Lipid-based nanocarriers for breast cancer treatment – comprehensive review

Abstract

Breast cancer is the second leading cancer-related disease as the most common non-cutaneous malignancy among women. Curative options for breast cancer are limited, therapeutically substantial and associated with toxicities. Emerging nanotechnologies exhibited the possibility to treat or target breast cancer. Among the nanoparticles, various lipid nanoparticles namely, liposomes, solid lipid nanoparticles, nanostructured lipid carriers and lipid polymer hybrid nanoparticles have been developed over the years for the breast cancer therapy and evidences are documented. Concepts are confined in lab scale, which needs to be transferred to large scale to develop active targeting nanomedicine for the clinical utility. So, the present review highlights the recently published studies in the development of lipid-based nanocarriers for breast cancer treatment.

• Breast cancer
• lipid
• lipid polymer hybrid nanoparticles
• liposomes
• nanostructured lipid carriers
• solid lipid nanoparticles

Introduction

Breast cancer is the most prevalent disease and holds second rank in the mortality rate of women (after lung cancer). In 2015, approximately 231 840 new breast cancer cases and 40 730 breast cancer deaths (40 290 women, 440 men) are estimated throughout the world. According to the World Health Organization (WHO), by 2050, it is expected that 27 million new breast cancer cases and 17.5 million breast cancer deaths will occur per annum (Anon., 2015). Globally, the burden and incidence rates of breast cancer are enormously increasing than the other cancers. Metastatic action of the breast tumors leaves the disease condition elusive and incurable (Aznar et al., 2013). Current therapies for breast cancer include radiation therapy - chemotherapy and endocrine therapy - has enriched the therapeutic effect but toxicity and side effects associated with these therapies are obstructing the clinical utility. Most of the cytotoxic drugs used clinically are chemotherapeutics administered into systemic circulation. Administering low molecular weight chemotherapeutics into systemic circulation exhibit rapid clearance, low pharmacokinetic profile and sub-optimal tissue distribution and a small fraction reach the tumor/tumor cell. Hydrophobic natured chemotherapeutics exhibit large volume of distribution leading to higher accumulation at healthy tissue site and causes toxicity. Chemotherapeutics are highly susceptible to develop multi-drug resistance (MDR) in the tumors. Exploration and development of a new technology are critical to treat breast cancer, which could effectively target tumor cells without killing healthy cells (Duo et al., 2012; Lammers et al., 2012).

Conventional breast cancer therapy

Chemotherapy

Chemotherapy is the treatment of cancers using cytotoxic drugs to kill the cancer cells or inhibiting their cell division. Even though there are no randomized-controlled trials for chemotherapeutics, still it has been widely used in the management of advanced breast cancer to improve the quality of life (Benner et al., 1994). Advantage of chemotherapy is reducing the tumor volume pre-operatively and avoiding the breast-conserving surgery. So, it has been applied to the treatment of lower-stage breast cancer (Fisher et al., 1997). Anemia is the common side effect of chemotherapy, it is a low red blood cell count that can cause fatigue and reduced quality of life. Chemotherapy is associated with many side-effects, such as pain, nausea, vomiting, hair loss, weight changes, fatigue and anxiety; however, the most commonly reported side-effect is fatigue (Groopman & Itri, 1999).

Radiation therapy

Radiation therapy/radiotherapy is a targeted treatment, which is the most effective way to kill the cancerous cells in the breast that may remain around even post-operation. Radiation can reduce the risk of breast cancer recurrence by about 70%. Side effects of radiotherapy treatment are confined to the treated area (König et al., 2015). Skin at the treated area turns red, dry, tender; itchy and other possible side effects cause limited oral intake, which may lead weight loss (Polisena & McCallum, 2000). Radiotherapy refers to the medical use of ionizing radiation for malignant tumors. The effects of radiation therapy are localized to the region being treated. This therapy injures or destroys cells in the area being treated (the “target tissue”) by damaging their genetic material, making it impossible for these cells to continue to grow and divide. Usually, a combination of all the three treatment regimes is used against any given cancer for the maximum benefit (Koch et al., 2010).

Hormonal therapy

Hormonal therapy is to treat hormone receptor-positive cancers. In this therapy, the amount of hormone can be reduced or blockade the hormonal action. Estrogen action may be blocked or its amount is reduced to decrease the risk of recurrence (Umesono & Evans, 1989). Hormone therapy includes aromatase inhibitors, selective estrogen receptor modulators and estrogen receptor down-regulators as well as surgical treatments, such as removal of ovaries and fallopian tubes. Tamoxifen use of at least five years is associated with a 12% reduction in recurrence and a 9% reduction in mortality over a 15-year follow-up period, in ER-60 positive and ER-unknown breast tumors. The benefits of tamoxifen appear to be optimized at five years, with current recommendations to discontinue adjuvant tamoxifen after five years. It can also be given for advanced-stage or metastatic disease to shrink or slow the growth of existing tumors (Aapro, 2001). Recent trials Arimidex-No lvadex study in postmenopausal women (ARNO-95) and the Intergroup Exemestane Study (IES) have shown benefits of aromatase inhibitors over tamoxifen for disease-free survival and complications (Kaufmann et al., 2007). Recently, it has been observed that some cases of breast cancer are resistant to hormonal therapy (in spite of ER positivity) either ab initio or while on treatment; and several mechanisms have been postulated to explain these, and many strategies are currently being tried to overcome this resistance.

Complications associated with conventional breast cancer therapies

Majority of cancerous diseases are treated with multiple drug therapies with a combination of two or more anti-cancer drugs that can exhibit synergistic or additive effects by different mechanisms involved in cancer progression. As briefed above, the chemo and hormonal therapies have demonstrated elevated efficacy still significant toxicities are associated with these therapies. Underlying mechanisms for the evolution of side effects and toxicities as the anti-tumor drugs are not site-selective and thereby cause non-specific cytotoxic action over healthy cells in the bone marrow, gastrointestinal epithelia and hair follicles (Tanaka et al., 2009). Serious side effects and complications are associated with the Tamoxifen, hormonal therapeutic include high risk for endometrial cancer by 2.4 times in women aged 50 years or older (Fisher et al., 2005) and thromboembolic complications by 1.9 times (Cuzick et al., 2003). Among the chemotherapeutics, conventional doxorubicin is associated with acute toxicities including myelosuppression, mucositis and alopecia. Irreversible congestive heart failure is one of the most serious, conventional doxorubicin-induced toxicity failure (Von Hoff et al., 1979). Even though targeted therapies demonstrated considerable positive effect by the results of multiple clinical studies, these therapies are associated with severe side effects. Severe heart complications include ventricular dysfunction and congestive heart failure, in addition to common flu-like symptoms are associated with Trastuzumab alone or in combination with chemotherapy (Slamon et al., 2001). Therefore, a safe, smart therapeutic approach is required for selective delivery of cytotoxic agents to the tumors, which are responsible for the breast cancer progression and metastasis and also improving the therapeutic index and efficacy/toxicity balance.

Application of nanotechnology for breast cancer therapy

Nanotechnology is no longer a new concept and creating higher impact in every part of the health care system. Nanomedicine-nanotherapeutics-nanotheranostics are the terminologies coined up because of the exploitation of the nano-sized particles in the field of health care. The keyword “nanoparticle” in the Pubmed resulted about 101 198 articles (search date: Jan 2015) inferring that the progression in the field of nanotechnology is very high. Remarkable progression has been witnessed by the fact that there are more than 150 ongoing clinical trials. Tremendous efforts and time have been spent to shift this technology from pre-clinical stage to commercialization stage (Zhang et al., 2013; Bertrand et al., 2014). Nanomedicine, which may be nanoparticulated (particle itself a therapeutic agent) or nanocarriers (carrier encapsulates the therapeutic agent) are intended to design in the range nanometers (nm) to several hundred nm as per the requirement and usage. Nanoparticles can easily permeate into the leaky vasculature-tumor tissue (where healthy tissue has a uniform vasculature) and can deliver the drug in the controlled manner at the site of tumor tissue, which is well known as Enhanced Permeability and Retention (EPR) effect. Nanoparticles include polymeric nanoparticles, liposomes, micelles, which are featured with encapsulation of poorly soluble drugs, site-specific drug delivery, elevating drug bio-availability, higher permeability across the biological membranes and controlled drug release. Most of the drugs intended for chemotherapy act on the surface receptor/within cytoplasm/within nucleus. Tumors are characterized by heterogeneity, such as aberrant expression, mutation of oncogenes/tumor suppressor genes, which cause modifications in cellular events, such as apoptosis, cell cycle arrest, adaptive resistance and metastasis. Drugs act while trafficking in the blood circulation and the concentration at the tumor site exhibits the therapeutic efficacy of the drugs (Tanaka et al., 2009). To attain high concentration at the tumor site, higher dosages are administered. Due to non-specificity systemic side effects and toxicities elicit. Drugs internalize into tumors mainly through the passive diffusion or active transport, whereas nanoparticles internalize into cells via endocytosis (Moghimi et al., 2005). Steps involved in the endocytosis are

1. Cargo enters into membrane invaginations which are pinched off to turn as membrane-bound vesicles, also known as endosomes. Cells consist of heterogeneous populations of endosomes furnished with endocytic machinery, which originate at different sites of the cell membrane.

2. The endosomes transport the cargo to various specialized vesicular structures, which enables to deliver the cargo towards different destinations.

3. Finally, the cargo is delivered to intracellular components and sent back to the extracellular milieu or delivered across cells (Sahay et al., 2010).

Endocytosis is broadly classified into phagocytosis and pinocytosis. Phagocytosis was originally discovered in macrophages (uptake of particles). Pinocytosis (uptake of fluids and solutes) is observed in all types of cells. It is present in four forms as per cell origin and function, such as clathrin-dependent endocytosis (also known as clathrin-mediated endocytosis) and clathrin-independent endocytosis. The clathrin-independent pathways are further classified as (1) caveolae-mediated endocytosis, (2) clathrin- and caveolae-independent endocytosis and (3) macropinocytosis. Clathrin- and caveolae-independent pathways are sub-classified as Arf6-dependent, flotillin-dependent, Cdc42-dependent and RhoA-dependent endocytosis.

Phagocytosis is a endocytic pathway where large particles are more likely to uptake and predominantly occurred in phagocytes, such as macrophages, neutrophils and monocytes. The uptake carried out by phagosomes formed by membrane invaginations, which later associate with lysosomes. And finally destructed by enzymolysis in lysosomes (Aderem & Underhill, 1999; Hillaireau & Couvreur, 2009). In clathrin-dependent endocytosis, nanoparticles interact with receptors present on the cytomembrane and cytosolic protein named clathrin-1 polymerizes on the cytosolic side of the plasma, where the nanoparticles are internalized. After wrapping the nanoparticles inside, the vesicle is detaches through the GTPase activity of dynamin, forming a clathrin-coated vesicles. Energy supply of actin transports clathrin-coated vesicles move towards inside the cells and the way is controlled by the cytoskeleton. The clathrin coat is disrupts in the cytosol (Harush-Frenkel et al., 2007; Rappoport, 2008). Caveolae-dependent endocytosis is a common cellular route which can bypass lysosomes and favorable for elevating concentration of drug and enhancement of therapeutic effect. Nanoparticles interact with receptors form flask shaped vesicles and pinches off by dynamin. Caveosomes associate with the multivesicular bodies, which maintain a neutral pH. The caveosomes containing nanoparticles move towards the endoplasmic reticulum. Nanoparticles present in endoplasmic reticulum penetrate into the cytosol and then penetrate into nucleus via the nuclear pore complex (Medina-Kauwe, 2007; Doherty & McMahon, 2009). Macropinocytosis is a peculiar mechanism of clathrin-, caveolae- and dynamin-independent endocytosis, which is triggered by transient activation of receptor tyrosine kinases by growth factors. The receptor activation generates a signaling cascade that brings changes in the actin cytoskeleton and triggers formation of membrane invaginations. The cell membrane engulfs the nanoparticles in the extracellular milieu, forms a macropinosome and delivers into the intracellular site (Jones, 2007; Mercer & Helenius, 2009). Clathrin- and caveolae-independent endocytosis cellular route depends on cholesterol and some lipid compositions. It is classified on the basis of effectors as Arf6- dependent, flotillin-dependent, Cdc42-dependent and RhoA-dependent (Doherty & McMahon, 2009; Iversen et al., 2011).

Among these caveolae-dependent endocytosis pathway has gained attention in nanomedicine, because it has the capacity to bypass lysosomes. In addition, the caveolae-mediated endocytosis is the remarkable transendothelial pathway and can be exploited for trans-vascular delivery of nanoparticles. Finally, nanoparticles aim to maintain balance between efficacy and toxicity of the chemotherapeutics. And this feature of nanoparticles drawn the attention of the oncology to deliver the chemotherapeutics for alleviating the disease and eliminating the toxicities (Haley & Frenkel, 2008; Mattu et al., 2013).

Lipid-based nanocarriers

Among the nanocarriers, lipid-based nanocarriers have great potential to solubilize, encapsulate and deliver active molecules in a programmed pattern to achieve bioavailability and avoid side-effects (Westesen & Siekmann, 1996; Yaghmur & Glatter, 2009). These drug carriers are made up of bio-compatible lipids, such as phospholipids, cholesterol and triglycerides. Numerous advantages of the lipid matrix make the lipid-based nanocarriers as an idealistic drug delivery system. Bio-compatibility and bio-degradability characteristics of these systems are prone to be less toxic as compared to other drug delivery systems, such as polymeric nanoparticles. (Fenske & Cullis, 2008; Liu et al., 2011). A number of recent reviews have provided perspectives on the use of various types of nanocarriers as therapeutic and diagnostic tools in cancer research. The emphasis is particularly laid on liposomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and lipid polymer hybrid nanoparticles (LPN) and their advantages, limitations are briefly summarized in Table 1.

Table 1. Advantages and limitations of lipid nanoacarriers.

Lipid nanocarriers	Advantages	Disadvantages	Reference
Liposomes	• Ease of formulation in pegylated (Stealth) liposomes • Stealth coating prolongs the systemic circulation time • Effective therapy for multidrug-resistant and other tumors • Capable of altering pharmacokinetics and bio-distribution • Reduces cardio-toxicities • Lipids used are compatible of parenteral administration • Can be used for drug resistant tumors	• Has stability issues • Ligands present in active targeted liposomes may affect the stability of the formulation	(Allen & Martin, 2004; Gabizon et al., 2006)
Solid lipid  nanoparticles	• Protection of drug from degradation • Has long-term stability • Reduces systemic toxicity • Feasible to load lipophilic and hydrophilic drugs • Easy scale up	• Inherent low drug loading capacity due to the crystalline structure of solid lipid • Drug expulsion • Unpredictable agglomeration • Higher incidence of polymorphic transitions	(Mukherjee et al., 2009; Patidar et al., 2010; Kaur & Slavcev, 2013)
Nanostructured  lipid carriers	• This drug delivery system developed to avoid the problems associated with the SLNs • Increased drug payload • Drug expulsion during storage • High water content of SLN dispersions	• Not feasible for thermo-labile drugs • Metal contamination during probe-sonication • Usage of large volume of aqueous phase may lead to higher dilution of dispersion	(Müller et al., 2002a; Iqbal et al., 2012)
Lipid polymer hybrid  nanoparticles	• Desirable particle size • Provision for surface modification • Higher drug payload • Entrapment of hydrophilic and lipophilic drugs • Amenable drug release pattern • High serum stability	• Lab-scale preparation is cumbersome • Not tunable for industrial scale production • Aseptic processing makes expensive	(Hadinoto et al., 2013; Mandal et al., 2013)

Liposomes

Liposomes are pioneers among the lipid nanocarriers designed until now. These colloidal carriers are made up of bio-degradable, bio-compatible, non-immunogenic natural phosopholipids, which can encapsulate water-soluble, fat-soluble, amphiphilic, biphasic insoluble drugs and cytotoxic agents into their aqueous core surrounded by unilamellar/multilamellar membranes (Figure 1) (Gulati et al., 1998; Lammers et al., 2008). Liposomes have gained the attention of conventional cancer chemotherapeutics because as a drug delivery system, they can elevate the concentration of drug at tumor sites and can decrease the drug concentration at normal tissues. Liposomes diminish the drug toxicities, such as cardiotoxicity and elevate the therapeutic effect by altering the drug pharmacokinetics and bio-distribution. Lipids used for the liposomal preparation are derived from natural origin egg yolk or soya bean oil, which is safe for parenteral administration (Allen & Martin, 2004). Myocet® and Doxil®/Caelyx® (Doxil in USA/Caelyx in Europe) were the first liposomal medicines approved by the regulatory bodies for the breast cancer treatment. Both Doxorubicin and liposomal formulations were successful in diminishing the key toxicities and cardiomyopathy, which are associated with free form Doxorubicin and showed improvement in pharmacokinetics. Doxil is the successor of Myocet where the prime most difference is Myocet is devoid of stealth coating, i.e. PEGylation (Poly-ethylene glycol). Pegylation provides steric stabilization, impairs reticulo-endothelial clearance to the liposomes thereby prolongs the circulation time and half-life of the drug. Lipid materials widely used in the preparation of liposome egg phosphatidylcholine, cholesterol, polyethylene glycol-distearoylphosphosphatidylethanolamine (PEG2000-DSPE), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dioleoylphosphoethanolamine (DOPE). Thin-film hydration is the most common and standard method for the preparation of liposomes (Forssen & Tökes, 1983; Waterhouse et al., 2001; Coleman et al., 2006).

Figure 1. (a) Liposome, (b) active targeted liposome, (c) solid lipid nanoparticles, (d, f) nanostructured lipid carriers, (e) lipid polymer hybrid nanoparticles.

Combination of two liposomal formulations of different drugs exhibited synergistic effects. Bee Jen Tan and co-workers studied the cytotoxicity of novel dihydrofolate reductase (DHFR) inhibitor M-V-05 into liposomes against the MDA-MB-231 and JIMT-1 breast cancer cell lines. DSPE-PEG and cholesterol in a ratio of (55:45 mol/mol) were used to prepare M-V-05-loaded liposomes. They were efficacious than the free drug DHFR inhibitor, Methotrexate, Carboplatin and liposomal Doxorubicin against the breast cancer cell lines. Combination of liposomal M-V-05 with a pegylated liposome formulation of Doxorubicin (molar ratio 1:3) exhibited synergistic cytotoxic action against the MDA-MB-231 cell lines and this adjuvant therapy should be investigated for the assessment of pharmacokinetic and toxicological parameters for the clinical efficacy (Tan et al., 2010).

Similarly, combinatorial drug encapsulation has exhibited higher cytotoxic potential than the single drug encapsulation. Waterhouse et al. (2005) estimated the serum drug levels of Doxorubicin liposomes and Trastuzumab liposomes, which were co-administered. Liposomal encapsulation of Doxorubicin (AUC_0–24 h 919.54 µgh/ml) has shown pharmacokinetic benefits than the drug alone (AUC_0–24 h 0.85 µgh/ml). No prominent change was observed in the peak serum drug concentrations or efficacy by the co-administration of liposomal Trastuzumab and liposomal Doxorubicin. AUC_0–24h of free drug and liposomal Doxorubicin was 0.77 and 919.88 2 µgh/ml, respectively, when administered along with Trastuzumab. Conjugating Trastuzumab to the liposomal Doxorubicin can exhibit better efficacy than combinatorial drug encapsulation of Trastuzumab and Doxorubicin. Morad et al. (2012) have developed Ceramide-Tamoxifen combinational liposomes for the treatment of chemoresistant and triple negative breast cancer. Dual drug encapsulation shows the better cytotoxic effects against the MDA-MD-468 cell lines by inducing the cell cycle arrest at G1 and G2 phases. Tamoxifen and its metabolites have promoted the lysosomal membrane permeability. So, addition of Tamoxifen in the drug regimen has synergistic action of Ceramide in targeting triple negative breast cancers. Another approach has been investigated for targeting matrix metalloproteinases by combinatorial drug loading of Epigallocatechin gallate and Doxorubicin into liposomes. In vitro studies indicated that Epigallocatechin gallate and Doxorubicin-loaded liposomes resulted in synergistic cytotoxicity against MDA-MB-231 cells. Hoechst staining and caspase-3 assay confirmed that Epigallocatechin gallate and Doxorubicin-loaded liposomes synergistically enhanced the apoptosis and tumor inhibition. MMP-2 and MMP-9 ELISA assay revealed that Epigallocatechin gallate and Doxorubicin-loaded liposomes showed higher MMP inhibition than the single drug-loaded liposomes and control groups. This combinational drug delivery approach can be further investigated for the pre-clinical utility (Ramadass et al., 2015).

Systemic toxicities associated with the chemotherapeutics were greatly reduced by liposomal encapsulation. Papa et al. (2013) investigated the cytotoxic behavior of Gemcitabine-loaded liposomes in vitro and in vivo using the breast cancer cell lines. At lower doses, Gemcitabine-loaded liposomes exhibited significant cytotoxicity than the drug alone against the MDA-MB-231 and 4T1 cell lines at 48 and 72 h. As per the phase III clinical trial in 2008; combination of Gemcitabine and Paclitaxel was effective in the treatment of advanced metastatic breast cancer and this evidence made the authors for the in vivo assessment of liposomal Gemicitabine and Paclitaxel in 4T1 syngeneic breast cancer model. Reduction of the tumor size burden and no prominent changes in body weight in the treatment groups inferred that the liposomal Gemicitabine and Paclitaxel did not show any systemic toxicity. Replacement of Paclitaxel with Nab-Paclitaxel in the therapeutic regimen may be effective in the breast cancer treatment.

Liposomal encapsulation of various chemotherapeutics afforded benefits over the free drug. Encapsulating Hydroxy urea and Artemisinin into liposomes improved the cytotoxicity against MCF-7 breast cancer cell lines. Using phosphatidylcholine and cholesterol (10:1) ratio, the liposomes were prepared by solvent evaporation method. Hydroxy urea-loaded liposomes (IC₅₀ = 43.78 ± 0.017 μg/ml) had higher cytotoxicity than the free drug (IC₅₀ = 69.11 ± 0.005 μg/ml) against the MCF-7 cell lines. Delayed drug release phenomenon attributed the improvement of efficacy (Alavi et al., 2013). Pegylation of Artemisinin encapsulated liposomes had a greater impact on the cytotoxicity. Cholesterol, phosphatidylcholine and PEG 2000 (1:5:14) were used to prepare the liposomes. Cytotoxicity was higher in Artemisinin-loaded liposomes (IC₅₀ = 1.58 ± 0.37 μg/ml) than the free drug (IC₅₀ = 2.7 ± 0.32 μg/ml) against the MCF-7 cell lines (Dadgar et al., 2013). Zhao et al. (2013) had synthesized liposomes with propylene glycol and trehalose to achieve better drug release and stability. Epirubicin-loaded liposomes were tested on the MDA-MB 435 and their mutant-resistant (MDA-MB 435/ADR) cell lines. In vitro MTT assay revealed that Epirubicin-loaded liposomes have significant cytotoxic action against the MDA-MB-435 cell growth, but also in MDA-MB-435/ADR cell growth and fluorescence studies showed higher uptake in the nucleus of the tumor cell via endocytosis. In vivo studies revealed that Epirubicin-loaded lipsomes have higher tumor efficacy than the Epirubicin free drug and also been safe for the normal cells. By the bio-distribution study, Epirubicin accumulation in the tumor was higher in Epirubicin-loaded liposomes group than the free drug group within 4 to 24 h.

Liposomes alter the pharmacokinetics of chemotherapeutics and prolong the half life and systemic circulation time. Gubernator et al. (2014) have developed Epirubicin-loaded liposomes by ethylene diamine tetra-acetic acid (EDTA) ion gradient method, which exhibited better efficacy than the free drug in the human breast MDA-MB-231 cancer xenograft model. Epirubicin-loaded liposomes had prolonged elimination (AUC 7.6487 µmole h/ml) than the free drug (AUC 0.0097 µmole h/ml).

Tumor cells are characterized by high temperature and low pH due to the excessive glycolysis cycles in aerobic and anaerobic environments. These characteristics can be exploited for the targeted delivery of nanomedicine. Zhang et al. (2014) designed thermosensitive liposomes for the targeted delivery of Doxorubicin at the tumor sites. Dipalmitoyl phosphatidylcholine (DPPC), monostearoylphosphatidylcholine (MSPC), DSPE-PEG2000 and egg yolk phosphatidylcholine were the lipids used for the preparation of the thermosensitive liposomes. In vitro drug release studies shown that drug release rate from the Doxorubicin-loaded thermosensitive liposomes was higher at 42 °C than the temperature at 37 °C, which signifies the temperature triggered drug release of liposome. In vivo tumor growth inhibition study was carried out by subcutaneous xenotransplanted tumor model of human breast cancer in nude mice. Docetaxel-loaded liposomes-treated group (10.0 mg/kg) had exhibited the lesser tumor volume (61.99 ± 26.88 mm³), less tumor weight (0.07 ± 0.03 g), and greater inhibition rate (94.3%) than the free drug-treated group. Inhibition rate of Docetaxel-loaded liposomes-treated group was about 1.3 times of free drug-treated group of same dose, which was statistically significant.

Gene therapy using small interfering RNA (siRNA) serves as one of the powerful therapeutic drug in tumor-targeted therapy. Even though siRNA is potent but the delivering to target tissues following systemic administration is a encumbering issue, which need to be overcome. It is a major challenge for developing a suitable delivery system for siRNA applications. Some researchers have developed siRNA encapsulated in liposomes for tumor inhibition. MDR1 is the gene that encodes P-glycoprotein (P-gp) and it exhibits over expression, which is a mechanism of drug resistance in breast cancer. Some of the researchers have developed siRNA-loaded liposome using cationic lipids. Plasma proteins cause agglomeration of positively charged complexes lead to faster elimination by reticuloendothelial system. By these information. Nourbakhsh et al. (2015) introduced a special class of PEGylated liposomes and named them as nanolipoparticles for gene delivery. Preparation of the nanolipoparticles was by detergent dialysis method similar to thin-film hydration method. Lipids used in the preparation were mPEG-DSPE/-DOTAP/DOPE. By the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis, at very low concentration (25 nM) siRNA-loaded nanolipoparticles have exhibited significant down-regulation of MDR1 gene expression. At 48 h, there was even significant downregulation of MDR1 gene by the siRNA-loaded nanolipoparticles. This effect was due to off target effects of luciferase control siRNA. Reversal of the MDR phenotype in MCF-7/ADR cells was confirmed chemosensitvity enhancement. SiRNA-loaded nanolipoparticles (100 nM) significantly elevated chemosensitivity in MCF-7/ADR cells. But its effect is low when compared with Oligofectamine, transfection reagent (positive control). This may be attributed due to the PEG, which interferes the interaction of nanoparticle and cell surface. MCF-7/ADR xenograft female BALB/c mice models were developed to confirm that siRNA may be delivered to the target cells in tumor tissue through systemic administration of siRNA-loaded nanolipoparticles. Intravenous administration of 1.6 mg/kg siRNA-loaded nanolipoparticles significantly gene silencing effect was observed, when compared with Oligofectamine and control siRNA. Developed drug delivery system can be exploited further by replacing the pH-sensitive lipids or thermo-sensitive lipids for effective targeting of tumors.

Likely the concept of tumor targeting, Gharib et al. (2014) targeted the breast cancers by the magnetic liposomes loaded with artemisinin and transferrin. Magnetic liposomes were prepared using soya phosphatidylcholine, DSPC, DPPC, cholesterol, magnetic iron oxide. In absence and presence of external magnetic field, time-dependent cytotoxicity was exhibited by the magnetic liposomes of Artemisinin and Transferrin against the MCF-7 and MDA-MB-231 cell lines. Ghanbarzadeh et al. (2014) investigated the anti-proliferative potential of fusogenic pH-sensitive liposomes of Rapamycin on MCF-7 cell lines. Fusogenic characteristics were generated to the liposomes by the cone-shaped lipid DOPE. Liposome preparation was carried out by ethanol injection method. Fusogenic liposomes of Rapamycin demonstrated dose-dependent anti-proliferation activity against the MCF-7 breast cancer cell line. In human umbilical vein endothelial cells, HUVEC cell lines - healthy cell lines, the fusogenic liposomes of Rapamycin exhibited lower cytotoxicity due to the pH variation. This evidence demonstrates that fusogenic property of the liposomes targets the tumor cells.

Targeted liposomal drug delivery

Active targeted nanoparticles, a novel nanoscale approach has been developing for the localization and cellular internalization in the tumor sites. Growth factors, hormones, receptor expression, protein up regulations are involved in signaling pathways for tumor development and metastasis have been identified for the site-specific targeting, internalization and localization of liposomes in the tumors therapeutic, diagnostic and theranostic approaches. Active targeted nanoparticles are the surface-modified or functionalized passive targeted nanoparticles are successful in tumor targeting and site-specific release of the chemotherapeutics (Phillips et al., 2010). Antibodies, peptides, nucleic acid aptamers, carbohydrates and some molecules are the ligands used to conjugate the nanoparticles for the active targeting of tumors (Gu et al., 2007). Important targets for the induction of apoptosis or inhibition of anti-apoptosis, angiogenesis, cell-cycle arrest and signal transduction in the breast cancer treatment have been summarized in Figure 2. In vitro and in vivo studies which have concentrated in active targeting of breast cancer by the liposomes have been summarized and reported below in Table 2.

Figure 2. Summary of targets for the induction of apoptosis or inhibition of anti-apoptosis, angiogenesis, cell-cycle arrest and signal transduction in the breast cancer treatment. EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptors; HER, human epidermal growth factor receptor; IKK, inhibitor of NF-B kinase; mTOR, mammalian target of rapamycin; NF-B, nuclear factor-B; TRAIL, tumor necrosis factor-related apoptosis inducing ligand; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Adapted from Schlotter et al. (2008).

Table 2. Summary of studies on active targeted liposomes to breast cancer.

Target ligand	Drug	Cell surface target	Reference
Siale^x	Merphalan	Lectins	(Vodovozova et al., 2000)
Vasoactive intestinal peptide VIP, a 28-amino acid mammalian neuropeptide	Radionuclide (Tc99m-HMPAO)	Vasoactive intestinal peptide (VIP) receptors	(Dagar et al., 2003)
17β-estradiol	Anti-cancer gene	Estrogen receptor	(Reddy & Banerjee, 2005)
Herceptin	Paclitaxel	HER2	(Yang et al., 2007)
Anti-transferrin receptor antibody	HER2-Si-RNA	Transferrin receptor	(Pirollo & Chang, 2008)
Anti-HER2 Fab’	PE38KDEL	HER2	(Gao et al., 2009)
Herceptin	Melittin	HER2	(Barrajón-Catalán et al., 2010)
Luteinizing hormone-releasing hormone analog	Mitoxantrone	Luteinizing hormone-releasing hormone receptor	(He et al., 2010)
Estrone	Doxorubicin	Estrogen receptor	(Paliwal et al., 2010)
Anti-HB-EGF monoclonal antibody	Si-RNA	Epidermal growth factor receptor	(Gao et al., 2011)
Peptide	PRDM14Si-RNA	MCF-7 cell specific	(Bedi et al., 2011)
Cetuximab	Doxorubicin	Epidermal growth factor receptor	(Mamot et al., 2012)
Fab’ of anti-HB-EGF monoclonal antibody	Doxorubicin	Heparin-binding epidermal growth factor-like growth factor	(Nishikawa et al., 2012)
Trastuzumab	Curcumin Resveratrol	HER2	(Catania et al., 2013)
Chlorotoxin	Doxorubicin	Matrix metalloproteinases	(Qin et al., 2014)

Most of the researchers exploited Herceptin antibody conjugation to target HER2. Anti-HER2 immunoliposomes loaded with drugs Melittin, Paclitaxel, Curcumin, Resveratrol showed predominant tumor targeting and internalization than the passive targeted liposomes (Reddy & Banerjee, 2005; Nishikawa et al., 2012). PE38KDEL, an immunotoxin, which faced toleration issues with polymeric encapsulation by PLGA has shown efficient internalization by loading into anti-HER2 immunoliposomes (Gao et al., 2009). Upregulation of estrogen receptor expression is observed in breast cancer cells and the estrogen or its derivatives (estrone) can be targeted to estrogen receptors. Conjugation of 17β-estradiol to anti-cancer gene-loaded liposomes have exhibited efficient targeting to estrogen receptor of MCF-7 breast cancer cell lines (ER + breast cancer cells) (Reddy & Banerjee, 2005). Targeting by estrone ligand has shown 6-fold higher accumulation of active target liposomes than the passive targeted liposomes (Paliwal et al., 2010). Nishikawa et al. (2012) developed anti-HB-EGF Doxorubicin-loaded immunoliposomes to target Heparin-binding epidermal growth factor, which is highly expressed in the breast cancer. In vitro cytotoxicity assays, the targeted and passive targeted liposomes shown dose-dependent inhibition and significant uptake in the Vero and MDA-MB-231 cell lines. By the in vivo studies, no marked difference in the therapeutic efficacy was found between the immunoliposomes and non-targeted liposomes. No quick tumor growth was found with the immunoliposomes. He et al. (2010) targeted luteinizing hormone-releasing hormone receptor, which is over expressed in breast, ovarian, endometrial, prostate, colorectal tumors. Conjugation of Gonadorelin (synthetic LHRH analogue) to the Mitoxantrone-loaded PEGylated liposomes shown better cytotoxic action than the free drug in the MCF-7 cell lines.

In the document entitled “Forward Look on Nanomedicine”, the European Science Foundation included in their definition; that the discipline of nanomedicine is not merely the use of nanometer-sized materials for the treatment but also as the diagnostic aids (Lammers et al., 2008). In regard with the latter aspect, the research has focused on imaging and monitoring of the in vivo fate and performance of tumor-targeted nanomedicines. Dagar et al. (2003) have chosen vasoactive intestinal peptide (VIP) receptors as cell surface target, which show hyper expression in the breast cancer cells of about 5-fold higher than the non-cancerous breast cells. Radionuclide Tc99m-HMPAO encapsulated in VIP-R grafted liposomes exhibited targeted imaging of the improved pharmacokinetics. By the bio-distribution studies, the peptide-conjugated liposomes shown higher uptake of 1.8-fold than the passive-targeted liposomes. Qin et al. (2014) targeted tumor metastasis inhibition by a Chlorotoxin-conjugated liposomes loaded with Doxorubicin hydrochloride. Earlier, Chlorotoxin was exploited on studies of brain tumors. It was proven for its extensive anti-metastatic action on glioma cells by virtue of its interaction with functional proteins, matrix metalloproteinases. Cellular uptake studies revealed that the chlorotoxin-conjugated liposomes had 1.8 times higher uptake than the passive targeted liposomes in the in vitro cell line of murine metastatic breast cancer, 4T1. Chlorotoxin-conjugated liposomes (IC₅₀ = 1.58 μg/ml) has exhibited higher cytotoxic action than the passive targeted liposomes (IC₅₀ = 6.49 μg/ml). However, the free drug Doxorubicin showed higher uptake and higher cytotoxicity than the liposomes, which may be due to its direct diffusion. Targeting of the Chlorotoxin-conjugated liposomes was evaluated in vivo using DiR, a near-infrared fluorescent dye. Two hours post-injection, the fluorescence intensity of Chlorotoxin-conjugated liposomes was higher than the passive targeted liposomes, which gradually escalated until 48 h. Chlorotoxin-conjugated liposomes had potent anti-metastasis effect on the lungs of BALB/c mice bearing 4T1 tumors. Reduction in the number of tumor nodules was observed in the lungs of the group treated with Chlorotoxin-conjugated liposomes, whereas no significant change in the amount of nodules was observed in the control group and passive-targeted liposomes. Comparatively, Chlorotoxin modified liposomes had higher anti-metastasis action than the drug alone and passively targeted liposomes.

Instead of antibodies, antibody fragments and peptides. Jain et al. (2014) performed liposome surface modification with Tamoxifen, an estrogen antagonist chemotherapeutic. Doxorubicin was encapsulated in liposomes and Tamoxifen was embedded on the bilayers of the liposomes. Tamoxifen played dual role, i.e. cytotoxic action and targeting moiety for ER +ve breast cancer cells. Loading Tamoxifen into liposomes was done by film hydration method and later Doxorubicin was post-encapsulated into the liposomes. In vitro cell line studies in MCF-7 cell lines revealed that Tamoxifen-Doxorubicin-loaded liposomes have shown higher cytotoxicity than Doxorubicin liposomes. By the cellular uptake studies, Doxorubicin alone was internalized in the cytoplasm whereas the Tamoxifen-Doxorubicin liposomes had higher compartmentalization in the nucleus of MCF-7 cells. Mice were treated with free drug Doxorubicin and Tamoxifen-Doxorubicin loaded liposomes at the dose 3 mg/kg/day. On the 4th day of the treatment, Tamoxifen-Doxorubicin loaded liposomes exhibited significant reduction in tumor volume when compared with the control group. Combinatorial drug-loaded liposomes shown superior performance in vitro cytotoxicity and in vivo tumor inhibition compared with Doxorubicin-loaded liposomes and Doxorubicin alone.

Solid lipid nanoparticles

In early nineties, Mueller et al. (2000) and Gasco (1993) have focused on the development of SLNs for the drug delivery. SLNs are bio-compatible; sub-micron sized colloidal drug delivery systems, which are designed by replacing the oil by solid lipid in the emulsions and shown in Figure 1. They provide higher entrapment efficiency, higher loading, greater surface area, simpler scale-up and manufacture and less toxic than the polymer, which potentiate the activity of the drug encapsulated in the lipid core (Muèller et al., 2000; Wissing et al., 2004; Mukherjee et al., 2009). SLNs exhibit sustained drug release and highly stable than the liposomes and even sterilization carried out during the manufacture of liposomes can be bypassed by the SLNs. The therapeutic utility of nanoemulsions is eclipsed by unpredictable drug release and of nanocrystals by poor solubilization of drug in biological fluids can be overcome by SLNs. Lipids present in the SLNs are highly purified triglycerides or waxes, calixarenes and sterols. A list of solid lipids employed in the preparation of the SLNs and preparation methods had been studied and shown in Table 3.

Table 3. Solid lipids used in the preparation of SLNs.

Solid lipid	Chemotherapeutic agent	Methods of preparation	Reference
Palmitic acid	Tamoxifen	Microemulsion and precipitation method	Fontana et al. (2005)
Stearic acid/Glyceryl mono stearate	Tamoxifen	Solvent injection method	Hashem et al. (2013)
Compritol® 888 ATO	Camptothecin	Modified olvent emulsification and ultra-sonication method	Acevedo-Morantes et al. (2013)
Trimyristin and soybean lecithin	Docetaxel	High pressure Homogenization method	Yuan et al. (2014)
Trimyristin	Paclitaxel	Homogenization method	Lee et al. (2007)
Stearic acid/Glyceryl mono stearate	Emodin	High pressure Homogenization method	Wang et al. (2012)
Compritol® 888 ATO	Tryptanthrin	Hot homogenization method	Fang et al. (2011)

Lipophilic drugs face solubility and bioavailability problems, which can be overcome by delivering through SLNs. SLNs are capable of loading of hydrophilic/hydrophobic compounds, controlled and extended drug release, bypass the reticulo endothelial system and deliver the chemotherapeutic at the site of action. Ingredients for SLNs preparation are approved by the Food and Drug Administration (FDA) and they are under Generally Recognized as Safe (GRAS) status (Wong et al., 2007; Kumar & Randhawa, 2013). History and background of SLNs are very short as the findings were lacking of clinical studies in the breast cancer treatment. Pre-clinical studies, such as in vitro cell line studies or in vivo animal studies for the SLNs in breast cancer treatment were promising, but this technology should become clinically significant. Some of the findings were discussed below.

Solid lipid nanoparticles exhibit controlled and sustained drug release irrespective to various pH environments. So, delay in elimination half-life alters the systemic circulation time of the drug. Fontana et al. (2005) formulated SLNs containing Tamoxifen citrate that were stable and showed the sustained release in different in vitro aqueous media (pH 7.4, 1.1 and human plasma). Tamoxifen SLNs exhibited prolonged release of intact drug in plasma, in turn, enhanced the drug bioavailability. In vitro anti-cancer studies on MCF-7 cell lines revealed that cytotoxic action of the drug-loaded SLNs was substantial compared with the free drug without affecting the bioavailability. Further efforts were made by Fahima et al. incorporating Tamoxifen into SLNs by solvent injection method. Using glycerylmonosterate, stearic acid, methanol, tween 80, polaxamer 188, the developed SLNs, showed higher encapsulation (90.40 ± 1.22%) and reasonable particle size (243.80 ± 12.33 nm). The SLNs containing the surfactant polaxamer 188 showed higher drug encapsulation might be the higher HLB value lead to formation of smaller-sized particles with effective surface area. Initial burst drug release followed by sustained drug release in the in vitro media by the SLNs enhanced the bioavailability of this poorly soluble drug. In vitro bio-availability studies revealed that C_max and AUC of Tamoxifen-loaded SLNs were 2 and 2.5 times higher than the free drug. Delayed attainment of T_max by the Tamoxifen-loaded SLN (10.00 ± 1.54 h) was due to sustained drug release from lipid vehicle and further it delayed the gastric emptying time. Intestinal lymphatic drug absorption might delay the in vivo metabolism and led to elevation of the relative bioavailability of the drug-loaded SLNs. Further investigation on clinical efficacy and safety studies needed to be carried out (Hashem et al., 2013).

Solid lipid nanoparticles encapsulate poorly soluble and hydrophobic natured drugs, which are facing bioavailability and cellular uptake limitations. Acevedo-Morantes et al. (2013) studied the anti-tumor activity of the Camptothecin SLNs on MCF-7 and MCF 10-A cell lines. Camptothecin is topoisomerase I inhibitor and unstable and hydrophobic in nature. The stability and bioavailability of the drug have been enhanced by loading into the lipid vehicle by super critical fluid technology. By the in vitro studies, the cytotoxic effect, cellular uptake and retention of drug-loaded SLNs was higher than the free drug. The lipid carriers showed sustained drug release and increased the transport of the drug to the cell by endocytosis pathway thereby the enhancement of cytotoxic action and cellular uptake.

Docetaxel is a widely used anti-tumor drug to treat a broad spectrum of solid tumors, such as non-small-cell lung cancer, locally advanced or metastatic breast cancer, advanced ovarian cancer and androgen-independent prostate cancer. It has dual mode cytotoxic action, where it exhibits G2/M cell cycle arrest and apoptosis at higher concentration whereas mitotic inhibition at low concentration. It has clinical limitations because of its serious side effects, neutropenia, myelosuppression, anemia and hypersensitivity, and its serious dose-limiting toxicity hinder its applications in cancer treatment. Yuan et al. (2014) made efforts to reduce its side effects and enhance its antitumor action by encapsulating Docetaxel in SLNs. Lipids used in the preparation were Trimyristin and soyabean lecithin and the SLNs were prepared by the high pressure homogenization. Docetaxel-SLNs effectively suppressed the growth of MCF-7 cells than the free drug. Docetaxel-SLN exhibited cell cycle arrest at G2/M stage and induced apoptosis at 5 nM dose. In vitro results have reflected similarly in the in vivo experiments in tumor-bearing nude mice. Myelosuppression is the major side effect of Docetaxel in clinical utility; authors were successful in reduction of myelosuppression toxicity by Docetaxel-SLN when tested in beagle dogs. Myelosuppression has close relation with the proliferation and differentiation of bone marrow cells. So, they performed hematopoietic colony-forming cell assay for investigating the capability of Docetaxel-SLNs reduce myelosuppression. The hematopoietic colony forming cell assays were carried out using primary mouse bone marrow cells in methylcellulose semi-solid medium containing Docetaxel-SLNs and Docetaxel-free drug to evaluate their myelosuppression toxicity. Primary mouse bone marrow cells were isolated and treated with 3 nM or 6 nM Docetaxel SLNs and Docetaxel-free drug for 9 days. At 6 nM dose Docetaxel-SLNs have significantly increased erythroid progenitors, colonies of granulocytes, macrophages. The erythroblasts colonies recovery is very low and not observed clearly. These evidences confirmed the possibility and proved that Docetaxel-SLNs have more potential in clinical therapy.

Paclitaxel is a potent anti-cancer drug prescribed for the solid and aggressive forms of ovarian, breast and lung cancer as well as AIDS-related Kaposi’s sarcoma. Its clinical use is limited by its low water solubility. Many researchers have developed the Paclitaxel-loaded SLNs in the treatment of other diseases, such as lung cancer and ovarian cancer (Muèller et al., 2000; Yuan et al., 2008). Lee et al. (2007) had formulated Paclitaxel-loaded SLNs by trimyristin. Trimyristin is a triglyceride with longer length of fatty acids showed adequate solubility and stability of Paclitaxel. Paclitaxel-loaded SLNs were stable during storage at 4, 25 and 40 °C without any drastic changes in the particle size (217.4 ± 32.82 nm) and zeta potential (−38.1 mV). The maximal inhibitory growth IC₅₀ of the Paclitaxel-Cremophor-based formulation, commercially available and Paclitaxel-loaded SLNs was similar in the MCF-7 cell lines.

Emodin, a Chinese herbal anthraquinone derivative, which has versatile biological actions, such as vasorelaxative, immunosuppressive and hepatoprotective activity. It is a promising cytotoxic agent by inhibiting cell proliferation, cell cycle arrest, induction of apoptosis and prevention of metastasis. Its application in cancer is limited due to its low aqueous solubility and poor cellular bioavailability. Wang et al. (2012) attempted to improve the solubility and elevate the cellular uptake of Emodin by encapsulating into SLNs. They developed SLNs, which exhibited biphasic release patterns, i.e. initial burst release followed by sustained release, which elevated the higher chemotherapeutic action than the drug alone. Cell cycle analysis revealed that emodin-loaded SLNs showed significant cell cycle arrest at G2/M phase in MCF-7 cells than the drug alone. SLNs unconditionally internalize into cells and led to higher cytotoxic action.

Fang et al. (2011) distinguished the efficiencies of the SLNs and NLCs loaded with Tryptanthrin against the MCF-7 breast cancer cells. Compritol and precirol were the solid lipids and squalene was the liquid lipid used for the preparation of SLNs. Glycerides with longer alkyl chains (compritol) have produced larger-sized nanoparticles than the glycerides with the shorter chains (precirol). Cell viability results have revealed that the NLCs have dose-dependent inhibition, and SLNs did not show the same behavior. Difference in the crystalline structures of SLNs and NLCs led to imperfections and sustained release of the drug. Lower melting behavior of NLCs than the SLNs contributed the higher cytotoxicity of NLCs.

Nanostructured lipid carriers

Nanostructured lipid carriers (NLCs) are the second generation of SLNs, which are a blend of different lipids, i.e. solid lipid matrix with a certain content of a liquid lipid (Wissing et al., 2004; Esposito et al., 2012). List of solid lipids as well as liquid lipids used and the preparation methods of NLCs are reported in Table 4 and NLCs are shown in Figure 1. It has various features, i.e. controlled drug release, site-specific targeting and drug accumulation at site of action. This carrier system has high tolerability due to presence of physiological and bio-compatible lipids (Müller et al., 2002b; Luo et al., 2011). NLCs exhibit higher drug loading capacity, lower risk of gelation and low drug leakage during storage caused by lipid polymorphism. It can prolong exposure of tumor cells to antitumor drug, EPR effect and subsequently increase the therapeutic effect of anti-tumor drug (Maeda et al., 2000; Müller et al., 2002a; Saupe et al., 2006). In NLCs, addition of liquid lipid to solid lipid creates a massive crystal disorder resulting imperfections in lipid matrix. This helps in the higher lodging of drug into the space thereby higher drug loading will be achieved (Davda & Labhasetwar, 2002; Gupta et al., 2004; Zhang et al., 2008b).

Table 4. Solid lipids and liquid lipids used in preparation of NLCs.

Solid lipid	Liquid lipid	Chemotherapeutic agent	Method of preparation	Reference
Glyceryl tripalmitate	Glyceryl tridecanoate	Quercetin	Phase inversion method	Sun et al. (2014)
Softisan 154	Olive oil	Tamoxifen	Melt emulsification method	How et al. (2013)
Glyceryl monostearate	Labrafil WL 2609 BS	Tamoxifen	Solvent diffusion method	Shete et al. (2013)
Stearic acid	Oleic acid	Doxorubicin/Paclitaxel	Solvent emulsification method	Zhang et al. (2008b)
PEG-DSPE	Oleic acid	Curcumin	Solvent diffusion method	Lin et al. (2015)
Stearic acid, Soybean phosphatidylcholine	Polyoxyl castor oil	Baicalein Doxorubicin	Emulsion-evaporation solidification method	Liu et al. (2015)

(Sun et al., 2014) formulated quercetin-loaded NLCs with the lipids, such as soy lecithin, glyceryl tridecanoate, glyceryl tripalmitate, vitamin E acetate, Kolliphor HS15 by the phase inversion method. This flavanoid molecule holds the limitations, such as low aqueous solubility and poor bio-availability, which were circumvented by encapsulating into NLCs. High encapsulation efficiency (95%) and sustained release of quercetin-NLCs promoted cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 cells (Sun et al., 2014).

Tamoxifen is one among the chemotherapeutics, which undergoes faster clearance (<24 h) through first-pass metabolism. NLC encapsulation benefited in prolongation of drug systemic circulation time, bypassing first-pass metabolism and improved absorption through lymphatic uptake. How et al. (2013) formulated Tamoxifen-loaded NLCs by the melt emulsification method using the lipids Softisan 154, olive oil, lecithin and 1% hydrophilic emulsifier (polysorbate 80), 4.75% sorbitol and 0.005% thimerosal. The structural disparity between the solid and oil lipids in NLCs resulted in higher drug loading capacity than the Tamoxifen-loaded SLNs. Incorporation of the Tamoxifen in NLCs has increased solubility and improved stability. Sustained drug release of Tamoxifen from NLCs will reduce the dosing frequency and side effects by the repetitive dosing. Shete et al. (2013) targeted intestinal lymphatic systems by the developed Tamoxifen-loaded NLCs. Tamoxifen-loaded NLCs showed a dose-dependent cytotoxicity in the MCF-7 (acinar epithelium) but its activity is lowered in the ZR-75-1 (ductal epithelium). Over-expression of MUC-1 gene in the ZR-75-1 cell lines might secrete a mucin layer hindered the internalization of NLCs leading to a decreased activity. Pharmacokinetic behavior of Tamoxifen-NLC shown significant improvement in C_max (2.71-fold) and longer t_1/2 (7.10-fold) compared with drug alone, which might be attributed by the drug–lipid association and intestinal lymphatic absorption. In vivo studies revealed that high drug concentration in the mesenteric lymph nodes clearly states that Tamoxifen NLCs were absorbed through intestinal lymphatic system.

Some researchers have exploited NLCs for active targeting of breast cancer cells. Zhang et al. (2008b) investigated the reversal action of the Doxorubicin- and Paclitaxel-loaded NLC against the ovarian-breast cancer, sensitive cell lines (MCF-7, SKOV3) and its multi-drug resistant variants (MCF-7/ADR SKOV3-TR30). Folic acid-conjugated NLCs has been targeted to the folate receptor, which is overexpressed in breast cancer cell membrane. Folic acid-stearic acid conjugation was carried out by carboxyl-amine reaction using a cross-linker 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide. Folate conjugation has shown enhanced internalization of NLCs into the breast cancer cells. Bio-compatible and nano-sized NLCs offered high cell membrane affinity thereby improved cytotoxicity. NLCs assumed to show competitive inhibition of P-gp activity in drug-resistant cells, which lead to high drug accumulation. Cytotoxicity of the Doxorubicin-loaded NLCs against the breast cancer was lower than that of Paclitaxel-loaded NLCs, which may be attributed due to the burst drug release behavior. Controlled release, high intracellular drug accumulation and reversal action of the Paclitaxel NLCs may serve as a candidate for the treatment of metastatic breast cancer.

Similarly, folate targeting was utilized by Lin et al. (2015) for the delivery of Curcumin. Folic acid-PEG-DSPE conjugant was synthesized for the active targeting of breast cancer cells. Cytotoxicity of folic acid-conjugated curcumin-NLCs has 3.5-fold higher than curcumin-NLCs. Tumor regression was observed in the folic acid-conjugated curcumin-NLCs group, the tumor growth was significantly inhibited when NLCs formulations were administered intravenously. This was attributed due to the active targeting potential of folic acid, which can internalize through specific folate receptors present on the surface of MCF-7 cells. Enhanced cellular uptake of NLCs by MCF-7 cells may be attributed though the cell membrane via endocytosis or direct penetration may enhance intracellular drug accumulation (Guo et al., 2014).

Liu et al. (2015) have actively targeted breast tumors using Hyaluronic acid for delivery of Baicalein and Doxorubicin. Cluster of differentiation 44 (CD44), a family of multifunctional trans-membrane glycoproteins, which is over-expressed in breast tumors, Hyaluronic acid is CD44 specific and has affinity at the N terminus of its extracellular domain. And thereby they have selected this major cell surface receptor for targeting with Hyaluronic acid. Baicalein, a bioactive flavenoid, obtained from the roots of Chinese medicine herb Scutellaria baicalensis Georgi. It exhibits broad anti-tumor action against breast, cervical and gastric cancers. But it is clinically limited because of extensive first-pass metabolism, poor bioavailability, shorter half-life (10 min), less water soluble and unstable (oxidized easily). They have selected Doxorubicin for the combination therapy to overcome multi-drug resistance, enhance the therapeutic effect and lower side effects. Baicalein and Doxorubicin encapsulated into NLCs by emulsion evaporation–solidification at the low temperature method. Hyaluronic acid was conjugated by the electrostatic attraction method. As it is a polyanion electrolyte molecule, it comprises of large number of negative charges and showed affinity on cationic NLC surface during conjugation. Cytotoxicity results revealed that Hyaluronic acid-conjugated Baicalein and Doxorubicin-NLCs have shown 12-fold dose advantage over Baicalein-Doxorubicin free drug. In vivo anti-tumor efficacy studies shown that Hyaluronic acid-conjugated Baicalein and Doxorubicin-NLCs exhibited significant tumor inhibition when compared with Baicalein-NLC, Doxorubicin-NLC and Baicalein-Doxorubicin free drug. This significant tumor inhibition may be attributed due to the presence of Hyaluronic acid, which could target CD44 receptors and enhance internalization of Baicalein and Doxorubicin. This developed drug delivery system is biocompatible, biodegradable, synergistic and reduces the systemic toxicity (Liu et al., 2015).

Lipid-polymer hybrid nanoparticles

Among the nanoparticles/nanocarriers, clinically cleared and commercially stepped are polymeric nanoparticles and liposomes for the breast cancer management. Pros and cons are associated with both the systems. Polymeric nanoparticles exhibit good structural integrity, tissue penetrable, various methods/polymer available for preparation, stable in biological fluids/during storage, ease of functionalization for active targeting and avoid RES clearance resulting in increased circulation time. Limitations associated with polymeric nanoparticles scale-up, organic solvent usage in the preparation and degradation; whereas, liposomes have been recognized as a superior drug delivery system due to its biocompatibility with biological components. They are tunable, bio-degradable, non-toxic, non-immunogenic, surface-modifiable. Drawbacks associated with liposomes are less stable during storage, drug leakage and sterilization (Hadinoto et al., 2013; Mandal et al., 2013). To mitigate the problems associated with both systems amalgamation of lipid and polymer brought novel drug delivery systems termed as lipid polymer hybrid nanoparticles LPN. Integrating architectural characteristics of polymer and bio-mimetic feature of lipid emerged into a hybrid drug delivery system and shown in Figure 1. Features of this drug delivery system are robustness, serum stable; exhibits sustained drug release, higher drug loading and tissue targeting (Zhang et al., 2008a; Liu et al., 2010).

Wong et al. (2006c) have made extensive efforts in engineering a robust drug delivery system for the treatment of multi-drug resistant breast cancer. Encapsulation of ionic and highly water soluble drug, Doxorubicin into a lipid carrier is challenging due to the low partition coefficient in the lipid phase. So, they encapsulated Doxorubicin into an LPN nanoparticulated system and studied their cytotoxic action on multi-drug resistant breast cancer cell lines MDA435/LCC6/MDR1 and its wild variant. Stearic acid and hydrolyzed polymer of epoxidized soybean oil was used for the preparation of the lipid-polymer hybrid carriers. Doxorubicin-loaded LPN enhanced the cytotoxic action, drug uptake and retention than the Doxorubicin-loaded SLNs. Same authors have carried out mechanistic studies of the Doxorubicin-loaded LPN against the multidrug-resistant breast cancer cell lines revealed that Doxorubicin entrapped in the nanoparticles bypass the membrane-associated Pgp and it is better retained within the Pgp-overexpressing cells than the drug alone. Elevated drug uptake in Pgp-overexpressing cells treated with Dox-PLNs was contributed by the phagocytosis (Wong et al., 2006b). Further, efforts were focused in the development of polymer-lipid hybrid nanoparticles simultaneous loading of cytotoxic drug-Doxorubicin and chemosensitizer-GG918 Elacridar. Dual drug-encapsulation approach has exhibited the enhanced MDR reversal action. Slow release of Elacridar GG918 due to its lipophilicity and higher association with lipid part of the nanoparticles did not show any deviation in the cytotoxic activity. Cytotoxicity studies, clonogenic assays and drug uptake studies revealed that the combinational drug therapy had superior and significant activity than the single drug therapy. Reasons for the enhanced drug uptake of the LPN are due to the higher accumulation prolonged retention and internalization by endocytosis in the tumor cells (Wong et al., 2006a).

Zhang et al. (2012) encapsulated a water-soluble drug Mitoxantrone into LPN by emulsification-ultrasonication method. Mitoxantrone LPN enhanced cytotoxic action than the free drug in the MCF-7 and MCF-7-MX (multi-drug resistant variant). Bio-distribution studies revealed that exposure of the drug by LPN to the heart tissue was reduced significantly, which has a great impact in the clinical use. Yang et al. (2013) formulated 10-hydroxycamptothecin-loaded LPN, which is actively targeted to breast cancer by conjugation with arginine–glycine– aspartic acid (RGD) peptide. Preparation of LPN was by lipid coating to the polymeric nanoparticles unlikely the lipid polymer amalgamation. Polymeric core degradation of LPN led to a sustained drug release; and engineering of the peptide ligand showed higher drug accumulation and significant toxicity in the breast cancer cell lines.

Conclusion

As addressed in this review, large quantum of work has been carried out and overwhelming evidences are available. The basic concept of lipid nanocarriers as a well tolerated carrier is confirmed and well documented. Lipids used in the development of liposomes, SLNs, NLCs and LPN are non-toxic, biocompatible and biodegradable with low or no immunogenicity. The lipids are able to form a nanostructure and it has been widely investigated as a nanocarrier for tumor-targeted drug delivery. Lipid-based nanomedicine has shown substantial therapeutic potential to treat a variety of diseases for both research and clinical applications. Physiochemical properties of drug and lipids can be optimized to customize the geometrical parameters, such as particle size, shape and encapsulation efficiency, and drug release behavior pattern. Characterization of the formulation is also a critical attribute, which controls the product quality, stability and release kinetics. These lipid nanocarriers exhibit passive drug delivery exhibits enhanced therapeutic effect. The presence of reactive functional groups in lipids facilitates for conjugation of proteins and some other targeting agents for tumor-targeted drug delivery. These lipid nanocarriers and their derivatives are conjugated with hydrophobic moieties and tumor-targeting ligands are found to exhibit a prolonged circulation time in blood and diminish uptake by the reticuloendothelial system (RES) due to their smaller size. Moreover, these lipid nanocarriers are able to target various portions of the tumor using targeting ligands and reduce the problems associated with multi-drug resistance. It is evident that lipid nanocarriers have tremendous potential as tumor-targeted drug-delivery for cancer therapy and they are fortunate as almost 40% of the new drug entities are lipophilic. In recent years, theranostic nanocarriers shown promissory results in the emerging field of personalized medicine, as they detect as well as monitor individual patient’s cancer at an early stage and deliver anticancer agents thereby enhanced therapeutic efficacy. Practically, non-invasive monitoring of the theranostic carriers enables clinicians have to rapidly fix on whether the regimen is effective in an individual patient or not. For these reasons, cancer theranostic functioned nanoparticles presents a novel promising strategy in breast cancer treatment. In this perspective, lipid-based nanocarriers conjugated/or encapsulated with targeting ligands and imaging agents would bring a great breakthrough by simultaneous diagnosis of tumors, targeted drug delivery with less toxicity and monitoring of treatment.

Coming in detail, the currently available data still do not have straightforward answers and need to address unmet clinical problems. Many strategies and systems have been designed for tumor targeting, but some of them achieved FDA/EMA approval. Caelyx/Doxil, the first nanomedicine (passive targeted system), developed for the breast cancer treatment have been approved by the FDA and have pronounced improvement in the safety of drug. Although, nanotechnology is promising several challenges are limiting the utility. Still, lacuna exists with the efficacy of these formulations and most of them have failed at early or late clinical trials because of misinterpretation of the basis in tumor-targeted drug delivery. Basics and concepts for the tumor targeting need to be emphasized and further investigations need to carry out for improving the efficacy and stability.

Pharmaceutical industries are concerned in design of a drug delivery system that could be sufficiently versatile and feasible for scale up of the formulation is very important. Success of a drug delivery system cannot be defined by the development only in academia research. Success comes by the guaranteed broad application of drug delivery system and technology transfer should be up-scaled from lab-scale to large-scale. Drug delivery is limited to the academia, which should reach clinical translation and need to be up scaled to industry. Lab-scale to large-scale approach is strongly recommended to bring nanomedicine for the clinical utility.

Acknowldgements

The authors are thankful to Shashank Tummala, Shashank Mulukutla, Research scholars, JSS College of Pharmacy, Udhagamandalam for giving valuable time and suggestions in preparation of this manuscript.

Declaration of interest

All the authors have no competing interests.