Encapsulation of Nutraceutical Ingredients in Liposomes and Their Potential for Cancer Treatment
ABSTRACT
At present, cancer is one of the major diseases in the world affecting numerous lives. There have been various approaches to combat the disease, particularly involving chemical inter- ventions (chemotherapy). However, owing to serious side effects of chemotherapy, employ- ment of natural supplements in cancer therapy has been long desired. Nutraceuticals are currently being studied as a medicament, to act as both preventive and curative measure. Nutraceuticals provide both nutrition and therapeutic benefits; besides, they are natural and biocompatible, and therefore pose no side effects. This facilitates their ready acceptance as dietary supplements with no requirements of special dosage and concerns over long-term usage. Nutraceuticals can be derived from the natural resources such as spices, fruits, vege- tables, and plants. However, nutraceuticals are vulnerable to environmental stresses that necessitate encapsulation for long-term storage and required bioavailability. The review col- lates the findings on encapsulated nutraceuticals in liposomes for cancer therapy. The article provides a coherent overview of the research conducted on liposomal administration of nutraceuticals to target various forms of cancer, explaining the advances made.
Introduction
In today’s world, cancer is the largest cause of mortal- ity that claims over 6 million lives each year. The death rate from cancer is increasing every year and according to World Health Organization (WHO) report, it will increase by 104% by 2020 (1). Over 100 distinct types of cancer have been identified till date, including breast cancer, skin cancer, lung cancer, colon cancer, prostate cancer, and lymphoma (2). From the upward trend of prevalence of cancer, it is presumed that at the present rate, cancer cases will affect 24 million per year worldwide by 2050. The cases of incidence of cancer are also not less. Epidemiological and laboratory data indicate that cancer is not only linked to genetics, but also with lifestyle, including dietary anomalies and envir- onmental extremities (3). Different types of therapies are in practice today to combat cancer including surgery, radiation therapy, chemotherapy, immuno- therapy, hormone therapy, and stem cell transplant.‘Chemoprevention’ is an emerging and extremely promising strategy for cancer prevention which employs chemical agents (synthetic or natural), singly or in com- bination, to restrict the occurrence of cancer in human cells. However, there are multitude reasons to preclude them from the current formulary, most asserting being their side effects. As a result, during the last two decades, efforts have been made to evaluate the chemopreventive role of chemical constituents in natural products (4). Therapeutically active chemical compounds in nature are commonly referred to as ‘nutraceuticals’, i.e., they pro- vide therapeutic value along with nutritional benefits. The term nutraceutical was coined from ‘nutrition’ and ‘pharmaceutical’ by Stephen DeFelice in 1989 (3). According to Mehta et al. (5), nutraceutical is a food or food constituent having preventive and/or curative roles against maladies. In the past decade, several nutraceuti- cals have been identified that have chemopreventive effects.
Several mechanisms are followed by nutraceuticals for cancer prevention, such as inhibiting cell proliferation and differentiation, inhibiting efflux transporters such as breast cancer resistance protein, P-glycoprotein, multi- drug resistance protein (MRP), or by reducing the tox- icity of chemotherapeutic drugs (Fig. 1) (6).However, for most of the promising drugs, the bio- availability gets compromised, owing to their hydro- phobic nature. Therefore, investigations discussed herein are focused on liposome-mediated targeted delivery of nutraceuticals for cancer therapy.Liposomes are reportedly known to be closed, continuous, vesicular structures composed mainly of phospholipid bilayers that incorporate hydrophilic molecules inside the aqueous core and lipophilic molecules in their bilayer (7,8). This encapsulation procedure offers several advantages such as higher encapsulation efficiency; ability to load lipophilic and hydrophilic drugs together; biocompatibility; provision of receptor-mediated site-specific targeting and triggering of controlled release (9). Liposomes are typically suitable for such drug delivery since they delay the clearance duration and increase the intravascular circulation time of encapsulated drugs, thereby altering their bio-distribution (10). Moreover, liposomes are modified with polyethylene- glycol (PEG) (pegylated liposomes), thereby decreas- ing digestion of liposomes by macrophages in the reticulo-endothelial system, and enhancing the reten- tion of drugs (11).
Albumin is a protein in human serum that helps in carrying drug molecules to their target sites, and releases the molecules at the target by fluid phase pinocytosis; whereas, liposomes release drug molecules by membrane fusion method (12). The drug encapsu- lated in the liposome is protected from metabolism and the drug molecule becomes active only after the release of the same from liposome (13). Generally mononuclear phagocytic cells in the reticuloendothe- lial system play a major role in uptake of liposomes; although bone marrow and lymph node uptake also occurs (13,14). This uptake is mediated by nonspecific association (or adsorption) of liposomes onto the cell surface, and subsequent endocytosis. Even though the regulating factors for the uptake of liposome are not clearly understood, researchers opine that the binding of liposome to the target cell itself maybe the control- ling step. Also, reports suggest that negatively charged liposomes (prepared with phosphatidylserine, phos- phatidylglycerol or phosphatidic acid) show faster and greater uptake by phagocytic (or endocytic) cells, compared with neutral liposomes (14).The present review would discuss the reports on liposomal encapsulation of several nutraceuticals that have been investigated to protect and enhance the bioavailability of the respective bioactives, along with their role in targeted delivery to alleviate cancer. At the outset, a brief overview on the key findings of researchers in liposomal delivery of nutraceuticals for cancer therapy has been presented in Table 1. The reports presented in the table have been discussed section-wise hereafter.
Curcumin is a natural polyphenol found in the rhi- zomes of Curcuma longa (turmeric). Curcumin reportedly exhibits antioxidant, anti-inflammatory, antineoplastic, and chemopreventive activities (12). It is a highly hydrophobic molecule (solubility of curcu- min in water is 0.0004 mg/mL at pH 7.3) and is poorly absorbed in intestine. Research suggests that after oral administration, curcumin is biotransformed to dihydro- and tetrahydro-curcumin. During intraven- ous and intraperitoneal administration, curcumin is excreted through bile secretion, after having been metab- olized as glucoronides of tetrahydrocurcumin and hexa- hydrocurcumin (15). Therefore, liposomal encapsulation is recommended for its delivery. Several investigations have focused on liposomal delivery of curcumin, and they have been presented as cases below.(16). NF-jB is activated by a broad range of agents, including various carcinogens, inflammatory cyto- kines (e.g., interleukin-1 [IL-1] and tumor necrosis factor [TNF]), and extracellular stress (e.g., ultravio- let light and cigarette smoke) (17). Li et al. (18) have employed electrophoretic mobility shift assay to detect NF-jB binding before and after curcumin- based liposomal treatment. Human pancreatic cell lines ASPC-1, BxPC-3, Capan-1, Capan-2, HS766-T, and MiaPaCa2 were utilized by these authors to investigate the effect of liposomal curcumin on cell proliferation and apoptosis, and on constitutive NF- jB activity and NF-jB-regulated genes expression. Exposure to liposomal curcumin for 72 h arrested pancreatic cell growth in all six cell lines. Liposomal curcumin decreased NF-jB binding and its effects were equipotent to that of free curcumin (Fig. 2). The liposomes suppressed the growth of both BxPC-3 and MiaPaCa2 tumors in murine models (dosage of 40 mg/kg of liposomal curcumin intra- venously three times in a week). The finding thereby established the chemopreventive effect of liposomal route of curcumin administration.Figure 2. Effects of free and liposomal curcumin on NF-jB in BxPC-3, Capan-1, and HS766-T cell lines. Cells were exposed to IC75 levels of free and liposomal curcumin and supershifts using anti-p50 and anti-p65 antibodies confirmed that the band shown con- tained p50 and p65 subunits of NF-jB (18).
The weak fluorescence of curcumin in organic solvents has been utilized by Kunwar et al. (12,19) to study the binding of curcumin to phosphatidylcholine (PC) liposomes (PC: cholesterol ¼ 2:1, w/w) and human serum albumin (HSA). Both liposomal and HSA vehicles have been examined for the transfer of curcumin to mouse spleen lymphocyte cells, EL4 lymphoma cell line (T cell lymphoma of mouse origin), and have been compared with the efficacy of aqueous DMSO vehicles.Liposomal vehicle was found to be capable of more sample loading and enhanced bioactive’s release into cells compared with HSA or aqueous-DMSO. For nor- mal mouse lymphocytes, concentration-dependent uptake of curcumin has been seen which was more efficient for liposome-mediated delivery. Comparison of fluorescence anisotropy of curcumin in aqueous solutions was with that of the curcumin present in PC liposome revealed significant increase in anisotropy (from 0.13 ± 0.01 in aqueous solution to 0.31 ± 0.01 in the presence of PC liposome). The data confirmed that there was sufficient entrapment of curcumin in the liposome gel phase (at neutral pH), wherein it experienced a restricted motion. The finding reflected that the uptake of curcumin was significantly higher in EL4 cells (~1.5 times higher) with all the vehicles, in comparison with spleen lymphocytes.
In another study, Thangapazham et al. (10) have observed anti-proliferative activity of curcumin lipo- somes (100–150 nm) with phospholipids:cholesterol:- curcumin in the ratio of 90:10:10 (w/w) on cancer cell lines. The authors worked on two human prostate cancer cell lines (LNCaP and C4-2B) employing tetrazolium dye-based [(3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide), MTT] assay. Curcumin liposome (5–10 lM) exhibited 70–80% inhibition of cellular proliferation, without affecting their viability; whereas, free curcumin evinced similar inhibition, but at 10-fold higher dose (i.e., >50 lM).These researchers found that LNCaP cells were more sensitive to liposomal curcumin compared with C4-2B cells. They also reported that liposome-loaded curcu- min was pharmacologically more potent in limiting cancerous cell growth than its free counterpart.Suppression of head and squamous cell carcinoma (HNSCC) which often necessitates surgical operation has been achieved with liposomal curcumin, in a dose-dependent manner by Wang et al. (20). The investigators studied two cell lines (CAL 27 and UM- SCC1). Their findings showed that curcumin liposome (prepared with 9:1 ratio of 1,2-dimyristoyl-sn-glycero- 3-phosphocholine and 1,2-dimyristoyl-sn-glycero- 3-phospho-rac-(1-glycerol), lipid: curcumin ¼ 10:1) resulted in suppression of NF-jB; but it affected neither the expression of phospho-AKT (pAKT), nor its downstream target, phospho-S6 kinase. The finding highlighted the effect of curcumin on the NFjB path- way by reducing expression of NF-jB-activated genes cyclin D1, cyclooxygenase-2, matrix metallo- proteinase-9, Bcl-2, Bcl-xL, Mcl-1L and Mcl-1S, in the curcumin-treated cell lines. In vivo treatment of HNSCC xenograft mice with liposomal curcumin (50 mg/kg) was then conducted by these authors employing 5-week-old female athymic nude mice (nu/nu; Harlan), four times a week, for 3.5 weeks, though tail vein injection. The results confirmed a nontoxic suppression of xenograft in mouse tumors treated with liposomal curcumin vis-`a-vis compared with the control mice as well as mice treated with empty liposomes.
The comparative evaluation of curcumin liposome and free curcumin, in vitro, by short-term (48 h) and long-term (2 weeks) assays has been reported by Pandelidou et al. (15). In the study, curcumin was reportedly incorporated in egg-phosphatidylcholine (EPC) liposomes (curcumin:EPC = 1:14 molar ratio). Colorectal cell lines of HCT116, HCT15, Colo205, and DLD-1 were employed for the short-term assay; while, HCT116, HCT15, and DLD-1 were used for the long- term cytotoxicity assay. A moderate growth inhibiting was achieved with curcumin liposome which was substantially enhanced with prolonged incubation. Highest activity of liposomal curcumin was obtained for HCT15 cell line (known to express multiple drug resistance (MDR) phenotype), suggesting the possibility of curcumin liposome therapy for the same.
In another study, curcumin liposomes have been shown to inhibit cervical cancer cells. HeLa (HPV type 18 positive cells) and SiHa (HPV type 16 positive cells) were employed to study the effect of curcumin liposome on cervical cancer cell lines by Saengkrit et al. (21). In their study, curcumin was encapsulated in cationic liposome prepared with didodecyl dimethyl ammonium bromide (DDAB), cholesterol and nonionic surfactant (Montanov82VR ). Four types of curcumin loaded liposomes namely, LS: liposome/ Montanov82VR ; LC: liposome/cholesterol; LSD: liposome/Montanov82VR /DDAB and LCD: liposome/ cholesterol/DDAB were formulated to investigate the cellular internalization of liposomes, their cytotoxicity to human cervical cancer cell lines, antitumor activities through apoptosis induction and evaluation of effect of curcumin liposomes on the changes in cellular morphology. Confocal microscopy and flow cytometry data revealed that cellular internalization was a time-dependent property of liposome, and formulation of cationic liposomes improved this capability of curcumin liposome. Also, the cytotoxicity of curcumin liposome was found to be more promin- ent compared to the free curcumin at the same con- centration (at the IC50 of curcumin, for HeLa 21 mM and for SiHa 16 mM). Cells treated with curcumin and curcumin liposomes have been reported to be double stained with Annexin V-FITC (AV-FITC) and propi- dium iodide (PI) for detecting apoptotic and necrotic cells. Similar to the results of cytotoxicity study, cells treated with curcumin liposome also manifested enhanced apoptotic capability compared with curcumin, owing to the high binding affinity between liposome and cell membrane, due to the presence of DDAB.
Therefore, the effect of curcumin-loaded liposomes in cancer treatment is significant and encouraging. The literature attests to the efficiency of the delivery method, with desirable release property.Resveratrol (trans-3,40,5-trihydroxystilbene) is a natural polyphenol commonly found in fruits, vegetables, and medicinal plants such as grapes, nuts, and polygonum roots (22). Resveratrol has been particularly studied in the past for its antioxidant, anti-inflammatory, anti- diabetic, anti-cancer, and neuro-protective activities (23). Wang et al. (24) have formulated mitochondria targeting resveratrol liposomes to combat the MDR nature of cancer cells, by targeting the ‘mitochondria apoptosis pathway’. As reported, liposomes were formulated with dequlinium-polyethylene glycol di-stearoyl phosphatidyl ethanol amine (DQA-PEG2000-DSPE) conjugate (lipids:- drug = 20:1). The authors also prepared mitochondria targeting coumarin liposomes that worked as fluores- cence probe in ‘laser confocal microscopy’, to ensure the reach of mitochondria targeting liposomes to the site, i.e., mitochondria of cancerous cells.The experimental data delineated that live cells had localization of mitochondria targeting coumarin lipo- somes (confirmed by fluorescent probe), confirming that these liposomes were actually transported into mitochondria of cancerous cells. However, the authors did not find any free coumarin or coumarin lipo- somes in the mitochondria. The finding thus proved that DQA-PEG2000-DSPE was able to target the lipo- somes to mitochondria of cancerous cells.Furthermore, in the same study, these authors carried out treatment of human lung adenocarcinoma A549 cells and MDR lung cancer cells (A549/cDDP cells) at various concentrations of free resveratrol (0–50 mM), resveratrol liposomes, blank mitochondria targeting liposomes, and mitochondria targeting resveratrol liposomes. Cell cytotoxicity was reportedly investigated employing combination of mitochondria targeting resveratrol liposomes (1, 5, 10 mM) and vinorelbine liposomes (0.01, 0.10, 1.00 mM). Strong inhibitory effects of mitochondria targeting resveratrol liposomes on both A549 and A549/cDDP cells and enhancement of the inhibitory effect of vinorelbine liposomes in the combination study were observed.
The authors hypothesized that the mitochondria targeting liposomes were endocytosed by the cellular mitochondria, wherein the liposomes triggered the release of cytochrome C that eventually resulted in cancerous cells’ death. The increased solubility of lipo- phobic resveratrol caused mitochondrial co-localiza- tion. The higher mitochondrial uptake of liposome observed, on the other hand, could have promoted the inhibitory activity of the mitochondria targeting resveratrol liposomes. The said liposome has also been reported to have shown inhibitory effects, in vivo on volume of tumors in the resistant A549/cDDP xeno- grafted nude mice, attesting to the antitumor activity.
In another study, Lu et al. (22) employed reverse-phase evaporation method with and without PEG2000- grafted DSPE-PEG2000 to formulate resveratrol liposomes. The experiment involved human hepatocellular car- cinoma (Hep G2) cells and HeLa treated with resvera- trol liposome and PEGylated resveratrol liposomes in different concentrations. The results indicated that encapsulation of resveratrol in liposome enhanced the effect of resveratrol to inhibit cancer cells, possibly owing to the increase in the concentration of resvera- trol in liposome. For Hep G2 cells, % occurrence of infected cells’ death was similar in both PEGylated (42.4% at 24 h and 56.23% at 48 h) and non- PEGylated (42.6% at 24 h and 55.7% at 48 h) resvera- trol liposomes. Higher effectiveness was observed in HeLa cells treated with PEGylated liposomes (40.5% at 24 h and 77.8% at 48 h) compared with non- PEGylated (36.1% at 24 h and 72.3% at 48 h) resvera- trol liposomes, at a concentration of 80 mg/mL.For improved bioavailability and enhanced chemo- preventive effects of curcumin and resveratrol, Narayanan et al. (25) employed combination treat- ment of liposomes against prostate cancer. Both cur- cumin and resveratrol were encapsulated individually in liposomes [5 mg of each/kg body weight (bw)] prepared with 1,2-dimyristoyl-rac-glycero-3-phospho- choline (nutraceutical:lipid = 5:1) and mixed (each at2.5 mg/kg bw) using a three-way adjuvant mixer. To determine the bioavailability, liposomes were adminis- tered to male B6C3F1/J mice (6–8 weeks age) indi- vidually and in combination, by oral gavages. Based on the high-performance liquid chromatography (HPLC) analysis, the authors reported that liposomal encapsulation enhanced the concentration of both curcumin and resveratrol in the serum and in prostate tissues. Also, co-administration of curcumin with resveratrol was found to enhance the concentration of curcumin in cancerous cells (1.5 folds for serum and about 3.0 folds for prostate tissue).
These authors also worked on genetically modified prostate-specific PTEN-KO (phosphatase and tensin homolog-knockout) mouse model to study the chemo- preventive effect of curcumin liposome co-adminis- tered with resveratrol liposome against prostate cancer
(25). This model resembles the multistep tumorigen- esis of human prostate cancer. Homozygous male PTEN-KO mice aging four weeks were treated with curcumin liposome (50 mg/kg bw), resveratrol lipo- some (50 mg/kg bw) and mix of both liposomes (each 25 mg/kg bw) by oral gavages, three times in a week for seven weeks. Examination of H&E stained DL prostate tissue sections revealed moderate inhibitory effects of individual liposomal curcumin and resveratrol; whereas, combination of both liposomes displayed strong inhibition and regression of adeno- carcinomas (Fig. 3).In vitro study was also conducted for the effects of free curcumin (10 mM), resveratrol (10 mM), and their combination (5 mM each) on murine PTEN-CaP8 prostate cancer cells for effects such as cellular growth inhibition, cell cycle regulation, detection of apoptosis, and androgen receptor (AR) gene silencing effects, by transfection assays (25). The results affirmed that the combination treatment had significantly decreased the cancer cells’ viability and increased the rate of apoptosis in the range of 55–62%. Cell cycle analysis further confirmed the finding, specifically describing the stage of cell cycle that was affected; the G1 peak was associated with a distinct pre-G1-peak, indicating presence of apoptotic cells (Fig. 4). Combination of curcumin and resveratrol resulted in cells that were deficit in target proteins’ AR and cyclin D1 levels, along with a strong increase in the expression of the cell cycle regulatory proteins (p21 and p27). Therefore, these cell cycle regulatory proteins could be modulated by these nutraceuticals. Further, western blot analysis evidenced a significant inhibitory effect of curcumin and resveratrol in combination with p-Akt (ser 473), AR, cyclin D1, and mTOR proteins in PTENCap8 cells, with a loss of Figure 3. Tumor growth inhibition. c1: untreated control with invasive adenocarcinomas, c2: cellular changes indicating the inhib- ition of adenocarcinomas with resveratrol liposome, c3: cellular changes showing moderate inhibitory effect on adenocarcinomas by curcumin liposome, and c4: cellular changes indicating a strong inhibition and regression of adenocarcinomas by combination treatment of curcumin liposome and resveratrol liposome (25).Figure 4. Cell cycle analysis. (A) Cells treated with DMSO as solvent control and (B) Cells treated with curcumin in combination with resveratrol (25).PTEN. The data suggested that these nutraceuticals can target multiple pathways of prostate carcinogenesis and restrict their growth.
Breast cancer is the second most common cancer worldwide and claims innumerable lives annually (26). Human epidermal growth factor receptor 2 (HER2), a transmembrane receptor tyrosine kinase, is generally over-expressed in breast and ovarian cancer patients, and is commonly targeted for breast cancer therapy. Immuno-liposomes are liposome with monoclonal or polyclonal antibody tagged to the liposomal surface that recognizes and binds to a specific antigen on the surface of the targeted tumor cells. Catania et al. (11) formulated immuno-liposomes (using trastuzumab, a humanized monoclonal antibody) with curcumin (6.5%) and resveratrol (5.8%) individually as well as in combinations (5.7 and 1.7%, respectively) to compare cytotoxic activity and selectivity of these immuno-liposomes against HER2 positive human breast cancer cells.As described by the authors, the study involved two human breast cancer cell lines (JIMT1 cell line, high-grade invasive ductal carcinoma positive to HER2 receptor and trastuzumab resistant; and MCF7 breast cancer cell line). The authors reported that the use of immuno-liposomes enhanced the efficacy of nutraceuticals against both cell lines compared with that of empty liposomes (control). The finding was more conspicuous in JIMT1 cells, and more promin- ent for curcumin than resveratrol. The combined immuno-liposomal formulation of curcumin and resveratrol was therefore more effective in mitigating growth of cancerous cells, evaluated against the immuno-liposome with curcumin or resveratrol alone. This observation further put strength to the premise that both these nutraceuticals were selectively target- ing cancer cells in liposomal formulation. In addition, the ImageStream (Amnis Corporation, Seattle, WA) technique divulged that there was 1.7 times higher incorporation of curcumin into the JIMT1 cell line using immuno-liposome.
Several fruits, vegetables, nuts, and wine contain the natural flavonoid, fisetin (3,30,40,7-tetrahydroxyfla- vone). This bioactive is known to have several pharmacological properties useful in prevention and treatment of cancer (27). The researchers have observed a 47-fold increase in relative bioavailability of liposomal fisetin in intra-peritoneal administration compared to its free form (28). Lewis lung carcinoma cells (female 8 weeks old C57BL/6J mice) were treated with free and liposomal fisetin. From cell cycle assess- ment, an increased duration of sub-G1 phase was demonstrated in both free and liposomal fisetin, indi- cating induction of apoptosis through both adminis- tration routes of the nutraceutical. The said authors have also reported that liposomal fisetin exhibited a higher in vivo antitumor activity in Lewis lung tumor bearing mice. The work also revealed a 3.3 days delay in tumor regrowth for cells treated with liposomal fisetin, compared with 1.6 days for cells exposed to free fisetin, for a 500 mm3 tumor volume, during the treatment tenure. Therefore, enhancement of antitu- mor activity of fisetin by liposomal encapsulation can be achieved.Effect of Crocin Liposome on Cancerous Cells
‘Crocin’ is a natural carotenoid and is the bioactive principle of saffron (Crocus sativus L.) and is responsible for its characteristic color. It protects human brain against oxidative stress and possesses antitumor and anticancer activities (29). Crocin has also been reported to have significant anti-prolifer- ation effects on human colorectal cancer cells (30).Few researchers have explored possibility of employing liposomal crocin as an anti-tumor and anticancer intervention. Liposomal crocin has report- edly been prepared by Mousavi et al. (29) using dehy- dration and rehydration method. HeLa and MCF-7 were considered samples for malignant cells, and mouse fibroblast cell line (L929) as nonmalignant control cell in this study. Both malignant and nonma- lignant cells were treated with free crocin (1, 2, and 4 mM) and liposomal crocin (0.5 and 1 mM crocin, with different lipid composition). Reports have showed decreased cell viability by crocin and liposo- mal crocin in malignant cells but not in nonmalignant cells (29). Their observation also proved that the lipo- somal formulations containing crocin had better cyto- toxic effects compared with the free crocin. The finding indicated that liposomal crocin reached inner cancer cells more effectively than free crocin. Scrutiny of cell cycle revealed that both free crocin and liposo- mal crocin induced a sub-G1 peak which is a bio- chemical marker of apoptosis; corroborating the anticancer potency of liposomal crocin.
Cyanidin-3-O-glucoside (C3G) is an anthocyanin found in fruits such as blackcurrant pomace, red raspberries, soybean seed coats, and peach. C3G has antioxidant and anti-inflammatory effects and inhibits proliferation and induces apoptosis in cancerous cells
(31). To improve the bioavailability of C3G, Liang et al. (31) have encapsulated it in liposomes (phospha- tidylcholine:cholesterol = 2.87:1.00), and administered to Caco-2 cells (human epithelial colorectal adenocar- cinoma cells). They found a concentration-dependent activity of both the samples, in which C3G liposomes inhibited tumor cell proliferation by 42.8% more than that by free C3G (at 0.20 mg/mL). Transmission elec- tron microscope analysis of Caco-2 cells showed cells treated with C3G liposomes to exhibit more changes in several organelles in comparison with the untreated cells. The changes in the number and morphology of mitochondria, increase in the number of fat droplets, and appearance of physalides in the treated cells were particularly significant. Moreover, flow cytome- try analysis reportedly revealed that C3G liposomes significantly enhanced the rate of cellular apoptosis rates at concentrations between 0 and 0.25 mg/mL.Genistein is a major active compound in soy and is a common phytoestrogen, owing to its ability to bind to estrogen receptors and influence on estrogen-regulated gene expression. Reportedly, genistein also has antitu- mor activities against several cancer types such as in breast, prostate, cervical, and ovarian carcinomas. Phan et al. (32) have optimized the ratio of hydro- genated soy phosphatidyl choline (HSPC), dioleyl phosphatidyl ethanol amine (DOPE), and cholesterol (Chol) for formulating genistein liposome. The optimized formulation was HSPC:CholDOPE = 72:8:20 M%, produc- ing liposome of diameter 161 ± 6 nm.In the reported study, authors treated murine 4T1 (breast carcinoma), human PC3 (adenocarcinoma- prostate), and OVCAR-3 (adenocarcinoma-ovarian) cells with different genistein formulations (blank liposome, genistein solution, genistein solution and liposome, and genistein liposome). Among them, genistein liposome exhibited minimum cell viability in contrast to other formulations. The authors also observed that genistein liposome induced higher damage to cancer cell mitochondria, which leads to specific activation of the mitochondrial pathway of apoptosis in human prostatic carcinoma cells. The liposomal genistein-induced DNA fragmentation at a concentration of 10 mg/mL that was almost 33% lower than that of the free genistein. Their inspection indicated strong depolarization of mitochondria by genistein liposome which resulted in activation of the apoptotic death mechanism in cancer cells.
Glucans are known to possess anticancer potency against colon, pancreatic, ovarian, and breast cancer. Halwani et al. (33) have formulated 1,3-b-glucan liposomes with different lipids [phosphatidylcholine (PC), dodecyl dimethyl ammonium bromide (DDAB), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dicetyl phosphate (DCP), and 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC)] and cholesterol (Chol); while working on human lung carcinoma epithelial cell line A549. b-Glucan liposomes prepared with DPPC:Chol = 6:1, DDAB: DPPC:Chol = 4:2:1
and DCP:DPPC:Chol= 4:2:1 displayed higher growth suppression of A549 cells relative to other formulations. The authors anticipated the mechanism of action of b-glucan to be that of a stimulated release of TNF-a from monocytes/macrophages. The authors further dis- covered that 1,3-b-glucan when encapsulated with doxorubicin in liposome significantly enhanced the growth suppression of A549 by doxorubicin.
Green tea (Camellia sinensis) contains the highest antioxidant potency among tea leaves which is mainly due to its high content of (—)-epigallocatechin-3- gallate (EGCG) having several therapeutic properties such as antioxidant, anti-tumorigenic, anti-inflamma- tory, and anti-angiogenic activities (34).In the research of Fang et al. (35), they treated basal cell carcinoma (BCC) cell line (expresses epithe- lial markers of keratin filaments and desmosomes) along with melanoma and colon cell lines with lipo- somes formulated with EGCG. The data revealed higher suppression of cell growth in all three cell lines compared with free EGCG. In vivo application of free EGCG, (+)-catechin and their liposomal encapsulates indicated highest deposition of drugs in the tumors for the liposomes prepared with egg phosphatidylcho- line:cholesterol (EPC):deoxycholic acid (DA) = 4:1:0.25 (w/w) (Fig. 5). The liposome system had the potential to serve as effective carriers of tea catechins and to increase the stability of EGCG inside the vesicles, in addition to activating higher EGCG accumulation within BCC cell line.Figure 5. Viability of basal cell carcinomas following treatment with EGCG in a hydroalcoholic solution or liposomes at various concentrations. F0: EGCG in in 15% ethanol (control), F1: EPC + Cholesterol (CH) = 4:1, F2: EPC + CH + DA = 4:1:0.25, F3: EPC + CH + Tween 80 = 4:1:1.64 (35).
In another study, de Pace et al. (34) have developed EGCG-encapsulated chitosan-coated nanoliposomes (CL-EGCG) to examine their anti-proliferative and pro-apoptotic effects on breast cancer cells (MCF7). As reported, for formulation of CL-EGCG, EGCG nanoliposome (soy lecithin:cholesterol = 4:1 molar ratio) was coated with 0.2% (w/v) of chitosan by stirring (using magnetic stirrer for 1 h at 4 ◦C). In this endeavor, human estrogen receptor-positive MCF7 breast cancer cell line was treated with free EGCG, EGCG liposome, CL-EGCG, void liposome, and void chitosan-coated nanoliposomes (V. CL). Liposomes were labeled with 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) to study the cellular uptake and localization of the same. The results showed that NBD-labeled CL had higher intracellular fluorescence intensity com- pared to the other liposomes. At 100 mM concentra- tion, CL-EGCG increased the cellular EGCG content in MCF7 cells by 34 times, compared with free EGCG. At 10 mM, CL-EGCG reduced the cell viability by 40%, and induced apoptosis in MCF7 cells by 27%. Therefore, this system can be used as biodegradable and biocompatible delivery system to increase bio- availability, solubility, stability, and payload of chemo- preventive agents such as EGCG.b-Cryptoxanthin is one of the major carotenoids in yel- low or orange fruits and vegetables. It has also been associated with anticancer activity against lung, bladder, breast, and colon cancer (36). b-cryptoxanthin lipo- somes have been reportedly formulated by researchers to enhance its anti-proliferative activity against human leukemia K562 cell line, when contesting the non- encapsulated compound (36). The results indicated that nanoliposomal formulation enhanced (P < .001) the anticancer efficacy of b-cryptoxanthin in comparison to its free form, in both dose- and time-dependent manners. Hoechst staining of K562 cells treated with b-cryptoxanthin illustrated that nanoliposomal b- cryptoxanthin had increased cellular apoptosis, evident by nuclear fragmentation and chromatin condensation. Therefore, the b-cryptoxanthin liposome can be used for leukemia therapies in the future. Berberine, an alkaloid isolated from Coptis chinensis, Hydrastis canadensis, and Berberidaceae, reportedlyhas anticancer potential. Researchers have formulated berberine liposomes to modulate the resistant mem- brane and mitochondrial proteins of breast cancer stem cells (CSCs), for acute and relapse conditions(37). Mitochondria-targeting DQA-PEG2000-DSPE was utilized to deliver the berberine liposomes to the mitochondria of breast cancer cells (MCF7 cells). Paclitaxel (known cytotoxic agent) was also encapsu- lated to investigate the effects of this compound on CSCs. From flow cytometry assay, the targeting ber- berine liposomes significantly increased the berberine content (geometric mean intensity 93.7 ± 1.49) in MCF7 and CSCs compared to the free berberine (geo- metric mean intensity 86.38 ± 1.07). The fluorescent microscopy indicated punctate distribution of the tar- geting berberine liposomes, confirming selective accu- mulation in the mitochondria triphosphate binding cassette (ABC) transporters ABCG2 and ABCC1 in MCF7 CSCs and exhibited strong inhibitory effects in both MCF7 cells and MCF7 CSCs. It also improved the effects of paclitaxel lipo- somes which is resistant to the MCF7 CSCs. Ma et al. (37) have further found that release of cytochrome C by the targeting berberine liposomes activated caspase- 9 and eventually initiated caspase-3, resulting in apop- totic reactions in MCF7 CSCs. The most significant efficacy in vivo was observed for the MCF7 CSCs xeno- graft mice treated with targeting berberine liposome along with paclitaxel liposome. Real-time imaging [Cyanine7 (Cy7) dye encapsulated in the targeting lipo- some] performed with xenograft mice revealed a higher accumulation of liposome in the cancer masses com- pared to the Cy7 liposomes or free Cy7.Among the edible mushrooms known worldwide, Flammulina velutipes has been extensively studied for its antioxidative, antivirus, anticancer, and immune modulatory properties. Yi et al. (38) have isolated the sterols (FVS) of this mushroom which mainly con- sisted of ergosterol (54.8%) and 22,23-dihydroergos- terol (27.9%). Their study revealed that, FVS liposome exhibited enhanced relative bioavailability of both ergosterol (162.9%) and 22,23-dihydroergosterol (244.2%) compared to the free forms. Human hepa- toma cell line HepG-2 and human lung adenocarcin- oma cell line A549 were treated with free FVS and FVS liposome. The tissue distribution study indicated fast and wide distribution of FVS liposome in differ- ent types of tissues. The concentrations of liposomal sterols were higher in liver and spleen compared to kidney, lung, heart, and brain; and after 2 h, this group of sterols exhibited rapid elimination. Therefore, further investigations are warranted to improve the distribution of FVS in other organs, espe- cially in lungs, especially since FVS demonstrated desirable anticancer activity against lung cancer cell line A549.MagnetoliposomesMagnetoliposomes differ from conventional liposomes in the context of aqueous core where the former contains iron oxide nanoparticles which play the role of navigation under the influence of external magnetic field to deliver and localize the liposomes to the target site. Magnetoliposomes can be used efficiently in cancer treatment through hyperthermia (39). Several researchers have worked on application of magnetoliposome in diagnosis and treatment of cancer (Table 2); however, reports on encapsulation of nutraceuticals in magnetoli- posomes for cancer treatment are very scant.Clares et al. (40) devised magnetoliposomes byencapsulating 5-fluorouracil, an anti-metabolite having inhibitory effect on colorectal cancer. They studied the influence of this formulation on human colon fibroblast CCD-18 and in human colon carcinoma T-84 cell lines. Indication of hyperthermia was also noted in the diseased state. The magnetoliposomes consisted of superparamagnetic magnetite (Fe3O4) particles embedded into a phosphatidylcholine (PC)- based multilamellar vesicle, and loaded with 5-fluo- rouracil. The authors reported that the heterogeneous structure of magnetoliposome was suitable for the selective 5-fluoro-uracil delivery and adequate heating characteristics for a hyperthermia effect, proving to be a strong alternative for medication for combined antitumor therapy against colon cancer.Aadinath et al. (9) have co-encapsulated curcumin and iron oxide nanoparticles to formulate curcumin- in-b-cyclodextrin-in-nanomagnetoliposomes to achieve the synergistic antioxidant potential of curcumin and iron oxide nanoparticles. The magnetoliposomes exhibited significantly higher antioxidant potency compared with the curcumin liposome and iron oxide nanoparticles owing to the synergistically enhanced radical scavenging property of magnetoliposome. Das et al. (41) have worked on nano hybrid based magne- toliposomes using leaf extracts of Datura inoxia and its liposomal encapsulation. More recently, aqueous magnetoliposomes (hydrodynamic diameter 150 nm) with magnesium ferrite nanoparticles has been investi- gated for enhanced therapeutic potential of curcumin(42). Though magnetoliposomes have better potential for directed and improved transport of drugs to the target organ, use of the same for delivery ofFigure 6. Mechanism of destruction of cancer cells and cancer stem cells by dual function liposomes; CSC: cancer stem cells (44). nutraceutical is rare and further investigations are required in this aspect to develop the technique to combat the malady. Efficacy of Liposomal Nutraceutical Delivery on Multidrug-Resistant CancerLiposomal nutraceuticals delivery has been pondered upon as a delivery method for therapeutics targeting multidrug resistant (MDR) ailments as well; with some work specifically extending on cancer therapy also. Considering the scope of the current disposition, we shall now focus on discussing the important findings in the field on liposome delivery of nutraceuticals on MDR cancer cells. The technique was first employed by researchers for encapsulation of topotecan with amilop- dipin to restrict drug outflow from resistant leukemia, ultimately resulting in inducing apoptosis (43).Basically, these liposomes function as dual carrier agents, providing the adequate exposure of the drug/ bioactive at the specific site, in addition to the advan- tage of prolonged targeted exposure of the drug at the desired site. The layout of action of dual function lip- osomes has been shown in Fig. 6. These aggregates have also shown evidence in limiting drug/bioactive efflux from the target cells, thereby rendering higher damage to cancerous cells, specifically targeting ATP binding cassette (ABC), mitochondria, stem cells, silencing resistant cancer genes, and in inducing apop- tosis and autophagy. In fact, these lipsomes are called double function liposomes, since they not only protect the core compounds of drugs/bioactive but also make the cancerous cells more vulnerable to the drug/bioactive exposure. They are also called ‘third- generation liposomes’. In fact, these co-encapsulate assemblies have shown effect in transcending the bar- riers posed by over-expressed ABC transporters (44).Nutraceuticals such as flavonoids derived from green tea polyphenols, catechins, quercetin, silymarin also have detrimental effect on MDR cancer cells (45,46). Few flavonoids such as tangeritin, quercetin, and naringenin regulate genes for MDR1 gene expres- sion in cancer (47). Quercetin and silymarin have been found to inhibit multidrug resistance-associated protein MRP-4 and MRP-5-mediated cellular trans- port (48). Nutraceuticals in combination with conven- tional drugs have also been explored as an alternative for possible enhanced activity. Combination of curcumin and doxorubicin nanoparticles has shown significant potency in preventing the growth of MDR cancerous K562 cells (49). Besides, nanoparticles have also shown potential as sources of nanomedicines to counter cancer (50).Few researchers have also suggested multi-targetedapproach to combat resistance in cancer cells, particularly involving the isoflavone ‘genistein’ to restrict cancer cells’ progression and sustenance. This phytochemical produces pleiotropic effects, by targeting multiple-cell signaling pathways on cancer, enhancing combat capability of liposomes (51). Similar anticancer functionalities exist for daidzein and glycitein from soy (52). In a recent endeavor, researchers have dem- onstrated more deleterious effect on cancer cells when treated with liposome-encapsulated resveratrol and pacli- taxel in drug-resistant cancer cells, in vivo (53). Nutraceuticals have also interestingly at times increased sensitivity of resistant cancer cells, such as capsaicin in KB-C2 cells (54), naringening, genistein, kaempferol increased K562 human leukemic cells (55). More surprisingly, EGCG has been shown to reverse the resistance of human carcinoma transplanted to nude mice, and make the cancer cells sensitive to doxorubicin, the anticancer drug (56). Conclusions Nutraceuticals have been extensively researched upon in cancer therapy off late. Since nutraceuticals are generally vulnerable to environmental factors, their encapsulation is a common recommendation, particu- larly for therapeutic purposes. Liposomal encapsula- tion has proven to be the most suitable form of entrapment of nutraceuticals, especially when admin- istered in cancer therapy. Researchers have accorded that liposomal encapsulation is a prominent and reli- able technique for enhanced stability of nutraceuticals, targeted delivery, and enhanced bioavailability. Anticancer potencies of major nutraceuticals in lipo- somes probed for anticancer activity including curcu- min, resveratrol, fisetin, crocin, EGCG, and sterols were discussed in this review. A scientific consensus was quite evident on the capability of nutraceuticals encapsulated as liposomes in restricting and/or arrest- ing various forms of cancer cell growth. The research findings discussed are both significant and promising, and moreover encouraging. As stated in the beginning, nutraceutical liposomes could act as both curative as well as preventive medicines, the preference shall understandably always be the former. For that, these formulations need to be included as food or food supplements. The recommendations from this disquisition includes greater research to highlight the activities of encapsulates, permutations in composition, and experimental conditions, explor- ing newer encapsulation approaches in this field such as that of ‘magnetoliposomes’ and on MDR cancer, in greater detail. This would be critical in gaining further scientific concordance, especially owing to the diver- sity in cancer types, newer types of cancers identified and their complexities, variable needs of targeted delivery and sustainability. As an epilogue, Fisetin it is rather heartening to conclude that liposome encapsulated nutraceuticals aid significantly in controlling cancer and its alleviation, and manifest evidence of being a trustworthy alternative to conventional chemotherapy.