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New Advances in Nanotechnology-based Diagnosis and Therapeutics for Breast Cancer: An Assessment of Active-Targeting Inorganic Nanoplatforms Priscila Falagan-Lotsch, Elissa M Grzincic, and Catherine J. Murphy Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00591 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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Bioconjugate Chemistry

New Advances in Nanotechnology-based Diagnosis and Therapeutics for Breast Cancer: An Assessment of Active-Targeting Inorganic Nanoplatforms Priscila Falagan-Lotsch*†, Elissa M. Grzincic†, and Catherine J. Murphy *† †

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States Corresponding Authors *E-mail : [email protected] Phone : 217.333.7680 Fax : 217.244.3186 *E-mail : [email protected] Phone : 217.333.3397

Abstract Breast cancer is a major cause of suffering and mortality among women. Limitations in the current diagnostic methods and treatment approaches have led to new strategies to positively impact the survival rates and quality of life of breast cancer patients. Nanotechnology offers a real possibility to mitigate breast cancer mortality by early-stage cancer detection, more precise diagnosis, as well as more effective treatments with minimal side effects. The current nanoplatforms approved for breast cancer therapeutics are based on passive tumor targeting using organic nanoparticles and have not provided the expected significant improvements in the clinic. In this review, we present the emerging approaches in breast cancer nanomedicine based on activetargeting using versatile inorganic nanoplatforms with biomedical relevance, such as gold, silica, and iron oxide nanoparticles, as well as their efficacy in breast cancer imaging, drug and gene delivery, thermal therapy, combinational therapy and theranostics in preclinical studies. The main challenges for clinical translation and perspectives are discussed.

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1. Introduction Breast cancer is the most commonly diagnosed cancer among females worldwide, making it a serious public health problem.1 Even though developed countries have reported an increase in 5-year survival rates2, breast cancer remains the second leading cause of death by cancer in American women.3 In the U.S., about 249,000 new cases of breast cancer are estimated in 2016 and more than 40,500 deaths are expected. Despite undeniable advances, the current diagnostic methods and treatment approaches such as surgery, radiotherapy and adjuvant systemic therapy endocrine therapy, chemotherapy with drugs such as anthracyclines (e.g. doxorubicin) and taxanes (e.g. paclitaxel), still have limited impact on the survival rates and quality of life of breast cancer patients.4 Nanotechnology-based approaches bring new possibilities for improving cancer patient care as supplements to traditional tools. Due to unique physicochemical properties dependent upon size, shape and surface chemistry (explored throughout this Review), nanoparticles (1-100 nm in size) are very attractive for cancer diagnosis and therapeutics. It has been more than 20 years since the first FDA-approved nanoparticle platform (nanoplatform) for drug delivery, a PEGylated liposomal doxorubicin (trade name: Doxil®) to treat different types of cancer (Kaposis’s sarcoma, metastatic ovary and breast cancers)5, became a milestone towards “the era of nanomedicine”. Since then, the number of studies and clinical trials focused on cancer nanotechnology has grown exponentially. However, the translation of nanotherapeutics from the bench to the clinic has been a difficult task. To date, besides Doxil®, only three more nanotechnology-based systems for solid tumor treatments have been approved by the FDA:

DaunoXome® (liposome-encapsulated

daunorubicin) to treat Kaposis’s sarcoma, Abraxane® (albumin-bound paclitaxel nanoparticle) and Myocet® (liposome-encapsulated doxorubicin) to treat breast cancer.6 At present, new advances in nanotechnology-based methods for breast cancer diagnosis and treatment are being explored in preclinical studies for future translation into clinical practice. Unfortunately, despite the high expectations, the impact of the currently available cancer nanomedicines on patient survival is considered only modest.7–9 W. Chan’s group recently reviewed data from published studies conducted during 2005-2015 regarding tumor targeting with nanoparticles (passively or actively targeted), and concluded that, on average, nanoparticles show poor delivery efficiency to solid tumors10. Although this data is still controversial11,12, it is clear that new strategies should be put in place. Compared to simpler methods of treatment and 2 ACS Paragon Plus Environment

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diagnostics, the path to clinical implementation is arguably more difficult for inorganic nanoplatforms based on their complex formulations and uncertainties with long-term side effects. In this review, the emerging approaches in breast cancer nanomedicine based on activetargeting and inorganic nanoplatforms with biomedical relevance, such as gold, silica, and iron oxide nanoparticles, as well as their efficacy in vitro and in vivo will be discussed along with perspectives on the future and then main challenges for clinical translation.

2. The Limitations in Current Breast Cancer Care and The Advent of Nanomedicine The current rates of breast cancer mortality can be assigned to two main factors: clinical parameters of the tumor (e.g. tumor stage and histological grade) at primary diagnosis and limitations in the current treatments such as the high systemic toxicity caused by anticancer drugs as well as the occurrence of drug resistance observed in some cases. The tumor size and how distant the tumor has spread are some of the most important factors in predicting the prognosis of breast cancer patients. When detected at early stages and before metastasis, the treatment is highly effective and breast cancer is considered a curable disease for more than 70% of cases.3 On the other hand, metastatic breast cancer is still considered incurable. Thus, early detection is crucial to reduce the breast cancer mortality rates and save thousands of lives. However, the current standard methods for breast cancer clinical detection such as mammograms that use X-rays, magnetic resonance imaging (MRI) based on contrast agents and ultrasound (used to detect tumors in dense breast) face many challenges. For instance, artifacts on mammograms may reduce the image quality leading to interpretation errors and not every breast tumor is detectable by this methodology;13 MRI presents low specificity as does ultrasound and, consequently, produces false-positive results.14 Therefore, approaches that are more accurate and sensitive for early-stage detection of breast cancer are urgently needed to positively impact the survival rates of patients Breast cancer is a very complex and heterogeneous disease that goes far beyond simple clinical parameters. Huge progress has been made in the understanding of the biology of breast cancer which has in turn contributed to important advances in the clinical practice over the years. The classification of breast cancer subtypes (luminal A and B, HER2-positive and triple negative basal-like) based on gene expression profiles provided new diagnosis and treatment perspectives, allowing for novel therapeutic alternatives.15 Analysis of the expression levels of three key 3 ACS Paragon Plus Environment


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biomarkers in breast cancer - estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) – has become an important clinical routine, helping to characterize the different subtypes of breast cancer since they have a direct impact on disease prognosis and treatment response prediction.16 Despite the implementation of more “personalized” therapeutics based on breast cancer subtypes, many challenges still have to be faced in order to decrease the current death rates due to the disease. Aside from the clinical parameters at diagnosis related to better or worse outcomes, the systemic drug toxicity caused by long anticancer drug circulation times and lack of tumor tissue specificity is another major challenge of the current therapeutics.17,18 The short and long-term side effects of traditional chemotherapies including osteoporosis, cardiomyopathy and heart failure, and neuropathy are well-documented.19,20 Unfortunately, these undesirable side effects have an impact on the treatment follow-up by patients. Additionally, drug resistance reduces the effectiveness of chemotherapy, leading to treatment failures and consequent mortality. The majority of patients with metastatic breast cancer develop drug resistance, with cancer stem cells present in tumors playing an important role. 21 The desirable early-stage detection, more precise diagnosis as well as more effective treatments with minimal side effects may be reached through the implementation of nano-based devices in cancer medicine. Nanoparticles can be designed to be employed as contrast agents to improve the already existing techniques such as MRI and allow the implementation of different strategies (e.g. photoacoustic tomography – PAT).22 Nanoparticles can be used in cancer treatment as anticancer agents as in photothermal therapy (PTT)23,24 or as delivery carries when loaded with one or multiple therapeutic agents as chemotherapeutic drugs, improving agent circulation halftime, tumor-specificity distribution and treatment efficacy.25,26 Further, nanoparticles can present multifunctional properties that allow their application in both cancer detection/imaging and therapy simultaneously (referred as theranostics)27–29 or even be designed to respond to certain specific stimuli (e.g. temperature, pH) that trigger drug release, providing a site-specific drug distribution.30–32


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3. The Potential of Inorganic Nanoplatforms for Breast Cancer Diagnosis and Therapeutics

The nanoplatforms currently approved for breast cancer treatment are based on organic nanoparticles. Properties such as biocompatibility, biodegradability and low polydispersity make these nanoparticles interesting for nanomedicine applications, especially drug/gene delivery. Moreover, extended circulation time in the body can be achieved by organic nanoplatforms, improving their chances of reaching the diseased sites preferentially and enhancing the efficacy of treatment.33,34 Organic nanoparticles include natural or synthetic polymers (e.g. chitosan, collagen, glycerol, PLGA), dendrimers (e.g. PAMAM) and lipid-based materials (e. g. liposomes, micelles). Inorganic nanoplatforms composed of nanoparticles widely used in biomedical research such as gold, silica and iron oxide, are more versatile than their organic counterparts since they offer additional advantages in properties and function, bringing new opportunities for breast cancer nanotherapeutics. While the core of these nanoplatforms is inorganic, there is always a ligand shell that provides colloidal stability and control of surface chemistry. Other types of inorganic nanoparticles (graphene, carbon nanotubes, quantum dots, upconversion nanocrystals, metalorganic frameworks, etc) are often studied in the field, but this review will focus on gold, silica and iron oxide specifically. Gold nanoparticles with different sizes and shapes (e. g. spheres, rods, shells) present a simple synthesis process and indeed are commercially available, are considered relatively inert, can have a broad variety of surface functionalization chemistries and provide fascinating tunable optical and thermal properties (resulting from surface plasmon resonance (SPR)) that can be used to ablate cells.35–37 Silica nanoparticles (silicon dioxide) are also considered relatively inert and, like gold, are readily synthesized in a large range of sizes, morphologies and surface chemistry.38,39 Silica can present nanometer scale pores (mesoporous silica) that can efficiently encapsulate high numbers of molecules (drugs, biomolecules or other nanoparticles).40,41 Iron oxide nanoparticles can also be made in many sizes and have sizedependent superparamagnetic properties.42 Consequently, inorganic nanoplatforms have been explored for drug delivery, gene therapy, imaging, photothermal therapy for tumor ablation, combinational therapeutics and theranostics. A schematic representation of typical nanoplatforms explored in cancer diagnosis and therapeutics is shown in Figure 1. 5 ACS Paragon Plus Environment


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Figure 1. Schematic examples of some common types of organic and inorganic nanoparticles explored in cancer therapeutics and diagnostics. Organic nanoparticles are ideal for packing with drugs and genes for delivery, while the intrinsic properties of many types of inorganic nanoparticles allow for new imaging modalities and therefore better contrast agents. Additionally, plasmonic and magnetic properties of some inorganic nanoparticles allow for cancer treatment via heat. Inorganic nanoparticles coated with or embedded within organic components can combine the best properties of both types of nanoparticles for next-generation combinatorial therapy and theranostics.

4. Passive vs. Active Targeting: What are the Targets?

Passive tumor targeting is the current strategy used by the clinically approved nanoplatforms for breast cancer treatment. The rationale for passive targeting is based on the enhanced permeability and retention (EPR) phenomenon, first described by Matsumura and Maeda in 1986 (Figure 2).43 Due to the special characteristics of tumors such as leaky vasculature, high angiogenesis rates leading to immature blood vessel growth, and poor lymphatic clearance, 6 ACS Paragon Plus Environment

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nanoparticles passively penetrate and preferentially accumulate in tumor sites.44 However, the EPR strategy alone is not sufficient to provide tumor targeting based on recent data.10,45 Indeed, EPR phenomenon lead to a modest 20-30% increases in delivery to tumor sites compared to noncancerous organs. The complexity of the tumor microenvironment poses physical and biological limitations to the EPR efficacy. Particularly in solid tumors (as breast tumors), the heterogeneity of tumor vasculature and lymphatic function lead to variations in vessel permeability; Dense collagen-rich tumor extracellular matrix, intratumoral pressure enhancing the resistance for nanoparticle diffusion, and nanoparticle clearance by macrophages present in tumor interstitium are among factors that compromise the EPR effect.46–48 Additionally, the EPR efficacy is affected by intrinsic properties of nanoparticles such as size, charge, shape and surface chemistry, reviewed elsewhere.49 A 30-year retrospective of the EPR effect was recently published by Maeda and coworkers, highlighting problems, solutions and the new knowledge in the field along with interesting future perspectives.50

Figure 2. Schematic of a) non-cancerous tissue and b) a tumor surrounded by non-cancerous tissue to depict the enhanced permeability and retention (EPR) effect. Cancerous tissue is characterized by poor lymphatic drainage, rapid angiogenesis, and leaky vasculature, and dense extracellular matrix; all of these characteristic allow nanoparticles to accumulate in tumors.

The active-targeting strategy involves the functionalization of nanoparticle surfaces with ligands selected to recognize and bind to overexpressed molecules on the surface or even in subcellular domains of tumor cells.51 Moreover, the targeting can be addressed to molecules in the tumor microenvironment (blood vessels, extracellular matrix, etc.) to repair its physiological 7 ACS Paragon Plus Environment


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abnormalities.52 The presumed benefit of actively targeted nanoparticles over passive nanoconstructs is related to the improvement in targeting specific cells as well as in cellular uptake and reduced side effects. A myriad of ligands such as antibodies, peptides, aptamers and sugars, have been used to guarantee the target specificity which may provide more accurate diagnosis and enhancement in efficacy and safety of nanotherapeutics. The use of actively targeted nanoplatforms has been assumed to create a huge potential for the improvement of cancer nanomedicine in both preclinical studies (explored below) and early clinical trials.53 The first FDA-approved active-targeting system (not nanoparticle-based) – an antibody against the HER2 receptor (trastuzumab; trade name: Herceptin®) in combination with paclitaxel chemotherapy – presented a remarkable success in the treatment of HER2-positive metastatic breast cancer (25% of the cases) related to poor prognosis.54 The antibody-chemotherapy combined led to significant increases of the median time to cancer progression and the overall response rates to the treatment as well as a significantly lower rate of death compared to the treatment with chemotherapy (paclitaxel) alone.54 This kind of result suggests that better insights into the biology of breast cancer as well as the signaling pathways driving the breast tumorigenesis, progression, maintenance and spread will yield a broad variety of potential candidates for nanoscale approaches (for more details about disrupted pathways and molecular markers in breast cancer see ref

55–57

). The discovery of additional altered genes involved in the

carcinogenesis process led to the rapid emergence of a novel active-targeting tool for nanotherapeutics based on siRNA (small interference RNA), able to silence genes in a very effective way.58 Examples of nanoparticle ligands and molecular targets mentioned in this review as well as their role in normal cells/tissues and in cancer physiopathology are summarized in Table 1. Despite the apparent gains of active targeting, still no nanomedicines for cancer diagnosis or treatment based on this approach have been approved at present in clinical practice.


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Table 1. Summary of nanoparticles ligands and molecular targets and their main physiological role in normal cells/tissues and in cancer physiopathology. Nanoparticle ligand Anti-HER2 antibody (trastuzumab; trade name: Herceptin®); antiHER2 aptamers

RGD peptides (cyclic)

Amino-terminal fragment of the urokinase-type plasminogen activator Folic acid

Molecular Target

Physiological role of the molecular target in normal cells/tissues and in cancer physiopathology Involved in cell proliferation pathways; related to cancer development and progression; overexpression in ~ 25% of breast cancer cases; HER2-positive cancers are related to poor prognosis.

Epidermal growth factor receptor 2 (HER2)

Cell transmembrane receptors involved in cell adhesion to the extracellular matrix; overexpression related to angiogenesis and cell survival; considered as marker of malignancy and cancer aggressiveness.

Integrin receptor αvβ3

Urokinase-type plasminogen activator (uPA) receptor

Cell membrane receptor part of the plasminogen activation system related to tissue reorganization; overexpressed in 60% to 90% of invasive breast cancer and tumor associated stromal cells leading to an increase of cell migration.

Folic acid receptor α (FRα)

Acts in cell metabolism pathways including DNA synthesis; related to cancer development and progression; overexpressed by a number of epithelialderived tumors including breast carcinomas.

Mucin 1 receptor (MUC1)

Involved in cell surface protection, cell-cell adhesion and several signaling pathways; overexpression in cancer cells is related to cell death prevention and metastasis; overexpressed in ~ 90% of human breast carcinomas.

Tamoxifen

Estrogen receptor (ER)

Related to cell proliferation pathways; overexpression in ~ 70% of breast cancer cases; ER positive cancers are related to good prognosis.

Nucleolin aptamer

Nucleolin

MUC1 aptamer

Nucleus protein involved in a variety biological processes such as ribosomal synthesis; overexpressed in cancer cells surface mediating cell growth and survival.

Ref

24, 60, 62, 69, 70, 76, 82, 91, 99, 115

63, 72, 79, 111

75

80, 104, 110

82

83

84, 95, 109



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Table 1. (Continued) Nanoparticle ligand

Molecular Target

Anti-CD44 antibody

CD44 receptor

γ-secretase inhibitor

Notch-gene signaling pathway

Glucose moiety

Transferrin

Glucose receptor

Transferrin receptor

c-myc aptamer

c-myc gene

Anti-TGF-β antibody

Tumor Growth Factor β receptor (TGF-β)

SMI#9 drug

Rad6 protein

Anti-CD150 antibody (TCR105)

CD105 receptor

Physiological role of the molecular target in normal cells/tissues and in cancer physiopathology Cell surface receptor involved in physiological activities of normal cells such as proliferation, differentiation, migration; overexpressed in tumor cells including cancer stem cells; related to angiogenesis, metastasis and drug resistance. Related to cell differentiation during the development and stem cells modulation (survival and self-renewal); pathway disrupted in many cancers including breast cancer; related to metastasis and drug resistance. Overexpression of glucose receptors is common in most cancer cells. The enhancement of glucose uptake is essential for cancer progression since this sugar is the main source of ATP generation in transformed cells. Transmembrane receptor related to growth regulatory pathways; overexpression in cancer cells is related to high proliferation rates and aggressiveness. Encodes a transcription factor that drives cell growth, proliferation, differentiation, cell death and stem cell self-renewal; essential for the carcinogenesis process; overexpressed in a majority of human cancers. Role in a variety of physiological pathways including cell growth, proliferation, differentiation, homeostasis and cell death; overexpression at later stages of cancer led to metastasis. Enzyme that plays a role in the DNA damage tolerance pathway (major mechanism of error-prone lesion bypass); overexpression related to breast cancer tumorigenesis, metastasis and drug resistance. Also known as endoglin, the receptor is part of the TGF-β receptor complex; crucial role in the angiogenesis process.

Ref

86

88

88

59, 61, 92

58

104

105

113


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5. Combining Active Targeting and Inorganic Nanoplatforms: How Much Does Active-Targeting Improve Outcomes? Emerging approaches focused on active targeting using inorganic nanoplatforms have been received with optimism in the nano-oncology field. Analyses of nanoparticle delivery to breast cancer tumors have shown an enhancement in tumor accumulation and efficacy when this approach is considered (details are below).59,60 To provide a brief comparison between passive and active-targeting nanoparticles, gold nanospheres functionalized with transferrin presented a 5-fold faster and a 2-fold higher tumor accumulation compared to the passively targeted PEGylated gold nanospheres in MDA-MB-435 orthotopic tumor xenografts;61 Anti-HER2 targeted-PEGylated iron oxide nanoparticles loaded with paclitaxel accumulated preferentially in the tumor site in a human HER2/neu+ SKBr3 breast cancer xenograft mouse model, about 2.5times more than non-targeted PEGylated nanoparticles;62 The uptake and accumulation of activetargeting mesoporous silica (MSNs) coated with cyclic RGD peptides were significantly higher in MDA-MB-231 breast cancer cells compared to MSNs without the active coating in vitro .63 In this section, we will discuss how actively-targeted inorganic nanoparticles (gold, silica and iron oxide) have been explored in preclinical studies (in vitro and in vivo) with emphasis on the applied strategies (targets, nanoparticle types) and their efficacy in breast cancer diagnosis and treatment. The goal is to provide a reader with quantitative metrics for “how much more” the targeted nanoplatforms improve their function, compared to appropriate controls.

5.1. Breast Cancer Imaging

Diagnosis by imaging, one of the most fundamental tools in the clinic, represents a fertile field for inorganic nanoparticles application. Nanoparticles can be implemented in imaging modalities such as MRI, ultrasound, computed tomography, positron emission tomography (PET), photoacoustic tomography (PAT) and optical imaging. 64–66 Inorganic metal nanoparticles can be engineered to be employed as contrast agents for traditional techniques applied in breast cancer imaging, MRI and ultrasound.67,68 By adding a targeting ligand in their surface, the desirable specificity lacking in the traditional approaches can be reached. Iron oxide nanoparticles are some of the most promising MRI contrast agents due to their tunable magnetic properties. Superparamagnetic iron oxide nanoparticles (SPIONs) 11 ACS Paragon Plus Environment


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can be tracked under a magnetic field in very small quantities. In studies focused on breast cancer diagnostics using nanoparticles, the conjugation of SPIONs with anti-HER2 antibodies presented in vitro and in vivo high targeting specificity and high imaging sensitivity, allowing the detection of very small tumors by MRI: A significant 4-fold increase in target selectivity to breast cancer cells overexpressing HER2 receptor was obtained by this nanoplatform loaded with 6 ± 2 molecules of anti-HER2 antibody per SPION compared to nanoparticles functionalized with a nonspecific antibody.69,70 In vivo analysis (BT-474 tumor model) showed that size is the determinant factor for SPION-anti-HER2 antibody breast tumor accumulation.71 The same success has been extended to in vivo MRI detection of prognostic molecules present in the breast tumor cells and neovasculature such as integrins (αvβ3). SPIONs functionalized with cyclic RGD peptides led to a 2-fold increase of SPION cellular uptake compared to nontargeted SPIONs.72 Multi-modal imaging approaches using targeted inorganic nanoparticle systems have also been explored for more accurate detection of breast cancer, avoiding false positive results and enabling the detection of very small tumors.73,74 A multi-modal system composed of iron oxide nanoparticles coated with the amino-terminal fragments of urokinase-type plasminogen activator (uPA) (8 to 10 molecules per particle) and loaded with the fluorescent Cy5 dye was developed by Yang and coworkers75 for combined MRI and optical imaging of breast cancer mouse mammary tumor 4T1 cells . The high internalization rates of these nanoparticles by those specific cells provided an amplification in the optical signal by 180-fold 48h after administration of the nanoparticles compared to the NIR imaging without the targeted contrast. The potential of mesoporous silica nanoparticles (MSNs) constructs for detection of HER2-positive breast tumors has been also evaluated. MSNs were conjugated with Herceptin on the external nanoparticle surface and FITC (fluorescein isothiocyanate, a common fluorescent dye) was bound in the internal pores. A comparison between cells treated with Herceptin-MSNs only and Herceptin-MSNs mixed with free Herceptin showed a 30% higher fluorescence intensity in cells exposed to Herceptin-MSNs only.76 New strategies for high-resolution bioimaging are also feasible by using active-targeting inorganic nanoplatforms. Employing the emergent non-invasive photoacoustic tomography (PAT), which combines high optical contrast and ultrasound resolution77,78, gold nanoparticles have shown a great potential as contrast agent for early diagnosis of breast tumor in vivo. PAT 12 ACS Paragon Plus Environment

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can be performed in highly sensitive angiography of tumor vasculature overexpressing integrins using gold nanostars conjugated with cyclic RGD peptides (∼ 210 peptides per nanoparticle). After 6h post-injection, the accumulation of RGD-gold nanostars in tumor vessels led to a significant 3.5-fold increase in the photoacoustic signal compared to nontargeted PEG-gold nanostars.79 Additionally, targeted nanoparticles conjugated with folic acid were used for a molecular detection of circulating breast tumor cells as a second contrast agent (after PEGylated SPIONs functionalized with uPA) for photoacoustic imaging in vivo.80 This approach provided a significant sensitivity improvement (up to 103-fold gain) in circulating tumor cell (CTC) detection, important biomarkers for breast cancer prognosis and therapy prediction.81 Jo and coworkers82 proposed a new highly sensitive platform for early-detection and prognosis of metastatic breast cancer based on dye-doped silica nanoparticles functionalized with dual aptamers that specifically recognize HER2 and MUC1 receptors in breast cancer cells. Although the work focused only on in vitro analysis, the strategy can be a promising tool for in vivo breast cancer diagnostic in the future. A summary of breast cancer imaging data is presented in Table 2.



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Table 2. Examples of active-targeting inorganic nanoplatforms evaluated in breast cancer imaging. Nanoparticle construct

Nanoparticle core size

Targeting strategy

Imaging method

Ref

SPIONs

3.5 ± 0.3 nm

anti-HER2 antibody

MRI in vitro and in vivo

69

SPIONs coated with chitosan and PEG

8 ± 2 nm

anti-HER2 antibody


70

10 ± 3 nm

RGD peptides


72

Paramagnetic iron oxide nanoparticles coated with dextran and PEG and Cy5 dye

10 nm

amino-terminal fragment of urokinase-type plasminogen activator (uPA)

MRI and NIR imaging in vitro and in vivo

Mesoporous silica nanoparticles coated with PEG

100 nm

anti-HER2 antibody

Optical imaging in vitro

Gold nanostars

55 ± 5 nm

RGD peptides

Photoacoustic imaging in vivo

Carbon nanotube core with gold layer coated with PEG

12 × 98 nm

Folic acid

Photoacoustic imaging in vivo

70 nm

biotion-HER2 aptamers and biotin-MUC1 aptamers

Optical imaging in vitro

SPIONs coated with 3aminopropyltrimethoxysilane (APTMS)

Silica nanoparticles coated with PEG and avidin

5.2. Breast Cancer Treatment

Drug Delivery The design of drug delivery systems is the main focus of much nanomedicine research. The long-standing dream, of course, is that with the proper delivery system, chemotherapeutic drugs will act only on tumor cells and not on normal cells and therefore require smaller doses and eliminate side effects. New advances have been made in preclinical studies to enhance tumor drug accumulation and consequent therapeutic efficacy by developing active-targeting inorganic drug delivery systems. The following in vitro and in vivo studies mentioned below, including their efficacy compared to the free drug, are summarized in Table 3. 14 ACS Paragon Plus Environment

75

76

79

80

82

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Gold nanoparticles conjugated with PEGylated tamoxifen enhanced about 3 times the drug potency in ER overexpressed breast cancer cells according to in vitro experiments.83 The surface modification of gold nanoparticles with the AS1411 aptamer led to a high affinity to the nucleolin overexpressed on the surface of malignant cells in vitro, providing an increased specificity and targeted delivery of doxorubicin (Dox) towards malignant cells, including MCF-7 breast cancer cells.84 Due to its high specificity, the AS1411-Dox-gold nanosystem increased breast cancer cell death by about 50% compared to Dox-gold nanoparticles modified with a nonspecific aptamer. Nucleolin is considered a very attractive target for cancer treatment since this protein is expressed only on cancer cell surfaces and not on their non-cancerous counterparts85. Anti-HER2 targeted-PEGylated SPIONs loaded with paclitaxel presented a 2.5-fold increased uptake in cells overexpressing HER-2 compared to cells with low levels of this receptor in a human HER2/neu+ SK-BR-3 breast cancer xenograft mouse model as well as a selective and increased breast cancer cell death in vitro compared to free paclitaxel.62 The same study also showed reduced paclitaxel toxicity for non-target cells when the drug is conjugated with nanoparticles compared to the drug free. Aires and coworkers86 developed a specific drug delivery system based on iron oxide nanoparticles conjugated with anti-CD44 antibody and gemcitabine (GEM) for CD44+ breast cancer cells therapeutics. The treatment with anti-CD44GEM-iron oxide nanoparticles (4 µM of GEM) decreased significantly MDA-MB-231 breast cancer cells viability (CD44+) in vitro compared to non-targeted GEM-iron oxide nanoparticles or lower doses of drug free (0.4 and 1 µM). However, no difference in cell viability was observed when the free GEM was delivered at the same concentration as in the nanoplatform (4 µM of GEM). A significant improvement in the therapeutic efficacy for triple negative breast cancer in a xenograft mice model was obtained after treatment with mesoporous silica (MSNs) coated with cyclic RGD peptides and arsenic trioxide (ATO). The tumor volumes were significantly smaller than tumors treated by ATO alone or ATO-mesoporous silica without targeting molecule and tumor weights dropped approximately 45% at the end of treatment. These results are in accordance with the in vitro assays in which a significantly higher cytotoxicity triggered by RGD-ATO-MSNs was observed when compared to the other systems.63 ATO is FDA-approved to treat hematological malignancies but presents poor efficacy for solid tumors treatment in addition to systemic toxicity. The encapsulation ATO in a nanodelivery system can overcome 15 ACS Paragon Plus Environment


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these limitations. However, the study did not show data about the biodistribution of RGD-ATOMSNs in vivo after injection nor the possible toxic impact of ATO on non-target tissues when conjugated with nanoparticles. MSNs have been also exploited to kill cancer stem cells (CSCs). CSCs represent a big challenge in cancer therapy and usually present a high glycolytic activity as well as a self-renewal ability sustained by the Notch signaling pathway, dysregulated in cancer.87 Taking advantages of these biochemical properties, MSNs coated with polyethyleneimine (PEI), targeted with glucose moieties and carrying the γ-secretase inhibitor DAPT (N-[N-(3,5difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) that disrupts the Notch pathway, reduced significantly the breast CSCs population in a xenograft mice tumor model just as well the treatment with free DAPT. However, considering the CSC fold reduction, the treatment with glucose-DAPT-PEI-MSNs presented the greatest efficiency leading to ~5.5-fold reduction on the cell number versus 2-fold observed by DAPT free treatment.88


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Table 3. Examples of active-targeting inorganic nanoplatforms evaluated for drug delivery in breast cancer therapeutics. Nanoparticle construct

Targeting strategy

Anticancer agent

Loading level

Dose

Efficacy compared to drug free

Citrate gold nanoparticles

ER modulator

Gold nanoparticles

Tamoxifen

12 000 tamoxifen ligands per particle

10 µM of the nanoconstruct

2.7-fold enhanced drug potency in vitro

nucleolin-aptamer (AS1411)

Doxorubicin

NAa

20.8 µM of Dox

NAa

84

SPION coated with PEG

anti-HER2 antibody

Paclitaxel

∼274 paclitaxel molecules per particle

1nM of Paclitaxel

~30% more effective in vitro

62

Iron oxide magnetic nanoparticles

anti-CD44 antibody

Gemcitabine

20 µmol gemcitabine per g Fe

4 µM of GEM

~30% more effective than 0.4 and 1 µM of GEM in vitro

Mesoporous silica

Mesoporous silica nanoparticles coated with PEI a NA - not available

RGD peptides

Arsenic trioxide (ATO)

glucose moieties

γ-secretase inhibitor (DAPT)

0.61 mmol/g

37.39 µg/mg

20 µg/mL of ATO (in vitro) 0.75mg/kg of ATO (in vivo) 1 µg of DAPT

83

~20% more effective in vitro; significant tumor growth regression in vivo

More effective reduction (3.5-fold) of the CSC breast tumor population in vivo

86

63

88


Ref


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Gene Therapy

The basic principle of gene therapy is the introduction of genetic material including DNA, mRNA, miRNA, siRNA and aptamers in cells to modulate gene expression. Gene delivery systems based on inorganic platforms are still in their infancy but have a promising future in nanomedicine. The use of these nanoplatforms as gene carries has been pointed out as an attractive alternative to overcome hurdles of the traditional viral vectors such as adverse immune responses.89 However, as W. Chan has pointed out, the immune response to nanoparticles has not been as thoroughly studied as it should be.10 Silica nanocontructs have been receiving a great deal of attention in gene delivery therapy. In 2009, Xia and coworkers90 demonstrated the maximization of the negative charged siRNA and DNA constructs delivery to cancer cells through the surface modification of MSNs with cationic PEI coatings. Along the same line, MSNs cores coated with cationic polymers (PEI-PEG) targeted with trastuzumab were engineered to deliver siRNA to silencing specifically the HER2 gene in HER2-positive breast cancer.91 In vitro analysis in 4 different breast cancer cell lines showed high levels of apoptosis only in HER2-positive cells triggered by nanoparticle constructs. In vivo analysis in an orthotopic mouse tumor model demonstrated a 60% reduction of HER2 protein levels in trastuzumab-resistant tumor cells after one dose of nanoparticles compared to saline controls and about 47% compared to the treatment with trastuzumab-targeted MSNs loaded with non-HER2 specific siRNA. Moreover, a significant tumor growth inhibition after multiple doses administrated over 3 weeks was observed. Additionally, the authors described a high reproducibility and a suitable scale up production of the construct.91 A therapy based on silica nanoparticles modified with transferrin loading the p53 gene, the most altered gene in human cancers, resulted in a 4-time gene transfer increase efficacy as well as significant growth inhibition of MCF-7 breast cancer cells compared to treatment with non-targeted p53silica nanoparticles. In vivo results demonstrated an efficient tumor growth inhibition in the group treated with transferrin-p53-silica nanoparticles. 92 The potential of ultrasmall gold nanoparticles for gene therapy has also been described It has been reported that the conjugation of nucleic acids with gold nanoparticles lead to an increase of nucleic acids stability and prevents their degradation by cellular enzymes.93,94 Gold nanoparticles of 2 nm diameter loaded with a specific oligonucleotide sequence (POY2T) were 18 ACS Paragon Plus Environment

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able to enter into nucleus of breast carcinoma cell lines and inhibit cell proliferation by regulating the expression of the c-myc gene that plays a key role in the carcinogenesis process (Figure 3). After 24h-treatment with POY2T-gold nanoparticles, the expression of c-myc gene significantly decreased by 40% in MCF-7 breast cancer cells compared to controls (non-treated cells) whereas the cell treatment with free POY2T led to only a 15% decrease in gene expression.58 Additionally, gold nanospheres (5 nm) functionalized with a nucleolin-target aptamer (AS1411) had an intense antiproliferative effect specifically on breast cancer cell lines (MDA-MB-231 and MCF-7) in a concentration about 20-fold less than that needed using the aptamer alone. Also, the nanoplatform AS1411-gold induced an expressive MCF-7 (80%) and MDA-MB-231 (88%) cell death, not observed when the cell lines were treated with goldnanoparticles alone or were conjugated with a control non-specific aptamer. A complete inhibition of tumor growth and tumor regression was observed after systemic administration of AS1411-gold in MDA-MB-231 xenografts mouse models. No systemic toxicity was detectable.95 All the reports focused on gene therapy discussed above are summarized in Table 4.

Figure 3. The potential of ultrasmall gold nanoparticles for gene therapy. a) scheme representing the intracellular distribution of 2 nm gold nanoparticles (Au) targeted with POY2T aptamer for gene therapy. b) TEM images of the nuclear localization of 2 nm Au-POY2T nanoparticles in breast cancer cells (MCF-7) after 24h exposure. c) Western blot assay showing the effective cmyc protein reduction by 2 nm Au-POY2T nanoparticles after 24 h MCF-7 treatment. Modified with permission from ref 58.



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Table 4. Examples of active-targeting inorganic nanoplatforms evaluated in breast cancer gene therapy. Nanoparticle construct Mesoporous silica nanoparticles coated with PEI and PEG Silica nanoparticles

Ultrasmall gold nanoparticles coated with tiopronin

Ultrasmall gold nanoparticles


Targeting strategy

Anticancer agent

Dose

Efficacy

1.25 mg/kg of siRNA

Decrease of HER2 protein levels by 47% compared to non-specific anti HER2nanoplatform and significant inhibition of tumor growth in vivo

anti-HER2 antibody

siRNA against HER2 gene

54 nm

transferrin

plasmid containing the p53 tumor suppressor gene

10 µg of the plasmid-p53

Significant growth inhibition of breast cancer cells in vitro and tumor volume reduction in vivo compared to treatment with non-targeted p53-silica nanoparticles

2.1 ± 0.6 nm

c-myc geneaptamer (POY2T)

Aptamer against c-myc gene (POY2T)

5 µM of the nanoconstruct

Significant downregulation of c-myc gene compared to free POY2T, leading to inhibition of tumor cell proliferation in vitro

Aptamer against nucleolin (AS1411)

200 nM of the nanoconstruct (in vitro) 22 µg of AS1411 (in vivo)

Significant antiproliferative and cell death induction effects in vitro compared to free AS1411, gold nanopartilces alone and gold nanopartilces + control aptamer; inhibition of tumor growth and tumor regression in vivo

47 ± 4 nm

5 nm

nucleolinaptamer (AS1411)

Ref 91

92

58

95


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Thermal Therapy One of the most ancient strategies to ablate solid tumors is based on the use of heat since tumors cells are less tolerant to high temperatures compared to the normal cells. Hyperthermia is characterized by the application of temperatures ranging from 41–47°C for about ten minutes in tissues leading to irreversible tissue damage.96 Nanoscale noble metals such as gold with different shapes (spheres, rods, shells, etc) have shown a tremendous potential as photothermal agents in cancer photothermal therapy (PTT). Their tunable optical properties that can be carefully optimized to enhance the light absorption from an excitation source (e.g. near infrared (NIR) lasers that can penetrate tissue somewhat) and convert it into heat, making gold nanoparticles ideal for thermal therapy applications.97,98 Since relatively lower energies are needed, heating leads to tumor ablation in a minimally invasive way. Loo and coworkers60 were the first to demonstrate how targeted gold-silica nanoshells improved the specificity and efficacy of PTT in breast cancer SKBr3 cells. The main results of this report as well as the following studies are presented in Table 5. The PTT strategy may overcome one of the major problems of current cancer treatments: tumor drug resistance. A successful localized cell death in HER2-positive drug-resistant breast cancer cells was reported after NIR light treatment with gold-silica nanoshells targeted with antibody against HER2.24 No effect was observed in non-tumorigenic breast epithelial cells (MCF10A) not overexpressing HER2 after nanoparticle exposure and irradiation. Another form of thermal therapy is based on the use of magnetic hyperthermia (MH), in which magnetic nanoparticles produce heat in the presence of an alternating magnetic field. The capacity of anti-HER2-iron oxide nanoparticles coated with dextran to selectively kill HER2positive breast cancer SKBr3 cells under an alternating magnetic field exposure was evaluated.99 The accumulation of anti-HER2-iron oxide nanoparticles was observed in SKBr3 but not in HER2-negative normal human mammary epithelial cells (HMEC). Due to the active-targeting, a significant increase in nanoparticle retention (up to 30%) was observed in HER2-positive cells compared to non-targeted particles, which led to a specific and more pronounced cell death effect after MH treatment. Recently, a biocompatible and highly specific PEGylated SPION conjugated with Herceptin was developed and emerged as a favorable nanoconstruct for MH application to treat HER2-positive metastatic breast cancer SKBr3 cells.100



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A related type of therapy, photodynamic therapy (PDT) which kills cancer by way of a photo-active drug, can be improved with targeted inorganic nanoparticles to deliver photosensitizer drugs, to change the wavelength of light near the photosensitizer (upconversion), or to enhance photosensitizer excitation via plasmonic effects.101


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Table 5. Examples of active-targeting inorganic nanoplatforms evaluated in breast cancer thermal therapy. Nanoparticle construct


Gold-silica nanoshells

120 nm

Gold-silica nanoshells

120 nm

Iron oxide nanoparticles coated with dextran

100 nm

Targeting strategy

Dose

Conditions

anti-HER2 antibody

3 × 109 nanoshells/mL

NIR laser (820 nm, 0.008 W/m2 for 7 min)

anti-HER2 antibody

1.71 x 1010 nanoshells/mL

anti-HER2 antibody

0.1 µg/µL of nanoparticles

Efficacy

Ref

Cell death only in cells exposed to antiHER2-nanoshells followed by NIR laser treatment in vitro. No cytotoxicity observed in cells treated with either nanoshells conjugated to a non-specific antibody or NIR light alone.

60

Specific cell death in HER2-positive breast NIR laser cancer cells exposed to anti-HER2(808 nm, 80 W/cm2 for nanoshells and treated with NIR in vitro. 5 min) No effect observed in breast cells with no HER2 overexpression.

24

Significant intracellular retention and cell Magnetic Hyperthermia death in HER2-positive breast cancer cells (35°C for 20 min; H exposed to anti-HER2-iron oxide ~36kA/m,f = 163 kHz) nanoparticles + heat compared to heat alone and non-targeted nanoparticles in vitro.

99



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Combinational Therapy Combinational therapies using multifunctional nanoplatforms to fight cancer have acquired special attention in the recent years. The combination of two or more anticancer agents or the combination of more than one therapeutic approach (multi-modal therapy) can synergistically improve treatment efficacy and decrease tumor drug resistance with reduced side effects.102,103 Synergistic treatment with different anticancer agents using nanoplatforms has been provided superior anti-tumor effects in preclinical studies (Table 6). A nanosystem composed by spherical gold nanoparticles functionalized with folic acid-BSA and combining two different anti-cancer drugs (methotrexate and anti-TGF-β1 antibody) was designed and successfully optimized for combinational therapy in metastatic breast cancer cells overexpressing TGF-β1 protein.104 As a result, the highest cytotoxic potential was obtained with the lowest concentration of methotrexate conjugated in the nanosystem. Moreover, the levels of free extracellular TGFβ1, associated with cancer progression and metastasis, were reduced by 30% in cells exposed to targeted nanoparticles compared to unexposed cells and by 10% compared to free anti-TGF-β1 antibody. A significant enhancement in triple negative breast cancer cells sensitivity to the cisplatin drug was observed when the treatment was combined with the exposure to mercaptosuccinic acid (MSA)-capped gold nanoconstructs loaded with SMI#9 (Rad6 protein inhibitor). The effective dose of cisplatin needed to inhibit the growth of 50% of cancer cells (4.9 µM) was at least 5 times less than in the treatment with free cisplatin (>25 µM) in vitro105 which may lead to a more tolerable treatment for patients in a still efficacious dose. To guide an optimal anticancer agent combination, mathematical approaches have been developed and validated aiming a more personalized therapy and better outcomes and safety for patients.106


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Table 6. Examples of active-targeting inorganic nanoplatforms evaluated in anticancer agent combinational therapy in breast cancer. Nanoparticle construct

Gold nanoparticles

Gold nanoparticles coated with MSA


100 nm

3-5 nm

Targeting strategy

folic acid

SMI#9 (Rad6 inhibitor)

Anticancer agent

Dose

Methotrexate + anti -TGF-β1 antibody

2.83mM methotrexate + 0.1 mg/mL Anti TGF-β1 antibody

SMI#9 + cisplatin

5 µM SMI#9-gold nanoparticles + 4.9 µM cisplatin

Efficacy High cytotoxic potential (~90% cell death) of conjugated methotrexate at low dose in metastatic breast cancer cells in vitro; decrease of TGF-β1 by 30% compared to untreated cells. Significant enhancement (> 5fold) in triple negative breast cancer cells sensitivity to cisplatin in vitro.

Reference

104

105



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Multi-modal therapy, such as combined hyperthermia and chemotherapy, has also been exploited to destroy cancer cells.107 Mild hyperthermia (~43 °C) can enhance the chemotherapy efficacy since the heat can disrupt cellular structures and increase cellular drug uptake. Additionally, a controlled drug release inside tumors is feasible. By increasing the temperature (46-47°C), cell death occurs leading to tumor ablation, as discussed previously.108 The conjugation of the multi-modal approach with targeted inorganic nanoplatforms has proven to be very effective in vitro and in vivo studies (Table 7). SPION loaded with Nucant, a nucleinreceptor target, and doxorubicin combined with hyperthermia showed a very strong synergistic anticancer effect (no tumor growth after 28 days) in breast tumor-bearing female athymic nude mice.109 A single platform composed of folic acid-targeted nanoparticles carrying cisplatin drug showed high accumulation into tumors and a synergistic effect in vivo to kill orthotopic triple negative breast tumors combined with PTT, which lead to virtually complete tumor eradication. No tumor growth was detected at day 20 post-administration of folic acid-cisplatin-gold nanorods followed by irradiation, significantly different from the growth rates (tumor volume > 600 mm3) observed in tumors treated in other conditions (Table 7). Additionally, the cotreatment suppressed the breast cancer metastasis to the lung by disrupting the peripheral tumor vasculature.110 A NIR light-sensitive targeted nanoparticles based on gold nanorods loaded with doxorubicin demonstrated a significant in vivo anticancer activity showing its potential for synergistic therapy in breast cancer treatment (Figure 4). Moreover, the encapsulation of doxorubicin in the nanosystem reduced significantly the systemic toxicity caused by the free drug.111


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Table 7. Examples of active-targeting inorganic nanoplatforms evaluated in multi-modal therapy (chemotherapy + hyperthermia) in breast cancer. Nanoparticle construct

SPION coated with DMSA

Gold nanorods coated with PGA

Gold nanorods coated with a NIR light sensitive chromophore moiety immobilized by β-Cyclodextrin


Targeting strategy

Multi-model therapy

Dose + Hyperthermia conditions

12 ± 3 nm

nucleolin antagonist (Nucant)

Doxorubicin + Magnetic hyperthermia

4µM of Dox + Magnetic hyperthermia (46°C for 30 min; H = 15.4 kA/m, f = 435 kHz)

length of ~ 50 nm

length of 41.7 ± 2.8 nm width of 13.2 ± 0.9 nm

folic acid

RGD peptides

Cisplatin + PTT

0.5 mg/kg of cisplatin + NIR laser (655 nm, 1.0 W/cm2 for 2 min)

Doxorubicin + PTT

5 mg/kg of Dox + NIR laser (808 nm, 800 mW for 5 min)

Efficacy

Ref

Strongest anticancer effect (inhibition of tumor growth) compared to Nucant-DoxSPION without hyperthermia and SPION, SPION-Nucant or SPION-Dox + heat in vivo.

109

Virtually tumor eradication and suppression of breast cancer metastasis to the lung in vivo.

110

Best inhibition efficacy on cancer growth, significantly different from free Dox and nanoparticle system without NIR irradiation; significant reduction of systemic toxicity compared to free Dox in vivo.

111



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Figure 4. Scheme representing the multi-modal therapy (chemotherapy and hyperthermia) to against breast cancer using a targeted inorganic nanoplatform. The design combines selfassembly of β-cyclodextrans with NIR light-responsive chromophores associated with doxorubicin, NIR light-absorbing gold nanorods, poly(ethylene glycol) for elongated circulation and cyclic RGDs for targeting. Upon NIR light irradiation, the chromophore is cleaved, releasing doxorubicin, and the gold nanorods both support this cleavage and heat up inside cells to induce photothermal therapy and to help break open the nanoparticle for drug release. Reprinted with permission from ref 111. Copyright (2016) Elsevier.


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5.1. Breast Cancer Theranostics Theranostic nanoparticle formulations combine diagnosis by imaging (especially considering metastases detection) and therapeutics in a single nanoplatform. Theranostics have been considered an important strategy to the development of personalized medicine because the target accumulation efficiency and drug release can be monitored in a real time way by imaging. This tracking enables the determination of a more accurate effective therapeutic dose to treat an individual tumor. Due to their NIR absorption and scattering properties, the potential of targeted gold nanorods as theranostic agents for imaging and PTT was demonstrated by the El-Sayed group.112 In vitro analysis of gold nanoparticles conjugated with fluorescent-labeled transferrin for breast cancer imaging and therapeutic purposes showed high cell uptake improving imaging and significantly reducing the laser power needed for an effective PTT.59 Hollow mesoporous silica nanoparticles (HMSNs) loaded with doxorubicin and targeted with TRC105 which binds to CD105, overexpressed in tumor neovasculature, are attractive candidate platforms for future theranostics. This nanosystem was successfully applied for dual modality imaging (positron emission tomography-PET and near-infrared fluorescence-NIRF) which provides more accurate information, and led to enhanced tumor targeted drug delivery in 4T1 murine breast tumorbearing mice.113 A nanocarrier composed of a FDA-approved SPION (ferumoxytol) conjugated with a prodrug azademethylcolchicine (potent vascular disrupting agent) activated by the metalloproteinase-14 (MMP-14), overexpressed in the tumor microenvironment, induced significant tumor-selective accumulation demonstrated by MRI and antitumor effect in breast cancer MMTV-PyMT cells in vitro and in vivo with no observed systemic toxicity.114 More recently, a novel and efficient in situ system based on PEGylated gold nanoparticles modified with anti-HER2 antibody and doxorubicin was proposed to monitor the dynamics of drug release in breast cancer SKBr3 cells by surface-enhanced Raman spectroscopy (SERS).115 This imageguided delivery gives us important information on the performance of delivery systems in realtime, crucial for the future clinical translation. The studies mentioned above in this section are summarized in Table 8.



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Table 8. Examples of active-targeting inorganic nanoplatforms evaluated in breast cancer theranostics. Nanoparticle construct

Gold nanoparticles coated with PEG

Hollow mesoporous silica nanoparticles conjugated with (64Cu) and ZW800 dye

Ultrasmall iron oxide nanoparticles coated with dextran Gold nanoparticles coated with PEG and cell penetrating peptide


25 nm

~65 nm

6.5 nm

30 nm

Targeting strategy

transferrin

anti-CD105 antibody (TRC105)

peptiderecognized by MMP14

anti-HER2 antibody

Theranostic approach

FITC- labelled transferrin + PTT

PET and NIRF + Chemotherapy

MRI + Chemotherapy

SERS + Chemotherapy

Dose/ Conditions

Efficacy

Ref

0.13 nM of gold nanoparticles + Laser light (530nm, various power densities for 5 min)

Significant enhancement of scattering-based optical contrast compared to no exposed cells and cell exposed to untargeted nanoparticles in vitro; significant PTT cell damage at 7 W/cm2, 2-fold less than needed to kill cells treated with non-targeted nanoparticles and more than 200-fold less than to kill no treated cells.

59

5–10 MBq of TCR10564 Cu-ZW800- HMSNs + 6.5 mg/kg of Doxorubicin

Significant 3-fold higher tumor targeting efficacy compared with the non-targeted group; significant higher optical intensity of tumor compared with the non-targeted group; significant enhanced tumor targeted drug delivery in vivo.

113

0.4 M (Fe) solution of nanoparticles (0.75mmol Fe/kg) + 0.29 mM of azademethylcolchicine (1.0 µmol/kg)

Significant cell death in MMP-14 positive breast cancer cells compared to cells treated with SPION alone in vitro; significant tumor selective accumulation and antitumor effect compared to cells treated with iron oxide nanoparticles alone in vivo. No difference compared to free drug.

114

SERS mapping image at 1275cm-1 Raman band + 5µM of Doxorubicin

Time-dependent Dox release (90.23%) by the intracellular GSH with 24 h of particle treatment; linear increased cytotoxicity (up to ~61%) at 24h after treatment with anti-HER2Dox-gold nanoparticles. No cytotoxicity in cells treated with non-targeted nanoparticles. 30

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6. Main Challenges for Clinical Translation and Perspectives

The reader will notice that in spite of the vast research literature on the topic, there is a lack of targeted inorganic nanoparticle-based nanodrugs currently available on the market. Even with the great potential to shift the current strategies in breast cancer care, the translation of targeted inorganic nanoplatforms to the clinic remains considerable challenge. Once inside the body, nanoparticles face complex biological barriers: opsonization by plasma proteins leading to a protein corona formation that can “hide” the surface ligand in targeted nanoparticles; phagocytic clearance by the mononuclear phagocyte system; and barriers posed by the tumor microenvironment, all impact nanoparticle delivery to tumor sites.116–118 Additionally, since clathrin-mediated endocytosis is a classical mechanism of nanoparticle cellular internalization in non-phagocytic cells, endosomal compartmentalization is another considerable obstacle, especially for those nanoplatforms designed for gene therapy.119,120 To reach the nucleus, the genetic material should successfully overcome the hostile environment of endosomes/lysosomes, an environment at low pH and rich in enzymes. The instability of the genetic material in the cytosol full of nucleases is also a barrier that should be addressed by preclinical studies. Indeed, the poor delivery efficiency of nanoparticles to solid tumors was recently described even when actively targeted nanoparticles were considered.10 Intrinsic characteristics such as size, shape, surface charge, and surface chemistry as well as cancer type and tumor model used in the studies had an influence in the efficacy of the nanoparticle delivery process.10 Based on the data presented in this Review, it is clear the great variability of experimental designs among preclinical studies, which overall cause a negative impact on the progress in the field. Therefore, the success of targeted nanoplatforms depends on the optimization of specific nanoparticle parameters with their performance being tested in appropriate models that mimic physiological conditions and tumor microenvironment, and the understanding of the nanoplatform-microenvironment interaction and target ligand-cell receptor-internalization. Advanced in vitro models have been developed to address these specific points, leading to an improvement of preclinical evaluation of nanomedicines and also, incorporating the 3Rs (replacement, refinement, reduction) requirement, overcoming ethical issues involved in animal experimentation. The 3D multicellular spheroids are described as good models to simulate the in vivo tissues and have been used to clarify the interactions between nanoparticles and biological 31 ACS Paragon Plus Environment


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systems.

121,122

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Sethi and coworkers123 recently developed a triple negative breast cancer 3D co-

culture spheroid mimicking the tumor milieu. Also, the 3D co-culture spheroid was implanted orthotopically in mice to represent the more complex in vivo conditions for evaluating an activetargeting multi-modal nanotherapeutics. This approach represents the next-step on the field and its replication is strongly encouraged. Further advanced technologies such as the production of organoids (miniaturized organs) formed by several cell types mimicking tissues as well as microfluid tissue/organ-on-chip devices are emerging,124–126 bringing good perspectives for nanotherapeutics and nanotoxicology assessments. Some limitations of the current in vivo models (such as mouse and rat) related to differences in physiology compared to humans can be overcome. However, a complete validation of these new methodologies is still ongoing. Addressing inorganic nanoparticles more specifically, the major concern for clinical translation is related to the baseline nanoparticle toxicity being not fully understood and in some cases controversial. The safety status is essential for the nanoplatforms approval by regulatory agencies, especially for those designed for imaging/diagnostics. Since nanoparticles exhibit altered properties in biological fluids, the prediction of their toxic potential in biological systems is difficult. The induction of toxicity by nanoparticles seems to change according to parameters such as size, surface coating, agglomeration state, protein corona composition, cell-type sensitivity, exposure time and dose.127–129 A general trend for nanoparticles toxicity can be observed, with small sized nanoparticles presenting higher toxicity compared to larger nanoparticles due to their increased surface area. For example, gold nanoparticles with 2 nmdiameter or below present catalytic activity (selective oxidation) not observed at larger sizes130 and high levels of cell death have been described after exposure to such small particles.131,132 However, this trend could not be confirmed by studies conducted by different groups using distinct cell types in which gold spheres nanoparticles with sizes ranging from 1.4-4 nm had no toxic effects as well as their larger counterparts.133–135 As discussed in this Review, the modification of nanoparticles surface with selected biofunctional ligands is an essential process to provide target-specificity as well as to improve their biocompatibility. Since the surface properties are strictly correlated with interactions nanoparticle-biological systems, the surface coating plays an important role in nanoparticle uptake by cells and consequently, toxic response. Studies have shown the influence of the surface physicochemical properties on the toxicity triggered by inorganic nanoparticles.136,137 32 ACS Paragon Plus Environment

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The impact of different types of surface-modified gold nanoparticles (20 nm) on human cells was evaluated previously by our group using the transcriptomic approach. The alterations in the expression levels of genes which play important role in pathways such as cell proliferation, angiogenesis, metabolism, inflammation, apoptosis and cell survival were in a surface-coatingdependent-manner. Our results suggest the great complexity that involves surface coating and nanoparticle-cell interactions.138 Taken together, the large majority of inorganic nanoparticle toxicity studies available in the literature is based on in vitro experiments focused on acute toxicity assessment at very high doses of particles, rather far from a realistic scenario of human exposure. Our group recently reported the long-term effects of a low dose of gold nanoparticles with different shapes and surface coatings in non-cancerous human cells after acute and chronic exposures. Although negligible cytotoxic, a long-term cell stress response induced by gold nanoparticles, especially after acute exposure, was observed. However, continual exposure does lead to cellular adaptation to nanoparticles at some level.139 Inorganic nanoparticles can accumulate inside the body mainly in liver and spleen (considering the most common intravenous injection route) which increases the chances of longterm effects, still largely unknown. For further clinical translation, the gap in the knowledge about long-term effects of nanoplatforms in more sophisticated in vitro models mentioned previously in this section and/or in vivo should be addressed as well as the limitations of the traditional cytotoxicity assays to evaluate nanoparticles, and the lack of standardized methods for assessing nanoparticle toxicity.140 It is also desirable to focus the efforts on a systematic evaluation of nanoparticles biodistribution in vivo considering different particle sizes and surface chemistries as well as the route of exposure and the elucidation of the cellular and molecular mechanisms involved in the nano-bio interactions.126,141,142 For colloidal gold in particular, there is a human population that drinks these solutions for alleged health benefits. We recommend that clinical studies be performed on a cohort of such people, matched with proper control populations, to learn what long-term consequences there are (if any) on human ingestion of nanoparticles. Additionally, the main technological challenge for clinical translation of actively targeted inorganic nanoparticles is related to the manufacture of these nanoplatforms on a large scale along with the required reproducibility. In general, nanoplatforms developed for preclinical 33 ACS Paragon Plus Environment


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studies are synthesized in small batches but large amounts of constant formulations, evaluation of stability, homogeneity and performance are urgently needed prior to the clinical trial stage. Yet, the measurement of targeting ligands on nanoparticle surfaces, important for reproducibility and even for controlled ligand-orientation, remains a tough task.143,144 Another consideration is the molecular display of targeting ligands on the surface of nanoparticles. W. Chan’s recent review of the literature estimates about 9 actively targeted nanoparticles out of a thousand appear to reach their target.10 But usually the nanoparticles are prepared with heterogeneous surfaces – a targeting ligand, perhaps PEG to improve circulation time, and then a web of proteins and other molecules that adsorb and desorb on variable time scales. M. Banaszak Holl and coworkers,145 using molecularly well-defined dendrimers, have shown that surface functionalization with ligands follows Poisson statistics; for instance, if one desired to have 4 targeting ligands and 5 drug ligands per nanoparticle, only a few percent of the nanoparticles in the pot would actually have that exact stoichiometry; and furthermore only a fraction of a percent would have the (presumably) proper molecular display of those ligands to interact with cells. These low numbers are reminiscent of the one W. Chan finds and lead us to ask: is it possible that the “right” nanoparticles are indeed finding their target very efficiently, but we chemists are actually not good at making large quantities of the “right” nanoparticle? Taking into account all the aforementioned hurdles and the years needed until the FDA approval there is only one inorganic nanoplatform clinically approved: Ferumoxytol, based on colloidal iron oxide nanoparticles, for treatment of iron deficiency anemia in adults. Previously approved nanoplatforms for imaging also composed of iron oxide nanoparticles such as Feridex I.V.®; Resovist® Gastromark™ were discontinued by FDA and the reasons remain unclear. Nevertheless, Ferumoxytol and others inorganic nanoplatforms are in current clinical trials for diagnosis and therapeutics of many types of solid tumors such as lung, prostate, head and neck, ovary and breast cancers (https://clinicaltrials.gov/). To date, only two active-targeting inorganic nanoplatforms are under clinical trials investigation. Aurimune (CYT-6091) and Cornell dots. Aurimune, a 7 nm PEGylated colloidal gold particle loaded with TNF-α, has successfully completed phase I and pharmacokinetic studies aiming the treatment of patients with advanced and metastatic solid tumors, including breast cancer (NCT00356980 and NCT00436410).146 This nanoplatform induced minimal side effects and presented specific accumulation in tumors and not in healthy tissues, especially when breast tissue was considered; Cornell dots (7 nm 34 ACS Paragon Plus Environment

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PEGylated silica nanoparticles loaded with Cy5 dye and radiolabeled RGD peptides for enhancing PET imaging)147,148, are currently being evaluated for imaging of melanoma and malignant brain tumors (NCT01266096) and visualization of lymph nodes with metastases in a real-time (during surgery) in patients with head and neck melanoma, cervical/uterine and breast cancers (NCT02106598).

Conclusions In this review, we have highlighted the preclinical emerging approaches in breast cancer nanomedicine based on active targeting using versatile inorganic nanoplatforms with biomedical relevance (gold, silica, and iron oxide nanoparticles) along with their reported efficacy in breast cancer imaging, drug/gene delivery, thermal therapy, combinational therapy and theranostics. Despite the optimism about the positive results observed in preclinical results, many problems should be solved before determining the clinical relevance of these nanoplatforms. Indeed, although the vast literature, only few nanoplatforms will be able to evolve to the clinical trial stage. Thus, efforts should be made to overcome/minimize the current barriers for the translation of targeted inorganic nanoplatforms from the bench to the clinic. The implementation of standard methods based on appropriate models for preclinical studies may certainly improve the number of inorganic nanoplatforms actively targeted under clinical trials. Furthermore, questions related to the long-term toxicity, biocompatibility and exact molecular display of these nanoplatforms should be specially addressed. Collaborations between academia and pharmaceutical companies are welcomed to benefit the development in the field and accelerate the clinical approval by regulatory agencies since the requirements needed may be more rapidly achieved. Nanotechnology offers a possibility for early breast cancer detection and more effective therapeutics to significantly impact the rates of breast cancer mortality. However, the full benefits of nanotechnology on cancer medicine are not yet achieved. Current nanoplatforms have not provided significant improvements in the breast cancer clinical practice as expected. The next phase of cancer nanomedicine is based on more personalized therapies arising by the rational design of nanoplatforms. In this way, the development of innovative strategies and advanced systems based on active targeting using versatile inorganic nanoplatforms may open new frontiers and perspectives in breast cancer nanomedicine.



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Competing Financial Interests: The authors declare no competing financial interests.

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