Antitumoral-Lipid-Based Nanoparticles: A Platform for ...

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Antitumoral-Lipid-Based Nanoparticles: A Platform for Future Application in Osteosarcoma Therapy Yolanda González-Fernández1,2,3†, Edurne Imbuluzqueta1,2,3†, Ana Patiño-García2,3,4 and María J. BlancoPrieto1,2,3* 1

Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Irunlarrea 1, 31008, Pamplona, Spain; 2Centro de Ingeniería Biomédica, University of Navarra, Irunlarrea 1, 31008, Pamplona, Spain; 3 Instituto de Investigación Sanitaria de Navarra, IdiSNA, Irunlarrea 3, 31008, Pamplona, Spain; 4Laboratory of Pediatrics, University Clinic of Navarra, Pio XII 36, 31008, Pamplona, Spain Abstract: Osteosarcoma is the most frequent primary bone tumor in the pediatric age group. Its aggressive local growth pattern and its high propensity to metastasize, mainly to the lungs, give the disease an unfavorable prognosis that has situated this disease as one of the leading causes of pediatric cancer death. Current protocols for osteosarcoma treatment are based on neo-adjuvant (pre-operatory) chemotherapy followed by surgical resection of the tumor and a new phase of adjuvant chemotherapy. Despite the progress that these protocols have made in imMaria J. Blanco-Prieto proving the outcome of the disease, the limited access of drugs to bone tumor and metastases, their indiscriminate distribution in the organism, the high required doses that cause intolerable toxicity and the development of multidrug resistance, still represent a major challenge. Nanotechnology has emerged as a new strategy to successfully address these problems by the development of nanoscaled drug carriers that present the ability to target the drug to the tumor cells, achieving high drug concentrations in the tumor area, while decreasing its presence in healthy tissues and therefore its potential systemic toxicity. This review summarizes the different lipid nanocarriers developed to deliver first and second-line anti-osteosarcoma drugs as well as emerging agents in the treatment of this disease. Moreover, it also discusses the potential of these nanocarriers for the treatment of osteosarcoma.

Keywords: Osteosarcoma, lipid nanoparticles, drug delivery, chemotherapy, doxorubicin, methotrexate. 1. INTRODUCTION Although historically pediatric cancer has not been one of the most frequent causes of death among children, the decreasing rates of mortality associated with other pathologies have situated cancer as the second leading cause of pediatric demises after domestic accidents such as intoxications, traumas and abuses. At the beginning of 2014, the American Cancer Society estimated that a total of 15,780 new cancer cases would be diagnosed, with 1,960 deaths among children and adolescents in the United States [1]. Unfortunately, approximately half of the patients who survive are estimated to relapse during the following years [2]. Bone tumors represent 5 % of total pediatric cancer, osteosarcoma (OS) being a critical and lethal disease that affects children during their first or second decade of life. It accounts for nearly 60% of such kind of tumors in children and young people [3], with an estimated incidence of 4 cases / year/ million persons in children from 0 to 14 years and 8 cases for the range of 15-19 years old [4]. Its main drawback is that in many patients it is accompanied by lung metastasis from the time of diagnosis and, although an early treatment is vital to control the disease, early diagnosis and treatment are not frequent in this type of cancer. Consequently, the 5year survival of patients with metastatic disease is still very low. Nowadays, the treatment of OS still represents a great challenge. OS therapy includes the surgical resection of all detectable tumor foci, as well as pre-operative and post-operative polychemotherapy. However, despite the advances made in chemotherapy protocols, local recurrence, systemic metastases and persistent chemo-refractory disease still constitute a major problem, and more *Address correspondence to this author at the Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain; Tel: +34 948425600; Fax: +34 948425649; E-mail: [email protected] † Both authors contributed equally to this work.

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than 70% of patients with metastatic OS and 20%–40% with nonmetastatic disease relapse or die [5]. One of the main causes of this therapeutic failure is the chemo-resistance to anti-OS therapy [6], which brings to light the urgent need for new less toxic and more effective therapeutic approaches. Nanomedicine has emerged as an attractive alternative to overcome some of the main drawbacks encountered in cancer treatment. In this sense, lipid-based particulated systems hold a great promise to improve both the effectiveness and safety profile of conventional antineoplastic agents. These vehicles, made of biodegradable and biocompatible lipids, allow medicine to be targeted to the affected areas, increasing the concentration of the drugs in the tumor cells, and consequently, reducing the systemic side effects [7]. Moreover, in comparison to other carrier systems such as emulsions, liposomes or polymeric nanoparticles, lipid-based nanoparticles present improved physical stability, relatively low cost manufacture, easy large scale production, the ability to entrap lipophilic as well as hydrophilic drugs and less potential toxicity owing to their composition and the possibility of organic solvent-free production [8]. This review describes the therapeutic benefits of encapsulating the anti-OS drugs currently used in lipid nanoparticles (LN), as well as the role played by emerging agents for OS therapy in the treatment of this disease. Moreover, the great potential these lipid carriers may have as a new strategy for treating OS is also addressed. 2. OSTEOSARCOMA OS is defined as a malignant neoplasm arising from mesenchymal cells that are able to produce an immature bone matrix, named osteoid (Fig. 1a), which presents varying degrees of mineralization and consequently, different tumor patterns. Moreover, due to their mesenchymal origin, OS cells possess the ability to differentiate into different histologic variants that produce fibrous, cartilaginous or bone tissue [9]. Depending on the predominant tissue differentiation of the tumor and the type of extracellular matrix © 2015 Bentham Science Publishers

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produced, the World Health Organization (WHO) classifies the conventional OS into three histological categories; osteoblastic, fibroblastic or chondroblastic subtype. In addition to these conventional OS, the WHO recognizes additional less common OS variants including small cell OS, telangiectatic OS, parosteal and periosteal OS and low grade central and high grade surface OS [3]. These different subtypes have distinct histological and radiological features that may vary significantly between cases and, more importantly, between areas in the same case. This tumor heterogeneity, together with the high genetic complexity of OS cells, makes it difficult to obtain a significant improvement in patient survival rates.

Fig. (1). a) Hematoxylin-eosin staining of an osteo-chondroblastic osteosarcoma, showing the tumor cells and the reactive malignant osteoid (black arrow). b) Plain X-ray showing an osteosarcoma of the distal femur. The white arrow indicates the Codman’s triangle. c) High resolution magnetic resonance showing the bone destruction due to the presence of an osteosarcoma of the distal femur. These images are courtesy of Dr. San Julian at the University Clinic of Navarra.

2.1. Age and Site Distribution The incidence rates of bone sarcomas present a bimodal age distribution. The first and main peak of incidence usually appears during the first and second decade of life. In these patients primary tumors occur in areas of large bones in the limbs with high growth potential, such as the metaphyseal area of the femur, tibia or humerus. They are more common in tall children and they appear at earlier ages in females than males, corresponding to their earlier pubertal growth spurt. A second small peak of incidence occurs in adults older than 65 years old. In this case the tumor is more frequently localized in flat bones of the pelvic or maxillary region [10]. However, despite the well-defined location of the primary tumor, OS is a highly invasive neoplasm that must be considered as a systemic disease, since at the time of diagnosis, 20% of patients present malignant cells in the lungs, distal bones or less frequently other regions of soft tissues, and in most of the remaining 80% of cases, the presence of undetectable micrometastases is assumed [11]. 2.2. Metastatic Process The development of the metastases present in most OS patients is the result of several events that conclude in tumor dissemination. Neovascularization, invasion, resistance to apoptosis and to the immune system, dormancy and activation are the main processes that lead tumor cells to form metastatic foci [12].

González-Fernández et al.

The first condition for the primary tumor to metastasize is its vascularization. This process is vital to provide the tumor cells with the oxygen and nutrients essential for their proliferation and expansion to distal sites. The hypoxia conditions of the growing tumor stabilize the hypoxia-inducible factor-1 (HIF-1), which, in turn, activates the pro-angiogenic vascular endothelial growth factor (VEGF), inducing the increase of vascular permeability and neovascularization [13]. Moreover, the activation of VEGF also upregulates the expression of anti-apoptotic proteins (Bcl-2, survivin) [14,15] and matrix metalloproteinases responsible for the degradation of the extracellular matrix, thus favoring tumor cell invasion of the bloodstream [16]. Unlike other types of cancer, which present a lymphatic dissemination, OS cells are spread through the hematogenous route. Once in the systemic circulation, disseminated tumor cells (DTC) produce immunosuppressive interleukins (IL-10) [17] or alter their membrane protein expression pattern to avoid their recognition and clearance by the natural killer cells and to evade the action of the immune system [18]. Moreover, they also acquire resistance to apoptosis (anoikis), which is vital for the successful dissemination of OS cells. The next step towards forming the metastases is the extravasation of the DTC into foreign tissues and their attachment and adaptation to the metastatic microenvironment. DTCs are larger than normal blood cells and tend to aggregate and form microemboli that get trapped in the blood capillaries. Interestingly, these aggregates are not distributed indiscriminately through the organism but disseminate to specific tissues, namely to the lungs in more than 80% of the cases, which suggests that there are specific molecules in the tissue microenvironment and DTCs that favor this specific location [12]. However, as mentioned in the previous section, in most patients the metastatic process is not evident from the first moment, suggesting that metastatic cells may remain dormant in the lungs until some factors induce them to proliferate. Dormancy in OS is not fully understood, but there is evidence indicating that some of these cancer cells may have stem-like cell properties and the ability to survive under stressful environmental conditions. The metastatic cells could undergo growth arrest, remaining dormant during lengthy periods of time while they express drug resistance proteins that make them less sensitive to chemotherapy [19]. Another hypothesis suggests that during tumor formation, metastatic cells could disseminate from the primary bone tumor to the bone marrow and remain dormant until their activation and posterior colonization of the lungs [20]. This state of dormancy is usually reversed by an increment of angiogenic factors and specific cytokines in the tumor environment, a process that could take place after the surgical resection of the tumor. Once awakened, metastatic OS cells begin to proliferate and acquire more resistance to apoptosis and chemotherapeutic drugs, giving rise to resistant and aggressive metastases in the lungs [12]. 2.3. Clinical Features and Diagnosis One of the main problems of OS is the lack of specific clinical signs and symptoms within its first phases. The primary and most common symptoms are localized and persistent pain and the swelling of the affected area. However, given the typical age of patients, the appearance of most of these symptoms is usually justified by traumas, tendinitis or typical child growth pains. Regarding nonspecific symptoms, most patients do not report fever or weight loss, anemia or loss of appetite and only if the tumor presents enough soft tissue involvement can a palpable mass be observed in the bone. As a result, the bone tumor is treated with analgesics and neglected for months, and so by the time OS is diagnosed, the cancer is already advanced [21].

Lipid Nanoparticles as a Platform for Future Osteosarcoma Treatment

The diagnosis of OS is based on the combination of both radiological and histological techniques. A plain radiographic image is usually enough to detect the presence of the tumor mass. Conventional sarcomas display lucent and sclerotic features as a result of the destruction of the normal bone tissue, which is replaced by a poorly mineralized osteoid tissue. The formation of the bone tumor is evidenced by the lifting of the periosteoum that leads to the appearance of a triangle named Codman’s triangle (Fig. 1b). Further evaluations include nuclear magnetic resonance (Fig. 1c), computed tomography and positron emission technology (PET) scans, which are useful to assess the extent of bone and soft tissue involved, evaluate the presence of metastases and monitor the patient’s response to chemotherapy [22]. However, even though the diagnosis of OS can be predicted by the radiographic appearance, a tru-cut biopsy of the lesion is mandatory to confirm diagnosis and to identify the tumor´s characteristics. Regarding laboratory analysis, OS lacks specific tumor markers and laboratory values are usually within the normal ranges. However, in most patients an increment in the levels of alkaline phosphatase and lactate dehydrogenase and the erythrocyte sedimentation rate is found. Extreme values of lactate dehydrogenase are associated with a poor prognosis, while the increase in alkaline phosphatase levels is related to a higher risk of relapse. Moreover, as the polychemotherapy administered for OS treatment can result in cardiac, kidney and liver toxicity, baseline assessments of their function should be performed in all OS patients [10]. 2.4. Treatment Until the 1960s, OS was considered a chemoresistant tumor and amputation was the only way to address it. Protocols used for the treatment of the disease were based on single drug therapies with methotrexate (MTX) or doxorubicin (DOX) [23] and although it was well-known that metastases were the main cause of patients’ demise, they were not treated specifically [24]. The concept of multidrug therapy for OS treatment was introduced in the 1970s when both high-dose MTX (HD-MTX) with leucovorin rescue and DOX were found to be effective in treating the metastatic OS. However, it was not until 1979 that OS started to be treated preoperatively. At that time, Rosen et al. conducted a pilot study to evaluate the benefits of pre-surgical chemotherapy and established

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the need for neo-adjuvant chemotherapy to improve the clinical outcome of OS patients [25]. From that moment on, all OS treatment protocols have been based on a pre-operative (neo-adjuvant) chemotherapy administered for 8-10 weeks, followed by the surgical resection of the tumor and 12-29 weeks of post-operative (adjuvant) chemotherapy. However, the ideal combination remains to be defined and the choice of the drug combination administered to the patients depends on each institution and national protocols. Neo-adjuvant chemotherapy usually combines DOX, HDMTX, cisplatin (CIS) and ifosfamide (IFO) (Table 1), which have been considered the most effective agents in OS therapy. This preoperative therapy plays an essential role in the treatment of OS since it offers the opportunity to examine the histologic response of the tumor to the administered drugs and assess their effectiveness. The degree of necrosis present in the tumor mass after the neoadjuvant treatment determines the prognostic value of the patients, those with more than 90% of tumor necrosis being considered favorable responders while patients with less than 90% of tumor necrosis are poor responders and show poor prognosis [26]. Moreover, this approach provides early treatment for the micrometastases and facilitates limb salvage procedures. The surgical management of OS is designed to completely remove the lesion with adequate margins in order to avoid relapse and tumor dissemination. In the past, bone sarcomas were treated by amputation with relatively poor functional outcomes, but nowadays limb salvage has become the standard of care and 80% of patients are treated with limb sparing reconstructions like endoprosthetic devices and biological reconstructions that maintain limb function [35]. If performed adequately, these reconstructions have not been shown to decrease the patient’s survival rate or increase their relapse rate in comparison to amputation. Regarding the post-operative chemotherapy, when the tumor responds favorably to the neo-adjuvant protocols, the same drugs and doses are maintained during the adjuvant treatment. However, when a poor response is observed, second-line drugs such as etoposide (ETO) in combination with IFO are usually added to the post-operative treatment protocol [26].

Table 1. Main mechanisms of action, adverse effects and resistance mechanisms of the first-line antineoplastic drugs used in osteosarcoma treatment. Drug

Administration Schedule 60-75 mg/m, IV or IA route.

DOX

Single bolus injection, short infusion or continuous infusion over 24-72 h

Mechanisms of Action

Main Adverse Effects

Resistance Mechanisms

Refs.

-Myelotoxicity -Cardiotoxicity

-Drug efflux by ABC transporters

[24,27-30]

-DHFR inhibition

-Nephrotoxicity -Myelotoxicity

-Overexpression of DHFR

-DNA alkylation

-Nephrotoxicity -Ototoxicity


[24,32,33]

-DNA alkylation

-Nephrotoxicity

-

[24,34]

-Topo II inhibition -Generation of free radicals -Disruption of membrane proteins

-Reduced RFC expression

10-12.5 g/m, IV route. MTX

CIS

IFO

4 courses in 10-14 days over 4-6 h with leucovorin rescue 100-120 mg/m over 2-4 h by the IV route or 150 mg/m by the IA route every 3-4 weeks 6-9 g/m in 4 courses and uroprotection with mesna


[24,31]

Abreviations: ABC = ATP binding cassette, CIS = cisplatin, DHFR = dihydrofolate reductase, DOX = doxorubicin, IA = intra-arterial, IFO = ifosfamide, IV = intravenous, MTX = methotrexate, RFC = reduced folate carrier.



Fig. (2). Illustration of the different lipid-based nanocarriers including lipid nanoparticles (solid lipid nanoparticles, nanostructured lipid carriers and lipid-drug conjugate nanoparticles) and lipid nanovesicles (liposomes, micelles, niosomes, transfersomes and ethosomes).

The use of these therapeutic regimens has drastically improved the survival rates from 10 to 70%. However, the 3-5 year survival of patients with metastatic disease is still 10-30%. In these patients, neither the use of second-line drugs nor the use of more aggressive treatments seems to improve the outcome of the disease [36] and, unfortunately, in those cases the post-surgical chemotherapy is just a palliative and potentially toxic treatment [37]. This therapeutic failure is the consequence of several factors such as acquired resistance to the administered drugs, systemic toxicity of the multi-agent protocols, inter and even intra-individual variability of the tumor and, above all, the tendency of this type of cancer to metastasize. In this context, nanomedicine, especially LN, may offer an attractive alternative to successfully address these problems by targeting the drug to the tumor cells, achieving high drug concentrations in the tumor area while reducing their presence in healthy tissues and therefore their systemic toxicity [7]. 3. LIPID NANOPARTICLES AS DRUG DELIVERY SYSTEMS Since their launch on the market in the mid-1980s, lipid-based nanosystems have been widely studied for their potential application to cancer treatment, and lipids have turned out to be attractive and versatile materials for drug delivery purposes [38,39]. However, despite the successful early development of liposomal formulations for the intravenous administration of antitumor drugs, such as liposomal DOX (Doxyl®, Caelyx®), the instability of liposomes in plasma due to interactions with plasma components represents a major limitation, since it results in the rapid release of the encapsulated drug [40]. Therefore, after the development of liposomes, research in this field has progressed from these first delivery vesicles to more sophisticated lipid nanocarriers in order to improve their performance (Fig. 2). LN are colloidal carriers made of biocompatible and biodegradable lipids that are solid at body temperature. They combine the advantages of other colloidal carriers while minimizing some of the problems associated with them. Like other carriers, these nanoparti-

cles may overcome the solubility problems of anticancer drugs and provide protection against their chemical, photochemical and oxidative degradation. However, the biodegradable and biocompatible nature of LN makes them less toxic than other systems. Besides, their easy scaling up for large scale production and the low cost of lipids compared to biodegradable polymers or phospholipids make them appropriate for use as potential drug delivery systems [41]. The main attractiveness of these systems as drug carriers for anticancer drug delivery resides in their excellent ability to enhance the oral bioavailability of drugs [42]. Oral delivery is the most convenient route of administration, and due to their small size and composition (lipids and surfactants), LN may present bioadhesive properties and possess the ability to promote oral absorption of the encapsulated drugs by enhancing their gastrointestinal and lymphatic uptake [7]. Owing to these favorable characteristics, research on LN is now an expanding field and the results obtained so far demonstrate the relevance of these platforms as a particularly promising alternative to conventional therapies [43]. The different types of LN are described in more detail below. 3.1. Solid Lipid Nanoparticles Developed in the mid-1990s as an alternative to the existing traditional carriers, solid lipid nanoparticles (SLN) are considered the first generation of LN [44,45]. They were developed with the aim of overcoming the major disadvantages of the traditional systems, such as low stability, drug leakage problems and toxicity issues, while maintaining their advantages. SLN are colloidal particles related to lipid nanoemulsions but with a matrix composed of generally recognized as safe (GRAS) natural or synthetic lipids that are solid at body temperature. The lipid matrix is usually composed of fats, triglycerides or waxes and provides protection for the drug from physicochemical degradation. Depending on the preparation technique and the materials used, SLN present sizes from 50 up to 1000 nm and they may be prepared without the need for organic solvent, which contributes to the good tolerability and low toxicity of these systems. Under optimized conditions they can incorporate



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lipophilic or hydrophilic drugs and, due to their ability to improve the bioavailability of water insoluble drugs, they have been successfully designed as carriers for the delivery of various anticancer drugs [7]. The main advantages of SLN include their physicochemical stability, excellent tolerability, easy production at large industrial scale, wide application spectrum and the controlled drug release. On the other hand, common disadvantages of SLN include their limited drug loading, which is subjected to the physicochemical characteristics of the drug, as well as to the solubility and miscibility of the drug in the melted lipid and the composition and crystallinity of the matrix [41]. Drug expulsion during storage is another common limitation of SLN. This phenomenon can occur during storage as a consequence of a rearrangement process of the low ordered lipid matrix that is transformed into a highly ordered lipid matrix characterized by a perfect crystal lattice with less space for drug accommodation [46]. 3.2. Nanostructured Lipid Carriers Nanostructured lipid carriers (NLC) are a new generation of LN developed to overcome the limitations associated with SLN. NLC are modified SLN in which the lipid phase contains both solid (fat) and liquid (oil) lipids, with the advantage that the majority of drugs have higher solubility in liquid lipids than in melted solid lipids [47]. Like SLN, NLC remain solid at room and body temperature. NLC share the advantages of SLN, such as controlled drug release, excellent biocompatibility and easy production at industrial scale while minimizing some of the limitations that hinder the use of SLN, including the drug loading limitation and drug leakage during storage. The matrix of NLC contains spatially incompatible solid and liquid lipids that form an imperfect matrix without a crystal arrangement that presents improved drug incorporation and reduced drug loss during storage. While SLN are composed of 0.1% to 30% (w/w) solid lipid dispersed in an aqueous solution, NLC are obtained by mixing solid and liquid lipids, preferably at a ratio of 70:30 up to a ratio of 99.9:0.1, with an overall solid content in the formulation that can go up to 95% (w/w) of solid (lipid and surfactant) incorporated in the aqueous phase. Therefore, NLC dispersions with higher solid content can be produced [48,49]. 3.3. Lipid-Drug Conjugate Nanoparticles Although both SLN and NLC present the ability to incorporate hydrophilic drugs, due to their high lipid content, partitioning effects during the production process can limit the loading capacity of these drugs in both types of carriers [50]. In order to overcome this limitation, lipid-drug conjugate (LDC) nanoparticles were developed. To prepare these nanoparticles, the hydrophilic drug is first transformed to a lipophilic molecule by its conjugation with a lipidic compound through a covalent linkage or by the formation of a salt with a fatty acid. Then, the LDCs obtained, which usually present a melting point around 50–100 °C, are melted or dispersed in an aqueous surfactant solution and formulated by high pressure homogenization to obtain LDC nanoparticles [51]. These nanoparticles can achieve drug loadings of 30 to 50% when the nanoparticle is composed only of the conjugate molecule [52,53] and solid lipids can also be additionally incorporated to obtain nanoparticles with a mixed matrix of LDC and lipid that present improved properties, such as higher permeability across the gastro-intestinal barrier and enhanced protection of the drug from chemical degradation [54]. 3.4. Non-Targeted and Targeted Lipid Nanoparticles: Passive and Active Targeting One of the most noteworthy problems associated with the use of antitumor drugs is their high systemic toxicity, mainly due to their unspecific distribution and accumulation in healthy tissues. Nanotechnology can tackle this problem by the passive and active targeting of the drug to the tumor tissue (Fig. 3). The main goals of targeting antitumor drugs with nanoparticles include improving the

Fig. (3). Schematic representation of the passive tumor accumulation of lipid nanoparticles due to the enhanced permeation and retention effect and the active targeting strategy using targeted lipid nanoparticles.

pharmacokinetics and pharmacodynamics of the drug, increasing drug concentration in the tumor tissues while diminishing its distribution to healthy tissues, and promoting the internalization and intracellular delivery of the drug into the tumor cells [55]. One of the characteristics that has attracted most attention towards nanoparticles is their ability to improve drug bioavailability and accumulation in the tumor tissue by their extravasation through the defective, leaky vasculature associated with tumor tissue. This effect, known as the enhanced permeability and retention (EPR) effect, allows the passive targeting of the nanoparticles to the tumors and is the consequence of a non-regulated tumor angiogenesis which, together with the poor lymphatic drainage system surrounding the tumors, contributes to the accumulation and retention of the nanoparticles in the interstitial fluid of the tumor [56]. The resulting selective accumulation of high drug concentrations in the tumor site reduces the distribution of the drug in healthy tissues, increases the maximum tolerated doses of the cytotoxic agents and minimizes their side effects, enhancing the efficacy of the antitumor drug while reducing its systemic toxicity [57]. On the other hand, the advances in surface-engineering technology have further improved the performance of LN, widening their targeting possibilities. Active targeting is usually achieved through the modification of the nanoparticle surface, which could be done by decorating the nanoparticles with targeting moieties that are specific to receptors that are overexpressed or selectively expressed in the tumor cells. To achieve this goal, LN can be coated with monoclonal antibodies [58], ferritin [59], folic acid [60] or mannose [61] among others. This targeting strategy allows, not only a more selective binding of the drug to the tumor cells, but also increases its internalization through specific interactions [62]. Therefore, taking into consideration that due to its architecture and structure, delivering drugs specifically to bone tissue is very challenging, the combination of passive and active targeting with antitumor drug-loaded LN holds great promise for the delivery of therapeutics to bone tumors and their metastases. The following sections outline the main non-targeted and targeted LN encapsulating currently used or promising drugs in OS treatment and their performance in different cancer models.


4. LIPID NANOPARTICLES FOR THE DELIVERY OF CONVENTIONAL DRUGS IN OSTEOSARCOMA THERAPY As stated before, the main antineoplastic agents used in OS treatment include DOX, MTX, CIS, IFO and ETO. These drugs are usually administered at high doses and by conventional routes and are unselectively and widely distributed throughout the body causing severe systemic toxicity. However, prolonged exposure to reduced drug doses can lead to the emergence of acquired drug resistances and can contribute to chemotherapeutic failure and favor the metastization process. This narrow therapeutic window makes these drugs ideal candidates for their incorporation into LN in an attempt to reduce their systemic toxicity while enhancing their overall efficacy. This section outlines the different LN described in the literature developed to carry and deliver first-line OS drugs to different tumor types. 4.1. Doxorubicin Lipid Nanoparticles 4.1.1. Doxorubicin DOX is an anthracycline antibiotic commercialized under the brand name Adriamycin that is widely used in cancer therapy for the treatment of a large spectrum of both solid and hematological tumors, such as adenocarcinoma, melanoma, leukemia, lymphoma or sarcomas [63]. Its mechanism of action is well characterized and is known to be multifactorial. DOX inhibits the enzyme topoisomerase II, which regulates DNA replication, transcription and repair among other nuclear processes, causing the breakage of the double-strand DNA and consequently cell death. Moreover, DOXmediated cell death can also be achieved by its intercalation in the DNA, forming DNA-DOX adducts that activate DNA damage responses, or by the oxidation of its quinone structure to an unstable semiquinone metabolite, a process that generates free radicals and peroxides that damage the DNA and the cell membrane [27]. However, despite being a potent chemotherapeutic agent, its narrow therapeutic window, the emergence of drug resistance and its severe side effects represent major limitations for the clinical use of DOX [64]. Resistance to DOX is closely related to the expression of drug efflux transporters such as p-glycoprotein (P-gp) (MDR1, ABCB1) and ABCC1 (MRP1) transporters among others, which hinder the intracellular accumulation of the drug and decrease its therapeutic activity. Moreover, these proteins are also responsible for the multidrug resistance (MDR) effect observed in most tumor cells [64]. In addition to the emergence of drug resistance, DOX-related toxicity is the main obstacle limiting its application, myelosuppression, mucositis and specially cardiotoxicity being the most severe side effects of this drug. The heart is the preferential target of DOX toxicity and although the molecular mechanisms behind DOX cardiotoxicity are not fully understood, they are related to the cumulative dose of the drug. In addition to cardiotoxicity, other DOX-related adverse effects include nausea, vomiting, alopecia or arrhythmias [29]. In order to overcome these limitations, several strategies have been developed with the aim of increasing the therapeutic index of DOX, such as adjusting the doses administered or changing the administration routes [24], protecting the organism against DOX induced cardiotoxicity by the concomitant administration of dexrazoxane [29], or including DOX in drug delivery systems. This last strategy has already been shown to minimize drug exposure to healthy tissues, to target the tumor increasing drug concentration at its site of action, or to reverse the MDR effect by bypassing the membrane efflux proteins. This, therefore, represents one of the most promising strategies to improve the therapeutic window of this drug [65]. 4.1.2. Doxorubicin Solid Lipid Nanoparticles In the last decades, DOX-SLN have been engineered as an alternative to conventional liposomes for cancer therapy. However,


since it is a hydrophilic molecule, the entrapment of DOX in lipid carriers has turned out to be a challenge. One strategy employed to overcome this barrier is the coordination of DOX with counterions. The resulting ion-pair complex presents a higher lipophilicity than the original drug, and thus increases the encapsulation efficiency of the drug in the different lipid-based systems [66]. One of the main goals of DOX encapsulation in SLN is to improve its bioavailability inside the tumor cells. Gasco et al. developed stearic acid DOX-SLN using hexadecylphosphate as a counterion and compared their efficacy to that of free DOX and liposomal DOX in human colorectal cancer, retinoblastoma and glioblastoma cell lines [67,68]. DOX-SLN produced a higher cytotoxicity than the other treatments in all cell lines. This effect was shown to be time and dose-dependent and the consequence of a faster and more effective internalization of the drug in the cells. Similarly, Kuo et al. prepared catanionic DOX-SLN containing cationic hexadecyltrimethylammonium bromide and anionic sodium dodecylsulfate as surfactants and tested their efficacy against a glioma cell line [69]. DOX-SLN exhibited a higher efficacy than DOX in solution inhibiting the proliferation of the tumor cells, which was attributed to an enhanced uptake of the carrier system with respect to free DOX. This increase in the antitumor efficacy due to improved bioavailability of the drug after its encapsulation has also been observed by other authors in DOX-sensitive and resistant human breast and ovarian cancer cells [70,71]. In these cases, the improved uptake of the encapsulated drug was thought to be the result of a different and more effective internalization mechanism of the nanoparticles and their ability to inhibit the P-gp efflux pump, which, together, contribute to overcome the chemoresistance of the cells. In addition to the drug that is incorporated, the components of the carrier itself can also improve the antitumor efficacy of the carrier, particularly by overcoming P-gp associated MDR effect. Ma and colleagues developed DOX-SLN using Brij®78 and D-tocopheryl polyethylene glycol succinate (vitamin E TPGS) as surfactants. DOX was ion-paired with sodium taurodeoxycholate (STDC) and sodium tetradecyl sulfate (STS) and the SLN obtained were evaluated in sensitive and P-gp overexpressing resistant leukemia cell lines [72]. Results demonstrated that while DOX-SLN were as effective as free DOX at inhibiting the sensitive leukemia cell line, they were almost ten times more effective against the resistant cell line. This increased efficacy was shown to be due to a higher retention of DOX inside the cells as a consequence of the Pgp inhibition by Brij®78. Moreover, the same group also demonstrated that the concomitant treatment of the cells with non-loaded SLN and free DOX was as effective as the treatment of DOXloaded SLN against a P-gp overexpressing DOX-resistant ovarian carcinoma cell line (NCI/ADR-RES). This demonstrated that the inhibition of the efflux proteins by the components of the SLN favored the efficacy of the antitumor drug [73]. Similarly, Siddiqui et al. used the same surfactants to prepare stearyl alcohol DOX-SLN and found that while free DOX was not able to inhibit the growth of NCI/ADR-RES cells, DOX-SLN exhibited a potent and concentration-dependent inhibitory effect on them [74]. In addition, these authors studied the effect of targeting the enzyme glucosylceramide synthase (GCS) on the response of NCI/ADR-RES cells to DOXSLN. In order to study this effect, non-loaded SLN were coated with a mixed backbone antisense glucosylceramide synthase oligonucleotide (MBO-asGCS) and administered together with DOXSLN. The results obtained showed that the co-administration of MBO-asGCS SLN and DOX-SLN sensitized the NCI/ADR-RES cells to the action of the drug improving the overall efficacy of DOX-SLN. The overexpression of GCS has been described to enhance the formation of lipid rafts [75] and consequently, to increase the number of receptors and membrane proteins involved in the MDR effect, such as P-gp. Therefore, this strategy of targeting GCS could be useful to improve the efficacy of chemotherapy in most


resistant OS cases, especially in metastatic diseases in which tumor cells overexpress lipid rafts [76]. Another surface modification made on SLN to actively target the tumor cells was the one performed by Jain et al. This group developed mannosylated DOX-SLN with the aim of selectively targeting the lectin receptors expressed on the surface of cancers cells [77]. When tested in human lung and breast cancer cell lines, these nanoparticles were more cytotoxic than non-mannosylated nanoparticles and DOX in solution as well as more efficiently taken up by the cells, promoted probably by the adhesion of mannose to the lectin receptors of the cells. Interestingly, mannosylated DOXSLN were less hemolytic than DOX and non-mannosylated nanoparticles, an effect that was attributed to the shielding of the drug on the one hand and to the masking effect of mannose over the charged amine groups present in the lipid matrix of the nanoparticles on the other. In addition, in vivo studies revealed a prolonged residence time, a slower clearance and therefore a higher bioavailability for mannosylated DOX-SLN in comparison to the nonmannosylated SLN and DOX after their intravenous administration to mice. Regarding the biodistribution of DOX, mannosylated nanoparticles were more effective at targeting the drug to the tumor and avoiding the heart and the kidneys, which, in turn, resulted in a better toxicological profile. This type of tumor targeting may be useful in the treatment of OS, as OS cells can overexpress several lectin receptors, such as Galectin-3, a carbohydrate-binding protein that is exponentially expressed as the disease progresses [78]. 4.1.3. Doxorubicin Nanostructured Lipid Carriers Despite the great advantages offered by SLN over DOX in solution, limitations in drug loading capacity or drug expulsion during storage led to the emergence and development of several DOXloaded NLC. Mussi et al. developed DOX-NLC with a lipid matrix containing solid compritol and liquid docosahexaenoic acid (DHA) or peanut oil and achieving drug encapsulation efficiencies close to 100% [79]. These NLC were evaluated for their potential to overcome drug resistance and to increase DOX efficacy in DOXsensible and resistant breast cancer cells (MCF-7 and MCF-7/Adr cells, respectively). DOX-NLC showed to be at least as effective as DOX in the sensitive cell line but significantly more active than free DOX and even liposomal DOX (Lipodox®) in resistant cells. This improved activity of the NLC in the resistant cell line was attributed to the nanoparticle-mediated inhibition of P-gp activity, since the encapsulation of the drug significantly enhanced its internalization in these cells. Interestingly DHA-containing NLC showed a higher antitumor activity than peanut oil-containing NLC, due to the described ability of DHA to sensitize the tumor cells to the action of anticancer drugs. Same findings were also observed in a more complex MCF-7/Adr spheroid model, in which DOX-NLC showed a higher internalization capacity than the rest of the treatments and DHA-containing NLC exhibited the most potent cytotoxic activity. These results demonstrate the great potential of NLC for penetrating into tumors that are poorly vascularized or difficult to access, as is the case with bone tumors. DOX-NLC have also been modified in order to achieve active targeting of the tumor cells. Zhang and colleagues modified the surface of the particles with the aim of targeting the folate receptors [80], which are highly overexpressed in several cancer cell lines [81,82]. They demonstrated that folic acid-decorated DOX-NLC were more effective at inhibiting the growth of both sensitive and multi drug resistant human breast cancer and ovarian cancer cells than the non-decorated NLC, probably due to the synergistic effect of improving their internalization and inhibiting the P-gp efflux pump [80]. Taking into consideration that folate receptors are frequently overexpressed in OS cells [83], this approach could also be used in OS treatment to target cancer cells and enhance drug uptake.


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In another study, Taratula and co-workers developed NLC decorated with a modified synthetic analog of luteinizing hormonereleasing hormone (LHRH) for the pulmonary delivery of DOX and SiRNA targeted to the resistance proteins P-gp, and Bcl-2 [84]. In an orthotopic murine model of lung cancer inhaled LHRH-NLC accumulated preferentially in the lungs, although to the same extent as the non-targeted nanoparticles. However, while non-targeted carriers distributed uniformly through the lungs, LHRH-NLC accumulated in the tumor tissue of the lung, sparing the healthy areas of the organ. Once inside the tumor cells, LHRH-NLC delivered the SiRNA in the cytoplasm and DOX in both the cell cytoplasm and nuclei. In OS treatment, where lung metastases are the main cause of chemotherapy failure, this targeting strategy and administration route could significantly improve the efficacy of the treatments and the outcome of patients with metastatic disease. 4.2. Methotrexate Lipid Nanoparticles 4.2.1. Methotrexate MTX is an antimetabolite of the family of antifolates. It is a structural analog of folic acid with immunosuppressive and antitumor activity, which has been clinically approved for the treatment of several pathologies such as psoriasis, rheumatologic diseases or cancer. Folic acid is an essential cofactor in DNA synthesis. Its reduction into tetrahydrofolate (THF) is catalyzed by the enzyme dihydrofolate reductase (DHFR) and allows de novo synthesis of purines. The main cytotoxic activity of MTX is related to the reversible and competitive blockage of the enzyme DHFR, reducing the THF pool and leading to the inhibition of DNA synthesis and cellular replication. MTX can enter the cell by two main mechanisms; through a carrier-mediated transport mechanism using the reduced folate carrier (RFC) or by a concentration and timedependent passive diffusion when it is administered at high doses. Once inside the cells, MTX is converted to active MTX polyglutamate by the action of the folylpolyglutamate synthetase (FPGS) enzyme, which adds glutamate residues to MTX. This process prevents the efflux of the drug across the cell membrane by ABC (ATP-Binding Cassette) transporters and therefore, polyglutamated derivates are retained in the cells for longer periods than MTX. Moreover these MTX metabolites are at least as potent DHFR inhibitors as the parent drug and they generally present higher affinities for folate enzymes as well as for other folate-dependent enzymes than MTX [31]. RFC-mediated active transport into the cells could be an advantage for cancer treatment with MTX as most cancer cells overexpress folate receptors. However, most resistant OS cells present a reduced expression of the RFC, which makes them resistant to the action of MTX. To overcome this limitation, high doses of MTX are given during OS therapy to favor the passive diffusion of the drug in a concentration-dependent manner. This affects cancer cells but also normal cells, which incorporate the drug by both passive diffusion and receptor-mediated mechanism. To avoid the toxicity of HD-MTX in normal cells, a MTX antidote, leucovorin, is given to patients after MTX treatment. Leucovorin enters the cells by the RFC, protecting normal cells, but not OS cells that present impaired MTX transport [24]. Nevertheless, resistance to MTX in OS cells could also be due to other several factors, which include increased levels of DHFR, loss of affinity to DHFR, reduced levels of FPGS or the overexpression of ABC drug efflux proteins such as P-gp [85]. Therefore, there is an urgent need for developing new treatment strategies in order to improve the therapeutic efficacy of MTX in OS cells while minimizing its toxic effects in healthy tissues. 4.2.2. Methotrexate Lipid Nanoparticles The use of drug delivery systems carrying MTX is a promising alternative to selectively deliver the drug inside the tumor cells


without the need for RFC and to overcome the active efflux of the drug from the cells, thereby improving its antitumor activity. Battaglia and colleagues developed highly loaded MTX-SLN using the hydrophobic ion-pairing strategy to neutralize the negative charge of MTX molecule and solve its solubility problems in the lipids [86]. In vitro studies in human and rat breast cancer cell lines demonstrated that MTX-loaded SLN were more effective at reducing the viability of the cells than the free drug and the ionpaired drug. Moreover, when intravenously administered, SLN improved the drug plasmatic concentrations, mean residence time and therefore the overall drug bioavailability. Regarding the biodistribution profile and targeting ability of the nanoparticles, in a breast cancer model in rats the administration of MTX-SLN achieved higher drug concentrations in the tumor area than the treatment with the free drug. Therefore, it was concluded that the use of SLN to carry MTX could make it possible to reduce the therapeutic doses in vivo, an aspect that would be very advantageous in the treatment of OS in which high drug doses are required. Interestingly, it was also found that the gastrointestinal absorption of MTX was enhanced as a consequence of its encapsulation into SLN, highlighting the great potential of this type of nanocarriers to improve treatment convenience. In another study regarding breast cancer, Zhuang and colleagues demonstrated higher cytotoxic activity of MTX in its encapsulated form. Besides, in a mouse model of breast cancer, the developed MTX-SLN presented a significantly superior efficacy in inhibiting the tumor growth than the corresponding free drug solution [87]. This improvement in the therapeutic activity of the drug after its encapsulation was also observed by Ruckmani et al., who evaluated the efficacy of MTX-SLN in a model of Erlich Ascite Carcinoma in mice [88]. Once intraperitoneally administered, the pharmacokinetics parameters revealed an increased half-life for MTX-SLN compared to free MTX, a lower elimination rate and a higher mean residence time. This improved pharmacokinetic profile resulted in a prolonged survival time for mice treated with nanoparticles compared to those treated with the same dose of MTX in solution. In addition to the entrapment of MTX in SLN, other authors have studied the feasibility of formulating it as a LDC nanoparticle. Vias et al. developed lipid-MTX conjugate nanoparticles for the oral delivery of the drug by conjugating it with stearic acid and incorporating it into LN [89]. The formulation of MTX in this type of carrier enhanced drug bioavailability and reduced MTXassociated gastrointestinal toxicity, even at the highest doses that are usually administered for OS treatment. Therefore, although they have been less widely studied, MTX-lipid conjugates should also be considered as an attractive alternative for OS treatment. 4.3. Lipid-Based Cisplatin Nanoparticles 4.3.1. Cisplatin CIS is classified as an alkylating-like agent because, although it does not have an alkyl group in its structure, it presents a mechanism of action similar to that of alkylating antineoplastic agents. Once inside the cytoplasm, CIS is hydrolyzed in an active product that reacts with the purine bases on the DNA and interferes with DNA repair mechanisms, causing DNA damage and leading to the blockage of cell division, apoptosis and cell death [33]. In OS, CIS is an essential component of most first-line therapy protocols and it is mainly used in combination with DOX, being reasonably effective in metastatic diseases. However, its intraarterial administration route and its severe side effects, mainly nephrotoxicity and ototoxicity or even secondary neoplasms, hinder the clinical use of this drug [24]. Therefore, as in the case of other first-line drugs in OS therapy, CIS entrapment into drug delivery systems could constitute a major advance for improving the conventional treatment, both in terms of efficacy and patient convenience.


4.3.2. Cisplatin Lipid Nanoparticles Tian and co-workers developed SLN carrying CIS by an emulsification-dispersion-ultrasonication method, paying special attention to the thermolability of the drug and its stability under high ultrasonic power [90]. Biodistribution studies in rats showed that CIS nanoparticles were targeted mainly to the liver while the free drug was mostly accumulated in kidneys, one of the target organs of CIS-associated toxicity. The same biodistribution tendency was also observed for the CIS-SLN developed by Doijad et al. [91], which also showed reduced heart accumulation in comparison to CIS in solution. Therefore, although the field of CIS incorporation into LN has not been studied in depth, the encapsulation of CIS appears to be a potential strategy to reduce the accumulation of the drug in non-desired organs and reduce its side effects. 4.4. Ifosfamide and Etoposide Lipid Nanoparticles 4.4.1. Ifosfamide and Etoposide IFO is a potent alkylating agent that is added to standard chemotherapy in combination with MTX, DOX and CIS for the treatment of high-grade localized, metastatic, and recurrent OS. It is a prodrug that is enzymatically metabolized by the cytochrome P-450 or chemically hydrolyzed in acidic media giving rise to metabolites that are responsible for both the anticancer activity and toxicity of IFO [92,93]. These metabolites react with the DNA molecules forming covalent bonds that trigger apoptosis [94]. The main adverse effect associated with IFO treatment is nephrotoxicity; hemorrhagic cystitis, Fanconi syndrome or acute renal failure being some of the other clinical complications deriving from the high IFO doses used during OS treatment [34]. This has led to the concomitant administration of IFO and mesna, an uroprotective agent that binds and inactivates the IFO metabolites responsible for the renal damage [24]. On the other hand, ETO is another potent drug used in cancer therapy. Although the use of ETO is not considered in all the OS treatment protocols, it is usually co-administered with IFO or DOX to enhance the antitumor activity of the polychemotherapy in the treatment of refractory and metastatic OS [11]. Although its mechanism of action it is not fully understood, it is thought to be due to the formation of ETO complexes with the DNA and the enzyme topoisomerase II, which progressively inhibit the synthesis of the DNA [95]. However, even though it is a second-line drug in OS management, the clinical use of ETO presents various challenges, such as its low solubility, poor bioavailability, emergence of resistance and appearance of systemic side effects, which, taken together, limit the efficacy of ETO in bone sarcomas [96,97]. 4.4.2. Ifosfamide and Etoposide Lipid Nanoparticles The main objective of entrapping IFO into LN is to protect it from its hepatic metabolism and hydrolysis in acidic media, thereby minimizing the generation of metabolites that are responsible for the side effects and avoiding the need for an antidote. Besides, the prolonged drug release achieved by this encapsulation could also decrease the doses required to achieve the therapeutic activity. With this aim, Pandit et al. developed IFO-SLN containing chitosan and glyceryl monooleate in the matrix and crosslinked with sodium tripolyphosphate [98]. The interaction among the different components of the nanoparticle allowed researchers to obtain sustained delivery of IFO and protected the drug from its degradation in acidic media. Moreover, in order to evaluate the suitability of the nanoparticles for their oral administration, permeability studies were carried out using Caco-2 cells. SLN loaded with IFO presented a high cellular uptake, probably by an endocytic pathway, and once inside the cells, the SLN localized in the cytoplasm, where it acted as a drug reservoir. Regarding ETO, its variable bioavailability after oral administration is one of the main challenges standing in the way of its clinical use. In order to overcome this limitation by leveraging the


lipophilicity of the drug, Zhang et al. designed pegylated ETO-NLC and studied their intestinal absorption across a rat intestinal membrane using the diffusion chamber method [99]. Interestingly, pegylated nanoparticles showed a higher transport across the intestine than both the non-pegylated ETO-NLC and the drug in solution, a fact that was thought to be due to a PEG-mediated opening of the tight junctions that could improve the paracellular transport of the nanoparticles. In concordance with this result, the oral administration of ETO-SLN to rats resulted in much higher drug plasma concentrations than the administration of the drug in solution, improving the relative bioavailability of the drug by 2 and 3-fold for nonpegylated and pegylated NLC, respectively. Moreover, the efficacy of the developed nanoparticles was also studied against human epithelial-like lung carcinoma cells, in which a great increase in the cytotoxicity was observed for ETO-NLC. Similarly, ETO-SLN have also been designed for the controlled delivery of ETO in tumor cells. Wang and co-workers studied the efficacy of ETO-SLN in a gastric carcinoma cell line and confirmed their superiority at inhibiting the cell growth [100]. This effect was attributed to the increased lipophilicity of ETO once inside the carriers, which favored its uptake by the cancer cells. Moreover, it was demonstrated that cell death was, at least in part, the result of the DNA damage caused by the drug, which was more pronounced in the cells treated with the SLN. It is noteworthy that SLN have also been shown to improve the efficacy of ETO in metastasized cancers. The ETO-SLN developed by Athawale and colleagues achieved higher drug plasma concentration than the drug in solution and accumulated preferentially in the most perfused organs, especially in the lungs, when administered intravenously [101]. Thanks to this pharmacokinetic performance, ETO-SLN reduced more effectively the number of metastatic lung colonies in a mouse model of melanoma and improved their survival rate. Taking into consideration that one of the main problems of OS is that it metastasizes through the hematogenous route to the lungs, this type of passive targeting could considerably improve the clinical outcome of those OS patients with metastatic disease. Finally, several attempts have also been made in order to actively target the drug to the tumor cells. Ghalaei et al., developed hyaluronate targeted SLN with the aim of directing the cells against the overexpressed CD44 receptors of the tumor cells [102]. As a result, the targeted nanoparticles were more internalized than the non-targeted ones in an epithelial ovarian carcinoma cell line and caused more toxicity in them. This approach could also be useful in OS treatment, as it has been described that CD44 receptors are also overexpressed in OS cells and have been found to be involved in the metastatic cascade [103]. On the other hand, Khajavinia and colleagues decorated ETO-NLC with transferrin, a protein that binds transferrin receptors, in an attempt at selectively targeting the drug to leukemia cells [104]. Cell uptake studies revealed that transferrin-conjugated nanoparticles were internalized more by acute myelogenous leukemia cells than unconjugated ones and showed higher cytotoxic activity. This tumor-targeting ability of transferrin has also been previously observed in OS cells [105], which indicates that this could be a good strategy for OS treatment. 5. LIPID-BASED NANOPARTICLES FOR DELIVERING EMERGING DRUGS IN OSTEOSARCOMA TREATMENT Despite the significant progress made in the survival rate of OS patients during the last decades, it appears that the therapeutic potential of conventional cytotoxic agents employed in the treatment of OS has reached a plateau and different combinations or higher doses are unlikely to improve patient survival. The narrow therapeutic window of conventional antineoplastic agents, the appearance of multidrug resistant phenotypes and the occurrence of secondary tumors and metastases are serious problems that dose intensification has not really solved. Moreover, due to its primary loca-


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tion and its complex genetic background, OS is relatively resistant to drugs that are efficient against other pediatric solid tumors [106]. These particular characteristics, together with the fact that no change in OS survival expectancy has occurred in the past decades, highlight the need to identify new chemotherapeutic agents that are active against this disease. A critical element for developing effective therapies is the identification of key biological mechanisms involved in aggressive OS. 5.1. Novel Targets and Strategies for Osteosarcoma Therapy The intensive investigations focusing on basic tumor biology and pathogenesis of OS, as well as on drug development, have led to the discovery of new therapeutic targets and a variety of active agents have emerged as promising alternative therapeutics for the treatment of this cancer (Fig. 4) [107]. In this sense, several signaling receptors and transduction pathways, as well as external factors related to the tumor microenvironment, have been shown to play a critical role in OS development and in its malignant behavior, and the investigation of agents that can inhibit these routes and interactions has attracted special interest. 5.1.1. Inhibition of Receptors and Signal Transduction Pathways As occurs in most cancers, many signaling molecules and pathways are activated, mutated, overexpressed or downregulated during OS development. Understanding these signaling pathways and identifying the critical pathways is vital to develop inhibitors that may aid in personalizing therapeutics to increase patient survival. An intense focus of study has been the mammalian target of rapamycin (mTOR) signal transduction pathway. This pathway is activated downstream of multiple receptor tyrosine kinase pathways, particularly through Akt, and is known to play an important role in tumorigenesis and to contribute to the chemoresistance of several cancers [108]. mTOR, a serine/threonine protein kinase expressed in most OS cells, is involved in protein synthesis and cell proliferation and differentiation, and it is associated with a poor prognosis in OS patients [109]. Rapamycin, also known as sirolimus, is a macrocyclic lactone antibiotic that specifically inhibits mTOR, preventing OS cell migration and invasion, as well as promoting their apoptosis [110-112]. The Pediatric Preclinical Testing Program (PPTP), a consortium of laboratories for investigating new agents with significant activity in several cell lines and pediatric preclinical cancer models, demonstrated a significant efficacy of rapamycin in delaying tumor growth in a murine OS xenograft model [113], and other studies have also proved the inhibition of OS lung metastasis development with rapamycin [114]. Currently, several clinical trials are ongoing with the aim of evaluating the role that several rapamycin analogs such as temsirolimus [115], ridaforolimus [116,117] and everolimus [118,119] may play in OS. Moreover, the possible benefits of the combinations of rapamycin and its analogs with other chemotherapeutic agents are also under study [120,121]. The alkyl-lysophospholipid perifosine is another inhibitor of the mTOR signaling pathway that acts through a mechanism different from rapamycin and its analogs [122]. Although the mechanisms by which it exerts the antitumor effect have not been fully elucidated, among other effects, perifosine blocks the activation of Akt/mTOR Complex-1 and induces the pro-apoptotic caspase-3 causing apoptosis in OS cells [123]. Moreover, it has been observed that perifosine significantly enhances OS cell sensitivity to the anti-OS drugs ETO and DOX [123], and a synergistic activity has also been found with rapamycin in other cancer cell lines [124]. Ongoing clinical trials are now studying the safety and effectiveness of perifosine as a single agent [125] or in combination with the rapamycin analog temsirolimus in children with recurrent pediatric solid tumors [126]. The implication of type-I insulin-like growth factor receptor (IGF-IR), a transmembrane tyrosine kinase with a central role in



Fig. (4). Schematic overview of the new targets identified in osteosarcoma development and associated investigational agents for therapeutic intervention, including: agents that target molecular pathways in osteosarcoma; that interact with the microenvironment of the tumor; and that present mechanisms of action different to those of conventional cytotoxics or that overcome the resistance mechanisms associated with them. Abbreviations: IGF-IR = Insulin growth factor receptor-1, Her-2 = Human epidermal growth factor receptor-2, HIF-1 = Hypoxia inducible factor-1, L-MTP-PE = Liposomal muramyl tripeptide phosphatidyl ethanolamine, mTOR = mammalian target of rapamycin, PDGFR = Platelet-derived growth factor receptor, PI3K = phosphatidylinositol 3-kinase, VEGF = Vascular endothelial growth factor, VEGFR = Vascular endothelial growth factor receptor.

normal bone growth, and its signaling pathway, is also being widely studied as a potential target for OS treatment [127]. IGF-IR has found to be overexpressed in most OS cell lines and clinical samples [128] and its signaling pathway seems to be important for the evolution of OS metastasis [129]. The PPTP showed that robatumumab (SCH 717454), a fully human neutralizing anti-IGF-IR antibody, significantly increased event-free survival in murine xenograft OS [130], and Wang et al. also observed tumor growth inhibition as well as an antiangiogenic effect in another murine xenograft OS model [131]. Since then, different anti-IGF-IR human monoclonal antibodies have been evaluated in clinical trials for their safety and activity against bone sarcomas. Cixutumumab (IMC-A12) has shown encouraging results in early phase clinical trials, and has been found to be well tolerated in children [132], although the activity of this monoclonal antibody was observed to be limited when used as a single-agent. Several studies suggest that IGR-IR inhibitors have modest activity as a single-agent in patients with relapsed and/or refractory OS [132,133], but ongoing studies aim to evaluate their impact in combination therapies. Interestingly, synergistic activity of IGF-IR and mTOR inhibitors has been described in murine xenograft OS models, achieving complete regression of tumors in 3 out of 4 studied OS cases [134]. In a further step, phase I and II clinical trials have reported significant clinical activity of these combinations, with a similar tolerability to that observed for the individual agents and with no drug-drug pharmacologic interactions [135,136]. These findings suggest that

targeting the different signaling pathways implicated in OS development could be an effective strategy for the treatment of this cancer. Similarly, IGF-IR inhibitors have also shown good tolerability in combination with conventional chemotherapeutics such as docetaxel [137] and synergistic activity with cyclophosphamide in murine models of xenograft OS [131]. Moreover, recent studies have shown that IGF-IR inhibitors enhance antitumor activity of DOX both in DOX-sensitive and -resistant OS cell lines [138,139]. Other tyrosine kinases which have been studied in OS are the human epidermal growth factor receptor-2 (EGFR-2 or Her-2) and the non-receptor Src tyrosine kinase. Src is a key molecule in osteoclast biology that has been found to be highly activated in highly-metastatic OS cell lines [140]. Dasatinib, a Src-targeting drug, inhibits cell adhesion, migration and invasion and induces apoptosis of OS cells in vitro [141] but failed to inhibit the development of pulmonary metastasis in a spontaneous metastatic mouse model and had no effect in the primary tumor [142]. In similar studies of the PPTP, no objective response to dasatinib treatment was observed in different murine OS xenograft models, although a statistically significant longer event-free survival was found in three out of the five studied models [143]. These findings reveal that the convergence and crosstalk of more than one pathway may be responsible for the development of metastasis and the progress of OS. Therefore, they highlight the need to block more than one signaling cascade, as well as to study the role of the inhibitors in combined therapies in order to achieve a therapeutic benefit in OS patients.


Currently, a phase I/II trial is ongoing in order to study the side effects, optimal dose and therapeutic activity of dasatinib given together with IFO, carboplatin, and ETO in young patients with metastatic or recurrent malignant solid tumors, including OS [144]. Regarding Her-2, several studies have associated its expression with a higher frequency of metastasis, a lower histologic response to preoperative chemotherapy and a worse prognosis [109,145,146]; however, its clinical significance remains controversial. Some researchers have found minimal membrane Her-2 expression in OS biopsy specimens and the Her-2 expressed in the cytoplasm was not correlated with the progression of the disease [147-149]. Trastuzumab, a humanized mouse monoclonal antibody against Her-2, has already been studied in a phase II clinical trial in patients with metastatic OS. Results showed no statistical differences in overall survival between the group of patients overexpressing Her-2 and treated with standard chemotherapy plus trastuzumab and the Her-2 negative group receiving only standard chemotherapy [150]. This lack of therapeutic benefit could be attributed to the negative prognosis effects associated with the overexpression of Her-2 and therefore, a randomized study of patients with Her-2-positive disease would be necessary in order to define the therapeutic potential of trastuzumab. Several pathways are being studied in the search for new molecular targets against OS; however more clinical trials and detailed studies are required before these potential new drugs can be included in the current OS protocols. 5.1.2. Interaction with the Tumor Microenvironment Exploration of the tumor microenvironment has become an important strategy against several cancers, as it might allow the identification of key parameters critical for disease progression that are less affected by tumor cell heterogeneity and subtype. The tumor microenvironment includes both cancer and stromal cells, and the communication network between them, via the secretion of growth factors and chemokines. This complex relationship leads to an altered extracellular matrix and the recruitment of new vasculature and stroma, which are vital to the survival, growth and metastatic potential of the tumor [151,152]. Therefore, therapies targeted at disrupting this interaction may be efficacious in OS. Solid tumors require new blood vessel formation for spreading and growth at both primary and metastatic sites and therefore, a potential target of therapy is to inhibit the tumor blood supply by inhibiting tumor angiogenesis [153]. VEGF is one of the most powerful promoters of new blood vessel formation, which, in turn, is the main exit route of tumor cells from the primary tumor site to the systemic circulation. In OS, VEGF expression has been correlated with increased local microvessel density, metastization and poor prognosis [154-156]. The anti-VEGF monoclonal antibody bevacizumab (Avastin®) is already approved by the FDA for its use in some adult malignancies and has shown encouraging results in OS, including synergistic activities with liposomal DOX and the mTOR inhibitor temsirolimus [120]. Currently there is an ongoing phase II clinical trial studying the benefits of including bevacizumab in combination with conventional chemotherapy for the treatment of OS [157]. Other antiangiogenic agents include the antibodies against VEGF receptors (VEGFRs). Sorafenib is an orally active multikinase inhibitor targeting tyrosine kinase VEGFR and plateletderived growth factor receptor (PDGFR) that has demonstrated tumor growth inhibition, antiangiogenic effect and reduction of metastatic potential in preclinical models of OS [158]. Moreover, sorafenib (Nexavar®), which is approved by the FDA for the treatment of kidney and liver cancer, has displayed clinical activity and an acceptable toxicity profile in a phase II clinical trial in patients with advanced and unresectable high-grade OS [159]. On the basis of the results obtained in preclinical models, in which combination of sorafenib with the mTOR inhibitor everolimus leads to boosted antitumor, antiangiogenic, and antimetastatic effects [160], several


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clinical trials have been planned to study the benefits of sorafenib in combination therapies [161-163]. The interaction between tumor cells and bone-resorbing osteoclasts has also an important role in the growth of bone tumors and the development of metastasis [164]. Bisphosphonates have an affinity for hydroxyapatite on bone surfaces and are delivered to sites of increased bone formation or resorption, where they exert an inhibitory effect on osteoclast-mediated bone resorption. For this reason, bisphosphonates are being widely studied for the prevention of bone tumor metastasis and primary tumor progression. Their mechanism of action, however, goes further and also involves osteoblastic cells, macrophages and tumor cells [165]. In vitro and in vivo OS models have demonstrated that the nitrogen-containing bisphosphonates, such as zoledronic acid and alendronate, inhibit tumor growth, progression and lung metastasis not only through the activation of apoptosis and inhibition of OS cell proliferation, but also by inhibiting tumor-induced angiogenesis and the invasive potential of the tumor cells [166-168]. Moreover, zoledronic acid also attenuates tumor-induced osteolysis both by direct inhibition of osteoclasts and through direct actions on tumor expression of osteoclast activators [166]. A recent study of the Children's Oncology Group (COG), the world's largest organization devoted exclusively to childhood and adolescent cancer research, has concluded that zoledronic acid can be safely combined with the conventional chemotherapy used to treat metastatic OS in patients with newly diagnosed high grade metastatic OS [169]. Similarly, in a phase II study conducted at Memorial Sloan-Kettering Cancer Center, the combination of conventional chemotherapy with the second generation bisphosphonate pamidronate has shown a favorable safety profile in adolescent OS patients and may improve the durability of limb reconstruction [170]. At the moment, there are two clinical trials evaluating the therapeutic effect of zoledronic acid as a single agent or as an adjuvant to chemotherapy in OS [171,172]. Finally, there is also evidence supporting the notion that zoledronic acid boosts the effect of the conventional antitumor drugs as well as the mTOR inhibitors, underlining the versatility of these agents [173-175]. Another strategy to exploit the interaction of tumor with the microenvironment to treat chemoresistant tumors is the use of inmunomodulators to stimulate the immune system and activate its tumoricidal function. In this field, muramyl tripeptide phosphatidylethanolamine (MTP-PE), a non-specific macrophage activator, has attracted special interest, and its liposomal formulation (L-MTP-PE) has been intensively studied for its role in the treatment of OS [176]. The latest studies by the COG concluded that the addition of MTP to post-operative chemotherapy resulted in enhanced overall survival in newly diagnosed patients with OS [177-179]. Owing to these favorable results, L-MTP-PE (mifamurtide or MEPACT®) was approved by the European Medicines Agency in 2009 for newly diagnosed, non-metastatic OS in conjunction with chemotherapy, being the first new drug approved for the treatment of OS since the introduction of IFO more than 20 years ago. 5.1.3. Overcoming the Resistance to Conventional Chemotherapy Resistance to chemotherapy is one of the main causes of treatment failure in OS, and understanding the mechanisms of resistance to current therapies is essential for the development of agents that circumvent these mechanisms. As discussed above, while MTX has been a cornerstone of OS therapy, tumor cells respond poorly to conventional doses of this drug due to its impaired transport into the cells, enhanced drug efflux from cells, reduced polyglutamation and therefore cellular retention, mutations in DHFR or increased expression of DHFR. In this context, intensive research has been focused on the development of new antifolates to overcome different aspects of MTX resistance [180,181]. Trimetrexate is a lipophilic DHFR inhibitor that enters cells by a non-energy-dependent process and therefore does not require the reduced folate carrier for entry into the cell. Recently, two clinical trials studying the clinical activ-


ity of trimetrexate in newly diagnosed and refractory or recurrent OS have been completed [182,183]. Early data in patients with refractory OS showed a 13% response rate in 38 patients and nonoverlapping toxicity for trimetrexate and methotrexate [184]. Another new antifolate drug is pemetrexed, a multitarget antifolate involved in DNA synthesis and folate metabolism [185]. However, despite its wider range of activity than MTX, several OS cell lines have shown a higher resistance to pemetrexed than to MTX [186]. Similarly, clinical trials with pemetrexed have shown little singleagent activity of the drug in patients with refractory, advanced or metastatic high-grade OS, but a good tolerability even in children and adolescents [187,188]. Recently, a good tolerability for the pemetrexed and CIS combination has also been demonstrated in refractory/metastatic OS patients [189], which indicates that pemetrexed could be included in combinational therapies for further therapeutic activity studies. Another phenomenon closely related to drug resistance is the increased efflux of cytotoxic drugs out of the tumor cells through specific energy-dependent transporters, which is the main cause of MDR. The best known drug efflux transporter is P-gp, a member of the ABC family which is involved, among other things, in DOX and MTX resistance in tumor cells, including OS cells [190,191]. In recent decades several ABC transporter inhibitors have been studied for their role in cancer and among them, curcuminoids and particularly curcumin have attracted special interest. Curcumin is a natural phenolic compound extracted from Curcuma longa that has been proved to reverse the MDR and sensitize cancer cells through the inhibition of P-gp and other ABC transporters [192,193]. Although this effect has not been studied in depth in OS, recent studies indicate that curcumin down-regulates P-gp expression in multidrug-resistant OS cells both in vitro and in vivo, increasing their sensitivity to several antitumor drugs and inhibiting their proliferation, invasion and metastization [194]. Interestingly, curcumin has also been demonstrated to possess intrinsic antitumor activity in several tumor cell lines, including OS cells. Curcumin induces caspase-mediated cell apoptosis of OS cells and inhibits cell migration and invasion through the inactivation of the notch-1 signaling pathway [195-197], these effects being found in OS cells but not in normal osteoblasts [198]. Currently, there is an ongoing clinical trial that aims at studying the safety and efficacy of curcumin in high grade relapsed or metastatic OS patients who are not receiving any other second-line chemotherapy [199]. As a complement to these strategies, and given the rising resistance of tumor cells to conventional cytotoxics, OS treatment research has also focused on the screening of cytotoxic drugs with a favorable efficacy versus side-effect profile and a mechanism of action different from standard first-line agents. The most widely investigated agents are gemcitabine and docetaxel. Gemcitabine is a fluorinated analog of the nucleoside deoxycytidine that in its active form inhibits ribonucleotide reductase and interferes with DNA chain synthesis [200]. Docetaxel is a semisynthetic taxane that stabilizes the microtubules, which results in the inhibition of mitosis and cell cycle arrest, and that also presents antiangiogenic properties [201]. The treatment with gemcitabine followed by docetaxel has shown synergistic effects in vitro and seems to be a welltolerated combination when administered to OS patients [202,203]. More importantly, retrospective reviews of children and young adults with refractory and/or relapsed OS have revealed a partial response in up to 50% of the patients, depending on the docetaxel and gemcitabine doses and cycles administered, and some patients even achieved a complete remission of the disease [202-205]. These are encouraging results that suggest that the gemcitabine-docetaxel combination could be considered for formal evaluation as a secondline therapy in patients with recurrent or refractory high-grade OS.


5.2. Lipid Nanoparticles with Promising Agents for Osteosarcoma Treatment Despite the clinical potential of the aforementioned molecules in the treatment of OS, several pharmacological obstacles limit their successful clinical translation. Poor water solubility, low stability, the induced systemic toxicity and the need to achieve high concentrations in the target area are some of the factors that hamper the success of these agents in cancer therapy. Formulation of these molecules in lipid nanocarriers is a promising strategy to improve their stability, limit the induced toxicity and achieve high concentrations of the agents in their site of action, and therefore, to further exploit their therapeutic potential. Moreover, the monoclonal antibodies that have shown a therapeutic potential in OS can also be used to decorate the nanoparticles in order to both target the cancerous cells and induce an antitumor response. The encapsulation of these potentially relevant new anticancer agents is still an emerging field and to date, only a few of these molecules have been encapsulated into lipid carriers, gemcitabine and docetaxel being the most extensively studied drugs. Interestingly, some of the lipid nanoformulations developed have already been tested in a wide variety of cancers, revealing great therapeutic potential for overcoming the problems currently associated with these molecules. The most representative examples of lipid nanocarriers containing potential drugs for OS treatment are discussed in more detail below. 5.2.1. Gemcitabine Loaded Lipid Nanoparticles Despite being a first-line treatment for cancers such as pancreatic cancer, gemcitabine presents a low bioavailability and suffers from rapid development of acquired resistance and an extensive first pass metabolism, which leads to a short half-life in the bloodstream [206]. Lipidic gemcitabine nanocarriers have shown several advantages over gemcitabine solution, especially in terms of stability and overcoming drug resistance, in several cell lines and murine xenograft cancer models. The effects of encapsulating lipophilic derivatives of gemcitabine in lecithin/glyceryl monostearate SLN on cancer cells resistance to gemcitabine have been extensively studied by Lansakara and co-workers. Nanoparticles containing different lipophilic monophosphorylated gemcitabine derivatives have been shown to be more cytotoxic than the original drug in gemcitabine resistant cancer cells that are deficient in deoxycytidine kinase or human equilibrative nucleoside transporter, or that overexpress ribonucleotide reductase M1 (RRM1) or M2 subunits [207]. Among the nanoparticles developed, stearoyl gemcitabine SLN were able to overcome the resistance related to the overexpression of RRM1 not only in cell culture but also in a murine xenograft model of gemcitabine resistant lung cancer [208]. This ability was attributed to the mechanism of entry of the SLN, via clathrin-mediated endocytosis, which targets the drug to the lysosomal compartment, where gemcitabine is hydrolyzed from stearoyl gemcitabine and exported out to be then efficiently converted into its active metabolite [209]. Pegylation of these nanoparticles further improved the in vivo performance of the drug, resulting in a higher accumulation of the drug in the tumor tissue and a prolonged blood circulation time. However, this favorable pharmacokinetic profile was not translated into an enhanced antitumor activity [210]. In another attempt to improve their antitumor efficacy, stearoyl gemcitabine SLN were conjugated to epidermal growth factor, a ligand for epidermal growth factor receptor (EGFR) [211]. The cellular uptake and cytotoxicity of these nanoparticles increased with higher EGFR density on tumor cells and SLN accumulated to a great extent in the tumors, which finally led to effective control of the tumor growth. Taking into consideration that most recurrent and metastatic OS express EGFR, this type of targeted delivery of the drug seems to be an interesting approach for increasing the efficacy of anti-OS therapies and could be a good starting point for developing lipid nanocarriers targeting different


aspects of OS cells and the tumor microenvironment. Other approaches with gemcitabine formulations such as self-assembling nanoparticles of gemcitabine-lipid conjugates have also led to improved drug activity. Recently, the self-assembling properties of stearoyl gemcitabine have been exploited to produce 340 nm particles after the emulsification of a drug organic solution in water [212]. These nanoparticles slowly released the drug and were more cytotoxic than the parental drug in pancreatic cancer cell lines, probably due to their more effective cellular uptake. Similarly, the Couvreur group showed that due to its amphiphilic properties, squalenoyl gemcitabine has the ability to form nanoassemblies of 130 nm in water, and this formulation strategy effectively overcomes the resistance mechanisms to gemcitabine. After its administration, both oral and intravenous, this gemcitabine formulation exhibited a more potent activity than the free parent drug in several gemcitabine resistant and non-resistant xenograft solid tumors and metastatic leukemia in mice, but its toxicity did not increase [213215]. The main reasons behind this higher efficacy were that nanoassemblies (i) protected the drug from degradation by deaminases, thus improving its half-life, (ii) improved the pharmacokinetic and biodistribution profiles of the drug, and (iii) led to much higher intracellular accumulation and retention of the drug in the cancer cells, due to their nucleoside transporter independent entry into the cells [216]. 5.2.2. Docetaxel Loaded Lipid Nanoparticles Apart from being a promising candidate for OS treatment in combination with gemcitabine, docetaxel is considered one of the most potent chemotherapeutic agents of the last decade. However, major problems associated with it include its systemic toxicity, mainly myelosuppression, and low water solubility. To deal with this, the available commercial docetaxel formulations (Taxotere®, Duopafei®) include a mixture of surfactants and alcohols to solubilize the drug, which also contributes to the overall toxicity of the preparation [217]. Lipid nanocarriers of docetaxel represent an advantageous alternative as they can improve the drug solubility, reduce its toxicity and preferentially accumulate in the tumor tissue by passive or active targeting [218]. In the last ten years, several SLN and NLC have been developed in order to improve the clinical use of this drug. Due to docetaxel’s lipophilicity, lipid nanocarriers have shown a high capacity to entrap the drug without needing to use alcohol and toxic co-solvents or evaporating them. Taken together with the more favorable pharmacokinetics and biodistribution behavior, this has contributed to reduce the toxicity of the treatment and to increase its tolerability and therapeutic window compared to commercial formulations [219,220]. Moreover, due to the EPR effect, these carriers demonstrated a significant selective accumulation of the drug in the tumors and a comparable or more potent in vivo antitumor activity than Taxotere® in murine xenograft models of ovarian cancer [220], colon carcinoma [221], breast cancer [222] and melanoma [223]. Importantly, while clinical formulations of docetaxel are administered intravenously, inclusion of lecithin in the formulation or surface coating of the nanoparticles with Tween® 80 or TPGS 1000 enabled effective oral administration of docetaxel carriers, leading to higher plasma concentrations than orally administered Taxotere® [224,225]. This effect was attributed to the protection of the drug from gastrointestinal degradation, the bioadhesive properties and ability to improve the nanoparticles’ affinity with the intestinal membrane of lecithin and the inhibition of P-gp-mediated efflux activity by the surfactants. Other surface modifications made to docetaxel lipid nanocarriers include their functionalization with antibodies against receptors such as Her-2 [226] and VEGFR-2 [227] which, as mentioned before, seem to play a major role in osteosarcomagenesis. Incorporation of an antiVEGFR-2 ligand was performed as a strategy of double-targeting both tumor cells and tumor neovasculature endothelial cells. Targeted nanoparticles accumulated to a greater extent in VEGFR-2 overexpressing cells than in deficient cells and achieved a much


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stronger antitumor effect than Duopafei® and non-targeted NLC. The higher docetaxel concentrations found in tumor tissue after treatment with targeted NLC suggest that apart from the EPR effect, which also allowed a high accumulation of docetaxel when encapsulated in non-targeted NLC, active targeting of the NLC with the anti-VEGFR-2 ligand also contributed to improve the therapeutic efficacy of the system [227]. On the other hand, surface decoration of LN with the anti-Her-2 antibody trastuzumab resulted in a higher uptake of the drug by breast-cancer cells overexpressing Her-2 than by the non-tumor breast-cell line, in which there was no significant difference in uptake between the Trastuzumab modified and the nontargeted nanoparticles [226]. These types of carriers with specific recognition properties represent a promising strategy for the selective targeting of tumors overexpressing different receptors and localized in areas with difficult access, such as OS, reducing thereby the exposure of healthy areas of the organism to a toxic concentration of antitumor agents. 5.2.3. Curcumin Loaded Lipid Nanoparticles Despite its remarkably good tolerability, as stated before, curcumin presents a low oral bioavailability due to its poor gastrointestinal absorption and stability, low solubility, rapid metabolism and rapid systemic elimination, resulting in suboptimal blood concentrations to achieve therapeutic effects [228]. In order to address these limitations, lipid-based nanocarriers have received particular attention due to their ability to encapsulate lipophilic drugs, their high intestinal permeability and good oral bioavailability. After studying the solubility of curcumin in different lipids, Sun et al. efficiently loaded curcumin into SLN by high pressure homogenization and showed that the encapsulation process greatly improved the chemical stability of curcumin [229]. Moreover, cellular uptake studies performed in a breast cancer cell line revealed that while curcumin in solution was both internalized and eliminated rapidly from the cells, curcumin SLN exhibited a progressive internalization into the cells, as well as a more prolonged cytotoxic activity. In another set of experiments, curcumin SLN were successfully developed and their cytotoxic effect on human leukemia, lung carcinoma and prostate cancer cell lines was studied [230]. The encapsulated curcumin showed a more potent antiproliferative effect than curcumin in solution and induced a significantly higher apoptotic activity, cell cycle arrest and caspase-9 and 8 activity, all these effects being attributed to the enhanced cellular uptake of curcumin after its encapsulation into the SLN. Moreover, pharmacokinetic studies after the oral administration of curcumin to rats revealed a significant improvement in bioavailability, by at least 32 times, for the encapsulated curcumin, along with a prolonged circulation time and reduced clearance from the systemic circulation [231]. This ability of SLN to overcome curcumin’s limitations has also been recently studied by Wang et al. both in vitro and in mice bearing human lung cancer xenografts [232]. The curcumin SLN developed presented a high stability in aqueous solutions, being able to protect the drug from its rapid degradation and biotransformation. Besides, curcumin nanoparticles were found to be at least 4 times more potent than the free drug, inhibiting the proliferation of two lung cancer cell lines, which correlated with the higher induction of apoptosis by the SLN. When administered to mice bearing human lung cancer xenografts, the curcumin concentration was greatly increased by the nanoparticles in all the tissues studied, especially in the tumor, where no detectable drug levels were found after the oral administration of curcumin in solution. Curcumin SLN significantly inhibited tumor growth compared to free curcumin, which failed to slow down the tumor progression. Immunohistochemistry studies confirmed that the mechanism behind this inhibition was mainly apoptosis, which is more favorable for tumor treatment than necrosis as it is less associated with local inflammatory reactions. Interestingly, apart from the tumor tissue, curcumin SLN yielded a high accumulation of the drug in the lungs, which indicates that SLN could boost curcumin’s antitumor effect in this organ and therefore


be useful for the treatment of lung cancer or lung metastasis derived from other cancers such as OS [232]. Finally, Zhao and co-workers have found that the co-delivery of curcumin (as a chemosensitizer) and DOX (as a chemotherapeutic agent) with LN results in a synergistic activity between them [233,234]. Experiments performed on the resistant BEL 7402/5-FU and HepG2 hepatocellular carcinoma cell lines showed an enhanced growth inhibition after treatment with DOX/curcumin LN in comparison to both DOX-LN and the combination of the free drugs, but a reduced cytotoxicity on the immortalized non-tumor cell line L02. Besides, when administered in diethylnitrosamine-induced hepatocellular carcinoma mice, DOX/curcumin-LN significantly reduced the tumor growth and the liver damage caused by diethylnitrosamine compared to DOX-LN treatment. This synergistic effect in vivo was attributed to the MDR reversal by curcumin and the consequently increased apoptosis and reduced proliferation and angiogenesis of the tumor after the DOX/curcumin-LN treatment. This ability of LN to simultaneously deliver different antitumor drugs appears as a promising strategy to improve current cancer treatment protocols. 5.2.4. Lipid Nanoparticles Containing other Potential AntiOsteosarcoma Drugs Other drugs that have also shown promising activity in OS treatment include alkyl-lysophospholipids and new antifolates. Although no studies regarding the encapsulation of new antifolates have been published to date, due to their high lipid solubility and their improved antitumor properties, MTX analogs appear as good candidates for delivery in LN. Although they were designed to overcome the most common resistance mechanisms of MTX, these antifolate analogs also present certain limitations that reduce their antitumor potential. Trimetrexate for instance, is a lipophilic quinazoline derivative which was developed in order to overcome MTX resistance mediated by impaired drug transport. However, despite being a more potent inhibitor of DHFR than MTX, as a P-gp substrate and due to lack of polyglutamylation it is not retained long within the cell, and it has been suggested that more prolonged exposures to this drug are required to produce optimal anticancer effects [180,181]. In this sense, encapsulation of trimetrexate in LN could provide sustained intracellular drug concentrations and therefore help to boost its DHFR inhibitory activity. Pemetrexed, on the other hand, is a thymidylate synthase inhibitor as well as a DHFR inhibitor. The superiority of pemetrexed over MTX seems to be related to its rapid polyglutamylation and the ability of pemetrexed to inhibit several key folate-dependent enzymes [235]. However, like MTX, permetrexed is transported into the cells via a carriermediated process and can therefore be affected by mutations in folate transporters. Taking advantage of their different mechanism of entry, LN could help to overcome impaired transport and lead to high intracellular drug concentration. Moreover, as already observed for MTX, LN could diminish the systemic toxicity generated by these new antifolate analogs. In contrast, alkyl-lysophospholipids, and in particular edelfosine, have been successfully entrapped into LN and tested in several cancer cell lines and mouse models. Edelfosine, which present a similar structure to that of perifosine (discussed previously, see section 5.1.1), is a prototypical member of the family of synthetic lipids known as alkyl-lysophospholipids, which have been intensively studied as potential antitumor agents against several malignancies. Despite its potent antitumor activity in cell lines, clinical trials with this drug have shown mild effects in vivo and present a dose-dependent hemolysis as a major side effect [236,237]. In addition, when administered orally, edelfosine presents low bioavailability and gastrointestinal toxicity [237]. The benefits of encapsulating edelfosine in SLN have been extensively studied by our research group [238-243]. Our work has shown that SLN have the ability to improve the relative oral bioavailability of the drug by 1500% while providing a protective effect against edelfosine toxic-


ity, even at doses as high as 120 mg of drug per Kg, at which the free drug was lethal [238,239]. The cytotoxic effect of the drug against several cancer cell lines was also considerably enhanced when encapsulated in nanoparticles [240-242] and in vivo, in a murine lymphoma xenograft model, the treatment of mice with nanoparticles every four days reduced the tumor weight more efficiently than the daily administration of edelfosine solution [238]. It is worth noting that while mice treated daily with edelfosine in solution presented an extranodal dissemination of the tumor, the administration of SLN every four days completely eradicated the metastization process of the tumor. Currently, the role of edelfosine SLN in the treatment of primary OS and its lung metastases is being studied by our research group. Preliminary work studying the cytotoxicity of this drug in primary human OS cells showed that edelfosine is more cytotoxic against the cells extracted from lung metastases than against the cells extracted from the corresponding primary bone tumor [243]. Interestingly, the encapsulated drug was five times more cytotoxic against the metastatic cells than free edelfosine, suggesting that edelfosine SLN could be a promising system for the future treatment and prevention of OS metastasis. On the other hand, a novel antitumor agent that has already shown good potential for the treatment of murine metastatic OS is honokiol, a natural compound derived from the Magnolia tree [244]. In the experiments by Steinmann et al. honokiol presented a high cytotoxic activity against several highly metastatic OS cell lines and an inhibitory effect on cell proliferation and migration in vitro. In mice bearing a subcutaneous tumor with spontaneous lung and liver metastasis, honokiol demonstrated antimetastatic activity by significantly reducing the metastization of OS cells to the lungs and liver [244], and although it did not inhibit the primary tumor growth, honokiol has been shown to inhibit the growth of a primary prostate cancer in the bone and induce the death of cells constituting the tumor microenvironment while partially restoring the normal bone histomorphology [245]. Although the mechanism of action behind the anti-OS activity has not been elucidated, honokiol has been shown to have a potent anti-angiogenesis activity and to target multiple signaling pathways, including the Src and EGFR signaling [246,247] the VEGFR signaling pathway [248] and the mTOR pathway [249] which, as mentioned before, play a key role in OS development. On the other hand, honokiol has also shown the ability to overcome chemoresistance in several cancer cell lines, to down-regulate P-gp expression and to boost the antitumor activity of other chemotherapeutic drugs [250-252]. However, despite the good anti-cancer properties of honokiol and its interest as a novel chemotherapeutic agent, the clinical use of this agent is hampered by its poor water solubility. Nanotechnology has provided the technology to address this problem and several drug delivery systems entrapping honokiol have been developed, including lipid-based systems [253,254]. Leveraging its lipophilicity, honokiol has been solubilized in lipid mixtures and entrapped with very high efficiency in lipid nanoemulsions [254]. Taking into consideration the fact that a lipidic emulsion is the first step towards obtaining lipid particles, we may presume that honokiol might also be efficiently loaded into LN, offering the advantage of more stable long-term storage. The developed honokiol lipid emulsion led to higher plasma concentrations than the drug in solution, as well as higher accumulation and retention in the tumor and the lungs. This improved pharmacokinetic profile of the lipid emulsion allowed greater inhibition of tumor growth, demonstrating that honokiollipid systems hold promise for clinical applications. Finally, over the last few years, multifunctional nanoparticles that enable simultaneous cancer therapy and diagnosis, so-called “theragnosis”, have been investigated. In this sense, Parhi and Sahoo have recently developed multifunctional LN containing the mTor-inhibitor rapamycin (as the chemotherapeutic agent) and quantum dots (as imaging agents), and have combined the theragnostic and tumor targeting strategies by decorating the nanoparti-


cles with the anti-Her-2 antibody trastuzumab [255]. The targeting approach considerably improved the internalization of the nanoparticles in Her-2 positive breast cancer cells without altering it in Her2 negative cells. As a consequence, when tested in Her-2 overexpressing breast cancer cells, the targeted nanotheragnostic system developed presented a higher cytotoxic activity than the untargeted nanoparticles. Moreover, targeted multifunctional nanoparticles exhibited a good imaging potential in breast cancer cells grown in monolayers and as tumor spheroids. Confocal studies showed high penetration ability of the targeted lipid nanoparticles into the spheroids, demonstrating the suitability of the system for tumor imaging and treatment purposes. Given the potential implication of the molecules integrated in this lipid nanosystem in the management of OS, these findings provide the basis for creating a useful tool for monitoring tumor response to antineoplastic agents as well as for early detection of metastasis formation. CONCLUSION Despite the great improvement brought about by the introduction of chemotherapy, there has been little advance in OS survival rates over the past decades. Moreover, the antitumor drugs and treatment protocols currently used for OS treatment have failed to achieve further clinical improvements, but do cause severe systemic adverse effects. These data suggest that new efforts need to be made in order to improve the prognosis for the 30% of OS patients who currently have a fatal outcome. LN represent a promising strategy to increase the therapeutic efficacy of conventional drugs by selectively targeting the drug to the cancer cells, thereby increasing drug bioavailability inside the tumor while sparing healthy tissues and the associated adverse toxic effects. As stated above, the encapsulation of the conventional anti-OS drugs DOX, MTX, CIS, IFO and ETO into different LN (SLN, NLC and LDC) has considerably improved their efficacy in the treatment of several cancers. The reasons behind this success are many and are related to the targeting properties of these carriers as well as to their ability to overcome the most common drug resistance mechanisms. Thanks to their small sizes, nanoparticles can be passively accumulated within the tumor tissue by a mechanism known as the EPR effect. Besides, the chemical modifications of the surface of these carriers and the addition of targeting moieties, have allowed more selective and efficient targeting of the drugs to the tumor sites. Once in the tumor microenvironment, LN allow a more efficient internalization of the drugs, either by increasing the lipophility of water soluble drugs, such as DOX or ETO, or by providing an alternative internalization pathway to those drugs that present an impaired uptake, as is the case with MTX. Moreover, the components of the carriers themselves also play an important role in the overall efficacy of the system by inhibiting the activity of the drug efflux pumps responsible for the MDR phenomenon. Therefore, taken together, all these properties associated with the encapsulation of the drugs into LN have enabled researchers to increase the therapeutic index of these conventional drugs in several cancer models without a concomitant increase in collateral adverse effects. On the other hand, several novel agents are being tested or have entered the clinical trial stage in an attempt to improve the current treatment for OS. Some of these agents are already being used as second-line drugs in many protocols (gemcitabine, L-MTP-PE and docetaxel) and others are offered as potential drug candidates since they target functional processes such as bone remodeling (bisphosphonates), tumor progression and metastization (monoclonal antibodies, rapamycin analogs and alkyl-lysophospholipids) and tumor angiogenesis (monoclonal antibodies and sorafenib), or because they present a mechanism of action that may circumvent the MDR phenotype of tumor cells and act synergistically with other antitumor drugs (curcumin). As is the case with conventional anti-OS drugs, these novel agents also present barriers that limit their appli-


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cation, but their entrapment into LN has been demonstrated to improve their performance in several tumor types. Taking into consideration the intra and inter-tumor heterogeneity of bone tumors, there is still a long way to go before we can obtain a single therapeutic tool that will be uniformly successful for all OS patients, but, in this sense, LN present great potential for simultaneously incorporating drugs with complementary mechanisms of action in their matrix as well as targeting moieties with therapeutic potential, such as monoclonal antibodies, on their surface. Beside this therapeutic approach, LN may also allow the codelivery of therapeutic and imaging agents in order to achieve both an antitumor response and a noninvasive, real-time monitoring of the tumor fate. Therefore, although most attempts have been focused on the delivery of single chemotherapeutic agents to the tumors, emerging aspects of cancer therapy with drug delivery systems involve the co-delivery of different drugs and imaging agents that may provide an integrative approach by enabling multimodal delivery with a single application. In summary, these biodegradable and biocompatible LN constitute an attractive and promising platform to passively or actively direct the antitumor agents to the desired site of action, increasing their bioavailability in the primary and metastatic tumor sites while decreasing the systemic toxicity associated to their use. Moreover, LN can be orally administered avoiding the intravenous route, and can thus improve OS patients’ welfare. CONFLICT OF INTEREST The authors declare that this article’s content has no conflict of interest. ACKNOWLEDGEMENTS The authors would like to thank Dr. San Julián of the Department of Orthopedics at the University Clinic of Navarra for the Xrays and high resolution magnetic resonance. This work is supported by the Asociación Española Contra el Cáncer (AECC) (CI14142069BLAN) and Fundación Caja Navarra. REFERENCES [1] [2] [3] [4]

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Received: August 14, 2015

Accepted: October 26, 2015


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