Sonobe T (2006) Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin. Int J Pharm 312(1–2):83–89 Sharma G, Anabousi S, Ehrhardt C, Ravi Kumar MN (2006) Liposomes as targeted drug delivery systems in the treatment of breast cancer. J Drug Deliv 14(5):301–310 Taylor KMG, Taylor G, Kellaway IW, Stevens J (1990) Drug entrapment and release from multilamellar and reverse-phase evaporation liposomes. Int J Pharm 58(1):49–55 Torchilin VP (2005) Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4(2):145–160 Tseng LP, Liang HJ, Chung TW, Huang YY, Liu DZ (2007) Liposomes incorporated with cholesterol for drug release triggered by magnetic field. J Med Bio Eng 27(1):29–34 Vemuri S, Rhodes CT (1995) Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharm Acta Helv 70(2):95–111 Verma S, Dent S, Chow BJ, Rayson D, Safra T (2008) Metastatic breast cancer: the role of pegylated liposomal doxorubicin after conventional anthracyclines. Can Treat Rev 34(5):391–406

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Published: Jul 23, 2018

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