Application of Nanomedical Technology in Breast Cancer Treatment A

Application of Nanomedical Technology in Breast Cancer Treatment A Isidora N. Tošić1,2, Momir M. Mikov1, Karmen M. Stankov3 A 1 Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 3 Department of Biochemistry, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia


INTRODUCTION
Malignant breast neoplasm is the most frequently diagnosed malignancy in women, representing the second leading cause of all cancer-related deaths [1,2]. As a result of continuous research and implementation of new therapeutic and diagnostic options fi ve-year survival of women breast cancer patients increased from 75% (1975-1977) to 90% (2005-2011) [3]. However, current strategies still largely rely on chemotherapy and other nonspecifi c approaches [3]. Cytotoxic nonspecifi c therapies do not provide successful tumor eradication since it is diffi cult to achieve high concentrations in the neoplastic cells due to a narrow therapeutic window of the drug. Th us, concentrations are similar between malignant and normal tissue, resulting in substantial systemic toxicity. Toxic adverse eff ects strongly aff ect patients' health, quality of life as well as patients' compliance [4,5]. In addition, if the malignant cells are not eff ectively eradicated, there is a considerable tendency for chemo-therapeutic resistance development [6]. If the malignant neoplasms establish resistance patterns, the treatment becomes much more challenging, greatly reducing the likelihood of favorable outcome [7]. Furthermore, advanced stages of breast cancer, particularly the metastatic phases are diffi cult to confront with use of conventional approaches, thus represent the prevailing cause of mortality [7]. An emerging area that shows high potential in overcoming these obstacles is the development of nanotherapeutics. Countless possibilities of nanocarrier structural alterations provide them with various characteristics and functions to satisfy versatile patient-specifi c medical needs.

Breast malignancies
Breast cancer alone accounts for 30% of all newly diagnosed malignancies in women in the United States, with an estimated incidence of 63 000 patients with in situ tumors and a total number of 271 000 newly diagnosed patients in 2019 [1]. In the last decade, the incidence is moderately increasing, partially due to the rising obesity epidemic [1,8]. Patients diagnosed in the early phases of the disease have promising chances for successful therapy. Nonetheless, metastases will eventually occur in almost every third patient fi rstly diagnosed with in situ cancer [9]. When the disease is diagnosed at advanced stages, therapeutic options are limited and fi ve-year survival decreases to only 26%. Th e most frequent mortality cause is metastatic dissemination in lymph nodes, bones, liver, lung and CNS [9].

Treatment approaches
Th e strategy for breast cancer treatment is most commonly multimodal and includes combinations of local treatments such as surgical methods and/or radiotherapy, and systemic therapy that comprises of chemotherapy, hormonal and targeted therapy [3]. Th e proper choice of therapy highly depends on the stage of the disease and the molecular type of cancer cells. Accordingly, local therapy has better outcomes in the treatment of early stages malignancies, whereas later stages, especially if metastases occur, require the addition of systemic therapy as the effi cacy of radiotherapy and surgical procedures are attenuated [2]. In addition, the determination of tumor molecular type is crucial for proper therapy choice. Survival of cancer cells is usually dependent on the specifi c molecule that is driving the oncogenic process and its inhibition results in cellular death. Th us, these specifi c molecules can serve as targets for both diff erentiation between the normal and malignant cells and for successful induction of cancer cell apoptosis. Th erefore, determining the molecular basis of breast cancer by immunohistochemistry and other molecular biology methods enables the prediction of the pharmacological agent that the cancer cells will respond to [3,9].

Molecular types of breast cancer
Depending on the expression of hormone receptors for estrogen (ER) and progesterone (PR), and human epidermal growth factor receptor 2 (ERBB2, also known as HER2), breast cancers are classifi ed into: hormone receptor positive (70% of patients), ERBB2 positive (15-20%) and triple-negative tumors which do not express any of the three molecular markers (15-20%) [9]. In accordance, hormone receptors positive and HER2 overexpressing breast cancers can be eff ectively treated with appropriate selective inhibitors such as small molecules and monoclonal antibodies. In addition to the most commonly used antibodies for HER2 inhibition such as trastuzumab and pertuzumab, they can be treated with both HER2/neu and epidermal growth factor receptor (EGFR) inhibitors lapatinib and neratinib [10]. However, basal or triple-negative breast cancer (TNBC) cells do not express any of the receptors that could currently be eff ectively targeted, therefore presenting the most challenging type to treat. Despite the relatively low prevalence, this is the most aggressive type which in addition lacks targeted therapeutic options, therefore having the highest mortality rates [7]. Th e standard therapeutic approach includes nonspecifi c chemotherapy which largely includes taxanes, anthracyclines and cyclophosphamide [11]. Even if the treatment is curative, the risk of relapse is higher than in other molecular types, and TNBC has a high tendency for metastatic dissemination. Th erefore, this is the molecular type of breast cancer with the greatest necessity for the development of new therapeutic methods.
Despite the targeting properties and specifi city of some of the therapeutic strategies, other pharmaceutics are defi ned by low specifi city with consequent modest eff ectiveness along with systemic toxicity which damage healthy tissue [12]. Moreover, even when targeted options are appropriate, they are commonly combined with a nonspecifi c agent. In addition, some agents can have undesirable pharmacokinetic (PK) profi le defi ned by suboptimal absorption and permeability through biological membranes, short circulation halflife, and low bioavailability, insuffi cient biodistribution in the target tissue and nonspecifi c eff ects with notable toxicity on healthy tissue [13]. Novel researches are directed towards overcoming these limitations and one of novel promising tool is the development of nanomedicine. Nanomedicine has the potential of overcoming some, if not all the listed constraints [14,15].

Nanomedicine
Nanomedicine represents a novel compelling form of pharmacotherapy in the treatment of numerous common diseases such as diabetes, cardiovascular diseases, infective diseases as well as various types of malignancies, including breast cancers [14][15][16][17]. It is defi ned as a biomedical application of materials with at least one dimension below 100 nm [18]. Incorporation of a therapeutic agent into a nanoscale carrier enables in vivo administration of various molecules which would be otherwise impossible due to extremely poor PK profi le, such as for siRNA, miRNA, genes, peptides, etc [19]. As for the cytotoxic agents that have modest but applicable PK features, they are protecting the active substance from degradation in circulation and enabling its controlled release, eminently aff ecting drug stability [20]. As a result, drugs do not exert very high blood concentrations and elimination half-life is prolonged [21]. For instance, Ran et al. showed that the maximum tolerated dose of a chemotherapeutic drug mertansine, clinically used for TNBC treatment, is 8-fold increased when incorporated into a nanocarrier [22]. Furthermore, nanoencapsulation enhances endo-and transcytosis of otherwise poorly permeable substances, resulting in augmented intracellular concentration [23,24]. Importantly, nanotherapeutic surface alterations provide the opportunity of preferentially delivering chemotherapy agents to malignant cells based on the diff erential expression of molecules on the plasma membrane [25]. As a consequence, higher concentrations are obtained at the target site, while minimizing the uptake in healthy tissue and reducing overall toxicity [25,26]. Nanomedicine is excessively investigated as a tool in breast cancer treatment, with currently 75 ongoing clinical trials testing new nanoscale formulations [27] and even greater numbers in the preclinical stage. So far, several formulations gained approval by the US Food and Drug Administration (FDA), most of which are indicated in oncology. Th ree of the liposomal formulations, Doxil®, Abraxane® and MyoCet® are already widely used in the treatment of breast cancer.

Liposomes
Liposomes are spherical particles with a hydrophilic core and a membranous lipid bilayer surface. Hydrophilic drugs, genes or RNAinhibiting molecules siRNA and miRNA can be encapsulated in their inner hydrophilic portion [19]. On the other hand, lipophilic drugs can be loaded in the lipid bilayer, thus extending circulation half-life with following reduction of systemic toxicity. In addition, coating with inert biocompatible polymers such as polyethylene glycol (PEG), reduces the recognition by the reticuloendothelial system, and elimination [26]. Doxyl®, the PEGylated doxorubicin liposome is widely used in breast cancer treatment for the listed benefi ts over a free drug. Th e limitations of liposomes mainly result from physiochemical instability, along with the ability to load only a certain number of lipophilic drugs, such as paclitaxel, into the single lipid bilayer [12].

Nanoparticles
Polymeric nanoparticles, such as micelles, capsules, colloids, and dendrimers, have been developed in parallel with the growing need for nanoscale carriers of greater capacity for hydrophobic substances including taxanes, cisplatin, and tamoxifen. Th ey consist of a hydrophilic shell and a lipophilic core, with the addition of amphiphilic molecules, polymers, and copolymers with PEG to prevent agglomeration [13]. Hydrophilic agents may be bound to the www.hophonline.org outer layer by covalent or hydrogen bonds, thus reducing the immunogenicity of certain anticancer drugs such as the ones of protein source [12]. In addition, alternatingly charged multilayered nanoparticles have a considerably higher capacity for molecule loading, and can accept external molecules up to 50% of the nanoparticle weight [28]. Another benefi t of these systems is the controlled drug release in response to certain factors such as pH, UV or temperature [29]. Lipid nanoparticles are similar, designed for loading the molecules of same affi nity, yet stabilized and solubilized by surfactants and emulsifi ers [30].
Carbon nanoparticles include carbon nanotubes and fullerenes depending on their structural organization into a tube or hollow cage shape. Carbon nanotubes are largely studied for their mechanical properties as tools in radiation oncology, biomedical imaging, as quantum dots and biosensors [31]. However, uncertainty regarding toxicity aspects of their administration remains to be revealed [31]. On the other hand, fullerenol derived nanoparticles are distinguished for their reactive radicals scavenging ability and anti-oxidative effect, while retaining a safe genotoxicity profi le [32][33][34]. Utilizing this feature might contribute to the safety aspects of certain chemotherapeutic agents in breast cancer treatment [35]. For instance, doxorubicin-induced oxidative stress may lead to cellular membrane lipid peroxidation in the heart, bone marrow, gastrointestinal tract, and other tissue, resulting in pronounced systemic toxicity and increased risk of congestive heart failure [36]. Coupling with an antioxidant carbon nanoparticle such as fullerenol C60(OH)24 could ensure a safer doxorubicin profi le, as proposed by in vivo studies [37,38]. In addition, several studies revealed fullerenol particles themselves to exert potent anti-tumor and anti-metastatic activity in murine breast cancer models [39,40].
Metal nanoparticles, including gold and silver nanoparticles are used to deliver a sensitizing agent during radiotherapy or as contrast enhancers, in addition to previously described features of nano-systems [41]. Compellingly, they can possess a peculiar ability to absorb near-infrared rays and emit absorbed energy in the form of heat during the process of photothermal ablation (PTA) [42]. Th e temperature of the surrounding tissue then increases to 50°C inducing irreversible cellular damage, resulting in complete breast 886 Volume 7 • Number 1 • April 2020 • HOPH tumor cells removal in vivo [43]. Similarly, certain polymeric nanoparticles, such as lipid-PEG layer coated polylactic-coglycolic acid (PLGA), are light-absorbing photosensitizers that generate reactive oxygen species when illuminated, therefore resulting in damage of the nearby tissue during the process of photodynamic therapy [44]. Th e external layer of these and previously described particles can be conjugated with a tumor-targeting molecule, thereby inducing selective damage to tumor cells [43]. Th is approach showed promising outcomes in breast cancer treatment and may represent a suitable non-invasive alternative for patients whose chemotherapy or radiation treatment did not produce any benefi cial effect.

Passive targeting of breast cancer using nanotherapy
Th e targeted eff ect on malignant cells using nanomedicine formulations can be active or passive. Retention of nanoforms in vascular tumor tissue is a common example of passive targeting. Breast cancers and other solid tumors develop their own vascularization which diff ers from the normal blood vessels, inter alia, by having greater permeability [45]. Th erefore, the leaky vasculature makes the tumor tissue highly accessible to nanoscale molecules [46]. Additionally, lymphatic drainage is reduced allowing prolonged retention in tumor tissue [47]. Although this mechanism is quite simple and nonspecifi c, it provides signifi cantly higher nanoparticle concentrations at the tumor site, known as the enhanced permeability and retention eff ect (EPR) [48]. Abraxane®, the FDA approved liposomal paclitaxel is a typical example of effi cient passive targeting of breast cancer cells. Th is eff ect strictly depends on the abnormal tumor vessel development which occurs in the initial phase of tumorogenesis, however early lymphogenic metastasis will hardly be targeted using this strategy [49]. Another method for nonspecifi c targeting is based on the hypoxic microenvironment of breast cancers and other solid tumors, resulting from insuffi cient tumor vascularization. Hypoxic microenvironment might further facilitate the development of somatic mutations and therefore contribute to cancer progression and metastatic behavior. Furthermore, the hypoxic setting might promote resistance to various anti-cancer drugs, at least partially www.hophonline.org by means of poor vascularization that results in insuffi cient drug accumulation [6,50]. Low oxygen state of tumors facilitates anaerobe cellular metabolism resulting in acidic pH of the surrounding tissue. Nanotherapeutics can be constructed to have greater affi nity and/or to be cleaved at lower pH conditions, thus releasing the drug, while having improved stability in the range of physiological pH [51][52][53].

Active targeting of breast cancer using nanocarriers
Active targeting implies complementing the surface layer of the nanocarrier with a molecule that has a specifi c affi nity to bind the structures aberrantly expressed on the surface of malignant cells. Th is type of targeted therapy is more advanced than passive targeting, as it can achieve greater specifi city and more eff ective selectiveness of drug delivery, followed by greater accumulation at the tumor site. Typical molecular structures that can be used to supplement the nanocarrier surface are monoclonal antibodies and ligands of the targeted receptors. For instance, CD44 cell surface adhesion receptor is highly expressed in a variety of malignant diseases, including breast cancer stem cells, thus nanocarriers complemented with its ligand, hyaluronic acid, exert tumortargeting properties [54,55]. Similarly, in targeting HER2 positive breast cancers, nanoparticles can be terminally complemented with trastuzumab [56]. Although antibody-covered nanoparticles express precise selectiveness and can be very eff ective, it is possible for large antibody structure to alter the physiochemical properties of the nanoparticle, modifying their PK profi le. Th erefore, researchers are also investigating the complementation of nanoparticles with peptides as targeting agents [57,58]. Th e potential of nanomedicine in the treatment of breast cancer is particularly important in TNBC, which currently lacks targeted therapeutic options. Since almost half of the TNBC has elevated EGFR expression, an appealing strategy for selective TNBC treatment is drug nanocarriers coated with anti-EGFR peptides and antibodies [59][60][61]. Additionally, folate receptor alpha (FRα) represents a promising target for selective TNBC treatment as it is expressed in 50-85% of metastatic TNBC [62][63][64]. In a nonmalignant manner, FRα tissue distribution is limited to a certain number of epithelial cells, with expression localized at the apical tissue margins hardly reachable by blood circulation [65]. Furthermore, oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is abnormally activated in a variety of malignant diseases including 70% of breast cancers and almost all TNBC cases and might serve as a potential target for designing selective therapy for this aggressive neoplastic disorder [66,67].

Nanotherapy in drug resistance prevention and management
Resistance to anti-cancer medications represents one of the most challenging obstacles in the treatment of breast cancer. Resistance patterns might evolve through various mechanisms, such as modifi cation of the receptor that is responsible for the drug uptake, mutation of the gene that encodes the drug-binding protein, enhancement of cellular effl ux etc [6]. Some of these issues might be solved using passive targeting nanocarriers, by ensuring higher drug concentration in tumor tissue and not permitting tumors enough time to develop the resistance mechanisms [68]. Another attractive method for resistance prevention and management is the application of combination therapy in a single nanoparticle. Th us, antitumor agents can be administered together with the inhibitor of the signal pathway responsible for resistance development, even if they are otherwise incompatible or either of them shows insuffi cient PK properties without an appropriate carrier. In the past decade the research was extensively focused on combining cytotoxic agents with RNA interfering molecules, siRNA and miRNA [69]. However, extremely low stability in circulation and inability to cross the plasma membrane disputes their use as free agents. With the employment of nanocarriers, treatment can be combined with siRNA to resistance-responsible gene [70] or as drug-sensitizing agents [71,72]. Moreover, all the components of combination therapy would be absorbed by the targeted cell at the same moment, enabling effi cient synergistic eff ect and preventing resistance development [73]. Aside from additive agents, RNA interference is tested as single nanoparticleconjugated therapy for the treatment of various malignancies [74]. For instance, Asik et al. successfully suppressed the growth of BRCA1mutated breast cancer using nanoparticles loaded with eukaryotic elongation factor 2 ki-nase (EF2K) siRNA in vivo, as its expression is upregulated in almost 80% of patients with this genetic alteration [75].

Nanoparticles in treatment of breast cancer metastases
In addition to previously described methods for targeted drug delivery, various strategies have been investigated for the treatment of certain metastatic malignancies. Ross et al. have shown in a mouse model of metastatic breast cancer that it is possible to deliver chemotherapy specifi cally to breast cancer bone metastasis, as they have changed their primary characteristics in response to specifi c bone micro environment [76]. Th ey found integrin β3 to be diff erentially expressed in bone metastases and have therefore designed docetaxelloaded nanocarriers conjugated with integrin β3 targeting agent. Hence, drug delivery to healthy tissue is reduced, which was verifi ed by unaltered markers of liver, renal, blood cells and other vital functions. In an opposite manner, free docetaxel treatment showed systemic toxicity in mice [76]. Th e diff erent study used a combination of two targeting molecules directed towards αvβ3 integrin and P-selectin, as they are expressed in diff erent stages of breast cancer metastasis. As these nanoparticles are targeting diff erent metastatic sites that overexpress either or both of the receptors, they have observed the accumulation of injected particles at 89% of the confi rmed metastatic sites [77]. Similarly, separate study effi ciently targeted breast cancer lung metastases using nanoparticles coated with integrin-binding exosomes for their navigation to tumor sites [78]. Breast cancer metastases in CNS are one of the most challenging to treat due to their localization and aggressiveness. Th e eff ectiveness of chemotherapy is usually suboptimal, as most of them have low permeability through hematoencephalic barrier. To address this complication, nanoparticles can be supplemented with polysorbate 80 (PS80) as it enhances the hematoencephalic barrier penetration resulting in more eff ective brain tumor burden reduction, as demonstrated in mice [79]. Caelyx® (Janssen-Cilag International NV) in Europe was the fi rst nano drug indicated for breast cancer treatment, which was approved by the FDA in 1995. It is a PEGylated liposomal formulation of doxorubicin with a diameter of approximately 85 nm. Doxorubicin has been an essential element of breast cancer treatment for decades. However, its systemic toxicity and cardiotoxicity in particular, significantly limit the extent of its clinical appliance [36]. Th erefore, incorporation of doxorubicin in PEGylated formulation reduces premature drug release, consequently attenuating systemic toxicity without aff ecting the antitumor eff ect. Th us, with this type of formulation, the incidence of cardiotoxic side eff ects is decreased fi ve-fold. Furthermore, the circulating half-life of Doxil® is approximately 74 hours, whereas it is approximately 5 minutes for free doxorubicin, at the starting dose of 60 mg/kg [80].

Currently available forms of nanomedicine indicated in breast cancer therapy
MyoCet® (GP-Pharm) is the second, however non-PEGylated liposomal form of doxorubicin approved for metastatic breast cancer treatment by FDA in 2000, although currently licensed only in Europe and Canada [36]. It has certain advantages over free drug application, yet its clearance rates are significantly higher comparing to its PEGylated analog Doxil® [81].
Abraxane® (Celgene Corporation) is a nanotherapeutic with albumin-bound paclitaxel, the so-called nab-paclitaxel. Like other taxanes, paclitaxel exerts its action by binding to microtubules, polymerizing tubulin and stabilizing tubular polymers, thus inhibiting their degradation during cell division, resulting in mitosis arrest. Th e clinical success of paclitaxel is confi ned by its low solubility in aqueous media. Th is limitation is solved by non-covalent bound to human serum albumin, the physiological carrier of circulating lipophilic components [82]. In addition, albumin has a binding affi nity for the vascular endothelial membrane protein Gp60, which in turn increases vascular membrane permeability and receptor-mediated uptake of circulating proteins into the interstitium. Th erefore, intratumoral tissue concentration of nab-paclitaxel is 33%, higher in comparison with free drug administration, resulting in a higher frequency of pathological complete response in breast cancer patients [83,84]. Abraxane® was approved by the FDA in 2005 and is currently widely used for the treatment of metastatic www.hophonline.org breast cancer and if cancer has relapsed within 6 months of chemotherapy. Additionally, its eff ects are investigated for neoadjuvant therapy and in the treatment of early stages of breast malignancies [85][86][87]. Innovative approaches are directed towards enhancing the activity of nab-paclitaxel, such as by conjugation with manganese dioxide which induces the oxygenation of hypoxic tumors, resulting in two-fold more eff ective tumor growth inhibition [88]. Certainly, as in other nanotherapeutic types, complementation with antibodies such as trastuzumab, bevacizumab or rituximab is being tested for enhancement of selectivity [89].

CONCLUSION
Breast cancer is the malignancies with the highest prevalence and mortality rate in women, representing one of the key public health problems worldwide. Diagnostic and therapeutic progression in the last couple of decades improved prognosis for patients. However, further advances and novel strategies are needed for providing greater success of the therapeutic outcomes along with reduction of systemic toxicity; which besides direct eff ects infl uences patient's compliance. With a better understanding of breast cancer molecular basis and development of nanotechnology, novel innovative approaches started being extensively explored. Nanomedicine application could overcome various restrains that characterize conventional chemotherapeutic agents. Possibility for the administration of unstable agents opened a new fi eld for pharmacotherapeutic consideration of siRNA, miRNA, genes, and peptides. Conventional therapies are being improved with encapsulation, as their pharmacokinetic properties are adjusted to better fulfi ll the safety and effi cacy criteria. New targets are investigated for designing selective agents to treat aggressive types, metastases and multi-drug resistance, which could hardly be overwhelmed with conventional approaches. Additionally, considering their multi-functionality and almost infi nite possibilities for modifi cation, they are intensively tested for utilization in imaging and diagnostics, theranostics, tissue regeneration, as sensitizing and contrast agents. Th erefore, nanomedicinebased research is widely distributed in the both pre-clinical and clinical setting and has promising chances of representing the future of breast cancer and other oncological treat-ments.

AUTHORS' CONTRIBUTIONS
Substantial contributions to the conception of the work were given by IT, MM and KS. IT has done the literature review and wrote the manuscript, which was critically reviewed by KS and MM for important intellectual content.