Nanotheranostics for personalized medicine

Advanced Drug Delivery Reviews 64 (2012) 1394–1416

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Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

Nanotheranostics for personalized medicine☆ Simona Mura, Patrick Couvreur ⁎ Univ Paris-Sud, Faculté de Pharmacie, 5, rue J.B. Clément, 92296 Châtenay-Malabry Cedex, France CNRS UMR 8612, Institut Galien Paris-Sud, 5, rue J.B. Clément, 92296 Châtenay-Malabry Cedex, France

a r t i c l e

i n f o

Article history: Received 29 March 2012 Accepted 15 June 2012 Available online 21 June 2012 Keywords: Theranostic Nanomedicine Drug delivery Non invasive imaging Cancer therapy Cardiovascular disease Personalized nanomedicine

a b s t r a c t The application of nanotechnology in the biomedical field, known as nanomedicine, has gained much interest in the recent past, as versatile strategy for selective drug delivery and diagnostic purposes. The already encouraging results obtained with monofunctional nanomedicines have directed the efforts of the scientists towards the creation of “nanotheranostics” (i.e. theranostic nanomedicines) which integrate imaging and therapeutic functions in a single platform. Nanotheranostics hold great promises because they combine the simultaneous non-invasive diagnosis and treatment of diseases with the exciting possibility to monitor in real time drug release and distribution, thus predicting and validating the effectiveness of the therapy. Due to these features nanotheranostics are extremely attractive for optimizing treatment outcomes in cancer and other severe diseases. The following step is the attempt to use nanotheranostics for performing a real personalized medicine which will tailor optimized treatment to each patient, taking into account the individual variability. Clinical application of nanotheranostics would enable earlier detection and treatment of diseases and earlier assessment of the response, thus allowing screening for patients which would potentially respond to therapy and have higher possibilities of a favorable outcome. This concept makes nanotheranostics extremely appealing to elaborate personalized therapeutic protocols for achieving the maximal benefit along with a high safety profile. Among the several systems developed up to now, this review focuses on the nanotheranostics which, due to the promising results, show the highest potential of translation to clinical applications and may transform into concrete practice the concept of personalized nanomedicine. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanotheranostics for personalized medicine . . . . . . . . . . . . . . . Nanotheranostics in cancer disease . . . . . . . . . . . . . . . . . . . 3.1. MRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. MRI and chemotherapy . . . . . . . . . . . . . . . . . 3.1.1.1. T1-weighted nanotheranostics . . . . . . . . . 3.1.1.2. T2-weighted nanotheranostics . . . . . . . . . 3.1.2. MRI and chemo/photothermal therapy . . . . . . . . . . 3.1.3. MRI and pro-coagulant therapy . . . . . . . . . . . . . 3.1.4. MRI and photodynamic therapy . . . . . . . . . . . . . 3.2. Optical imaging . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Optical imaging and chemotherapy . . . . . . . . . . . 3.2.2. Optical imaging and photodynamic therapy . . . . . . . . 3.2.3. Optical imaging and photothermal therapy . . . . . . . . 3.2.4. Optical imaging and gene therapy . . . . . . . . . . . . 3.3. Combined MR/optical imaging and therapy . . . . . . . . . . . . 3.4. Ultrasonography . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Ultrasonograpy and chemotherapy . . . . . . . . . . . . 3.5. Combined photoacoustic/optical imaging and photothermal therapy

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Personalized nanomedicine”. ⁎ Corresponding author. Tel.: + 33 1 46 83 53 96; fax: + 33 1 46 83 59 46. E-mail address: [email protected] (P. Couvreur). 0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2012.06.006

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4.

Nanotheranostic in cardiovascular diseases . . . 4.1. MRI and anti-angiogenic therapy . . . . . 4.2. MRI and anti-restenotic therapy . . . . . 4.3. Optical imaging and photodynamic therapy 4.4. Ultrasonography and thrombolytic therapy 5. Concluding remarks . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The term "nanomedicine" has been proposed to refer to the application of nanotechnologies to the biomedical field for controlled delivery of drugs as well as for diagnostic purposes [1]. In the past decades, several nanosized carriers, made of different materials such as lipids, polymers, carbon or metal have been designed and widely characterized with the aim to overcome the limitation of the traditional drug delivery modalities [2–12]. For example, drug loaded nanocarriers were found to be able to accumulate at the tumor site by “passive targeting” through the enhanced permeability and retention (EPR) effect, due to the large pores present at tumor endothelial vessels (compared to healthy tissue) and to a reduced tumor lymphatic drainage [13–15]. Noteworthy, relying only on the EPR effect and therefore on the variable tumor anatomy, passive targeting would, in some cases, not allow a sufficient amount of drug to reach the target site. For this reason, in order to improve the treatment specificity, passive targeting has often been associated to a so called “active targeting” obtained by decorating the surface of the nanocarriers with molecules able to recognize specific ligands expressed by target cells [5,16–20]. This approach offers the advantages to improve drug therapeutic efficiency and to dramatically reduce toxicity and unspecific side effects, thus leading to possible future evolution in the management of severe diseases. Mainly, nanomedicines have found wide applications in the treatment of cancers [21], however neurodegenerative [22,23] and inflammatory/autoimmune diseases [24,25], diabetes [26,27], as well as lung [28], and cardiovascular affections [29,30], can benefit from the use of nanosized carriers for drug delivery. Nevertheless, despite extensive efforts on developing targeted drug delivery systems, up to now, only very few products have achieved success in the clinic and reached the marketplace. Successful examples include liposomal formulation of doxorubicin (Doxil and Myocet) and paclitaxel (Abraxane and Xyotax) [31–34]. Aside from therapeutic interventions, actively and passively targeted nanomedicines have been employed in recent years also as imaging tools which hold great promises both in preclinical research and in clinical settings [35–42]. The currently accessible imaging techniques include magnetic resonance imaging (MRI), optical imaging, ultrasonography (US), positron emission tomography (PET), computer tomography (CT) and single photon emission computed tomography (SPECT). The combination of the variety of available nanocarriers with different imaging contrast agents (i.e. paramagnetic metal ions, superparamagnetic iron oxide (SPIO) nanoparticles (NPs), Near Infra Red (NIR) probes, radionuclides) led to the development of versatile platforms which, enabling single or multimodal imaging, are extremely interesting for both detection and diagnosis of diseases [11,43]. Indeed, in vivo application of these nano-based imaging agents allows achieving an enhancement of the signal to noise ratio in the targeted tissue compared to the surrounding health one. The increase of the imaging resolution enables to discover also small lesions which are undetectable with traditional methods [44]. Moreover, imaging technologies allow following the biodistribution of the nanocarriers, to determine their mechanism of action and to monitor in real time the disease progression [45–47]. However, these nanocarriers loaded with contrast agents do not usually offer therapeutic effects. To handle the rapid proliferation of severe diseases such as cancer, neurological and cardiovascular diseases, there is a need for improved diagnostic and therapeutic strategies allowing early detection and treatment. Recent advances in nanoscience and biomedicine and the convergence of these disciplines have now expanded the ability to

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design and construct “multifunctional” nanoparticles, combining targeted therapeutic and diagnostic functions in a same entity [48–53]. Thus, theranostic nanomedicines emerge as an alternative to the separate administration of diagnostic probes and pharmacologically active molecules. The various imaging modalities currently available offer the possibility of a longitudinal study which enables to monitor changes at the target site in response to the treatment and to gain insights on disease progression and efficacy of the intervention at an early stage [54]. The efficient combination of a therapeutic agent with an imaging molecule in a single nanomedicine and the extreme versatility of such so called “nanotheranostic” platform would therefore contribute to the development of optimized and individualized treatment protocols, offering the opportunity to perform a “personalized nanomedicine”. Theranostic nanomedicines have shown interesting results during in vitro studies but there are still some challenges for their application in vivo and in the clinical treatment of patients. A simple research on PubMed® database reveals that more than 400 articles have been published in the last decade within this field. Most of them describe the preparation and physico-chemical characterization of nanotheranostics with sometimes an in vitro evaluation on cell culture but without any proof of concept in vivo. Other are describing in vivo data related to either the therapeutic or the imaging function but not a combination of both. Therefore, they may not be considered as real theranostic systems. The most relevant of these studies, which are too far from the personalized medicine, will not be discussed in this review but are just summarized in Table 1. Noteworthy, the number of papers dealing with the in vivo evaluation of real theranostic nanodevices (i.e. combining therapy and imaging) is progressively increasing (around 15% of currently published articles). It is evident that extensive in vivo investigation is needed before the clinical application of the nanotheranostic concept. This is the reason why, in this review, we have mainly focused our attention on the nanotheranostics which have demonstrated some preclinical relevance to potentially make shorter the step for their introduction in clinical trials. Here, we have chosen to classify nanotheranostics as function of their imaging properties. In the first part, nanotheranostic for non-invasive imaging and treatment of cancer will be discussed, and then attention will shift to cardiovascular diseases and in particular to atherosclerosis. Detailed description of nanocarriers and imaging modalities was out the scope of this review, but the reader can refer to excellent articles published in the last years [43,49,52,55–64]. 2. Nanotheranostics for personalized medicine The expression “personalized nanomedicine” refers to the use of nanosized carriers to elaborate optimized treatment protocols tailored to each specific patient. In the simplest definition, personalized medicine consists to administer “the right drug to the right patient at the right moment” [101,102]. With the aim of developing a patient specific therapy, pharmacogenomic, pharmacoproteomic and a wide variety of -omic strategies have been developed in the last years [103–109]. These different techniques allow a detailed genetic and molecular profile of each patient, contributing to identify the molecular biomarkers which would affect the evolution of the disease and the response to treatments. Thus, personalized medicine is not only limited to the study of biomarkers and genetic polymorphisms [105,110], but relies also on the development of strategies for the detection of a disease and the prediction of the therapeutic response. In this perspective,

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Table 1 Promising examples of nanotheranostics. In these studies, despite extensive physico-chemical characterization and/or in vitro investigation, no clear-cut proof of concept of combined imaging and therapeutic activity was however provided in vivo. Nanocarrier

Imaging modality

Contrast agent

Therapeutic activity

Drug

Reference

Doxorubicin [65]a Doxorubicin Camptothecin [66]a Recombinant tissue-type Plasminogen Activator [67]a (rt-PA) Nanoparticles MRI Iron oxide Chemotherapy Doxorubicin [68]b Nanoparticles MRI Iron oxide Chemotherapy Paclitaxel [69]b Nanoparticles MRI Iron oxide Chemotherapy Docetaxel [70]b Nanoparticles MRI Iron oxide Chemotherapy Curcumin [71]b Nanoparticles MRI Perfluorooctylbromide Chemotherapy Doxorubicin/Paclitaxel [72]b Nanoparticles MRI Iron oxide Gene therapy siRNA [73]b Liposomes MRI Gd3+ Chemotherapy Doxorubicin [74]b Wormlike-polymer vesicles MRI Iron oxide Chemotherapy Doxorubicin [75]b Micelles MRI Iron oxide Chemotherapy Doxorubicin [76]b Micelles MRI Iron oxide Chemotherapy Doxorubicin [77]b Gold nanoshells MRI Iron oxide PTT [78]b Nanoparticles Optical Imaging Gold nanoparticles PTT [79]b Nanoparticles Optical Imaging Indocyanine green Chemotherapy Doxorubicin [80]b Upconversion nanoparticles Optical Imaging Lanthanide-doped nanocrystals Chemotherapy Doxorubicin [81]b Dendrimers Optical Imaging Fluorescein Chemotherapy Methotrexate [82]b Gold nanoparticles Optical Imaging Gold nanoparticles Chemotherapy Paclitaxel [83]b Gold nanoparticles Optical Imaging Plasmonic nanobubbles Mechanical cell damage Plasmonic nanobubbles [84]b Gold nanoshells Optical Imaging Gold nanoparticles PTT [85] Gold nanorods Optical Imaging Gold nanorods PTT [86]b Gold nanorods Optical Imaging Gold nanorods PTT [87]b Echogenic liposomes US Liposomes Thrombolysis Recombinant tissue-type Plasminogen Activator [88] (rt-PA) Nanoparticles MRI/Optical Imaging Iron oxide/Cy5.5 dye Chemotherapy Noscapine [89]b Nanoparticles MRI/Optical Imaging USPIO/Rhodamine B Chemotherapy Methotrexate [90]b Nanoparticles MRI/Optical Imaging Iron oxide/cyanine dye Chemotherapy Paclitaxel [91]b Gold nanoshells MRI/Optical Imaging Iron oxide/gold nanoshells PTT [92]b Nanoparticles MRI Iron oxide Chemotherapy paclitaxel, rapamycin, carboplatin [93]c Nanoparticles MRI CoFe2O4 PTT [94] c Nanoparticles MRI Iron oxide Chemotherapy Mitoxantrone [95]c Micelles MRI Gd3+ Chemotherapy Paclitaxel [96]c Micelles MRI Gd3+ Chemotherapy Doxorubicin [97]c Liposomes MRI Gd3+ Chemotherapy Doxorubicin [98]c Micelles/microbubbles US Perfluoropentane Chemotherapy Doxorubicin [99]c Micellar hybrid nanoparticles MRI/Optical Imaging SPIO/quantum dots Chemotherapy Doxorubicin [100]c

Polymersomes Nanobialys Echogenic liposomes

a b c

MRI MRI US

Maghemite nanoparticles Manganese (III) Liposomes

Chemotherapy Chemotherapy Thrombolysis

Only physico-chemical characterization. Physico-chemical characterization and in vitro investigation. Physico-chemical characterization, in vitro investigation, in vivo assessment only of imaging or therapeutic function.

theranostic nanomedicines, which integrate therapeutic and imaging agents in the same nanocarrier, could contribute to develop a personalized approach in the management of grave diseases. Being conceived for non-invasively surveying the evolution of a disease during the treatment, nanotheranostic will, in other words, drive toward the personalization of clinical treatments which would reflect the specific characteristics of the disease in each patient. Non-invasive monitoring of drug accumulation at the target site may enable to screen patients who are likely to positively respond to the treatment (characterized by high accumulation of the nanomedicine) from others who would need a different therapeutic option. Moreover, the evaluation of the accumulation also in healthy tissues would allow determining the risk of patients to develop side effects. The treatment, therefore, could be optimized in order to achieve the highest therapeutic efficiency along with the best safety profile. The possibility of having early feedbacks on the effectiveness of the treatment offers an important advantage permitting a better management of the disease, thus increasing the possibility of remission. Indeed, treatment can be tuned in real time without waiting for traditional end points, such as the tumor wrinkling. Assessing the accumulation at the target site, nanotheranostics enable to predict the effectiveness of a treatment and may also provide a justification for the failure of the drug targeting approach in certain diseases. For example, it is well known that cancer treatment takes advantage of the passive drug accumulation in the tumor due to the previously mentioned EPR effect [111]. The efficacy of anticancer drugs such as Doxil® (doxorubicin-loaded nanoparticles) and Abraxane® (albumin-bound nanoparticles form

of paclitaxel), which are already approved for the therapy of solid tumors (i.e. ovarian, breast cancer and Kaposi Sarcoma) [31–33,112,113] is based on this mechanism. However, the efficiency of the EPR effect is not completely understood and individual differences are observed at the different stages of the disease with, additionally, a high variability among patients. Therefore, the idea that “one fits all” and that a single therapeutic agent may be used for the treatment of all patients is not conceivable. A scarcely EPR effect seems to be the cause of the absence of response in the treatment of solid tumors such as the pancreatic adenocarcinoma [114], and the diffuse-type gastric carcinoma [115]. For these high malignant tumors, various chemotherapeutic agents have shown high efficiency in vitro but they failed in vivo. This discrepancy is probably due to the physiology of the tumor which includes fibrosis and hypovascularization which oppose to drug diffusion [116,117]. Moreover, significant differences can be observed also in the same tumor type, probably correlated to inter-patient variability, related to the density and structural integrity of the tumor neovasculature. Thus, a rigorous evaluation of the extent of the vasculature leakage and of the drug accumulation into the tumor allows predicting the outcome of the treatment [31,118,119]. A proof of concept of this strategy has been provided by Karathanasis et al., which have used iodine-labeled liposomes in prediction of the therapeutic response to doxorubicinloaded liposomes treatment, in a rat breast tumor model [120]. Good or bad prognosis animal groups were created by measuring the X-Ray signal enhancement which reflected the tumor accumulation of i.v. injected iodine-labeled liposomes. After treatment, the evaluation of

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the tumor growth rate confirmed the previsions: a slower tumor grow rate was associated to the highest signal enhancement in the tumor and therefore to a leakier vasculature. This study represents a clear example of how theranostic nanomedicine could facilitate the personalized treatment of breast cancer. Indeed, clinical translation of this protocol would enable to prescreen patients, predicting which ones would have a positive outcome to the treatment due to an incomplete vasculature formation and a more important EPR effect. For potential nonresponder patients another optimized and personalized option might be considered thus avoiding the rigor of this treatment. 3. Nanotheranostics in cancer disease Due to the dramatically high number of death caused every year by cancer [121], much effort has been done to improve the traditional treatment of cancer by developing theranostic nanomedicines. It is probably in the oncology field that the possibility to combine imaging and therapy rises the more useful and interesting opportunities for developing personalized medicine [122]. Indeed, it is obvious that a strategy which would enable the early diagnosis and therapy, the prediction of the therapeutic efficacy as well as the follow up, would provide significant benefits in the management of cancers, thus facing the heterogeneity of the disease and the individual differences [123]. Although the proof of concept of the effectiveness of certain theranostic nanomedicines has been provided, many of them are far from the clinical trials. 3.1. MRI MRI is a powerful non-invasive technique which offers the possibility of deep penetration into soft tissues, high spatio-temporal resolution associated to a relative ease of use [49,124]. MRI signal is physically based on the interaction of certain nucleus (generally protons) with each other and with the surrounding molecules within an applied magnetic field. The different T1 (longitudinal) and T2 (transversal) proton relaxation times in the different tissues generate the endogenous contrast. However, exogenous contrast agents are usually used to increase the contrast and the signal/noise ratio, thus improving the detection sensitivity. Contrast agents act by shortening the T1 or the T2 relaxation time thus leading to a bright (positive) or dark (negative) contrast enhancement. Paramagnetic metal ions such as Gd3+, manganese (Mn2+ and Mn3+) linked to different molecules or NPs are used as T1 contrast agents, while iron oxide NPs are the most largely used T2 contrast agents [124]. Gd3+ is a MRI contrast agent FDA approved and currently, several Gd 3+-based contrast agent formulations exist in the clinic. However in recent years, iron oxide NPs have found widest application due to the higher magnetic moment which enables to reach a highest resolution and soft tissue contrast. Since the superparamagnetic properties of iron oxide NPs allow them to provide an enhancement of the contrast, it enables the non invasive monitoring of their tissue distribution and accumulation. Moreover, in addition to their magnetic properties, iron oxide NPs can be used for the entrapment of various drugs [125–127]. Therefore, they are extremely attractive for developing theranostic nanodevices for personalized nanomedicine [95,128]. 3.1.1. MRI and chemotherapy Various nanodevices have been proposed for the conception of nanotheranostics. They include multifunctional nanoparticles, polymeric micelles, as well as liposomes often enriched with iron oxide NPs or paramagnetic metal ions. 3.1.1.1. T1-weighted nanotheranostics. Polymeric micelles made of self assembling block co-polymers are characterized by a hydrophobic core that can load hydrophobic molecules and an external hydrophilic layer which assures the colloidal stabilization in aqueous media. Due to these properties, polymeric micelles have also attracted interest for

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the development of multifunctional theranostic nanodevices, combining therapeutic drugs, targeting molecules and contrast imaging agents [19]. Several kinds of micelles have been developed as theranostics for the diagnosis and the treatment of cancer, even if the validation of the systems in vivo is far to be entirely completed. They open, however, exciting opportunities for the development of novel strategies for improving the management of cancer therapy. Indeed, as already mentioned, monitoring in a real time the distribution of the polymer micelles in the body and their accumulation at the target site, would enable to predict the effectiveness of the treatment and to optimize the therapy according to the specific characteristics of the disease in each patient [66,77,100,129]. An example of multifunctional cancer-targeted drug loaded micelles with MRI contrast properties has been proposed by Kaida at colleagues [130]. In this construction, poly (ethylene glycol)-b-poly(glutamic acid) block copolymer has been used to form micelles, showing a mean diameter of around 30 nm, which encapsulated in their core both Gd 3+ and the anticancer drug DACHPt (oxaliplatin derivative). The potential of Gd-DTPA–DACHPt-loaded micelles as imaging and therapeutic systems has been evaluated on an orthotopic human pancreatic (BxPC3) xenograft model using as control free Gd-DTPA and free Oxaliplatin. It was found that intravenously injected Gd-DTPA–DACHPt-loaded micelles highly accumulated in the tumor area leading to a strong enhancement of the MRI contrast signal while no enhancement was observed following administration of free Gd-DTPA. The effectiveness of the treatment has been monitored by MRI correlating the signal enhancement at the tumor region, the accumulation in this area of Gd-DTPA–DACHPt-loaded micelles and the size of the tumor. It was observed that Gd-DTPA–DACHPt-loaded micelles allowed an accumulation of Gd-DTPA up to seven times higher than the GdDTPA free in solution which also showed a faster metabolism. The subsequent release of the encapsulated drug led to a significant reduction in the volume of the orthotopic tumors. Since these micelles showed an important anticancer efficacy and a significant increase of the tumor's MRI contrast, they may also represent a suitable strategy for early detection, treatment and monitoring of tumor pancreatic lesions, small liver metastasis and peritoneal dissemination. Indeed, in this study, the excellent contrast resolution of the MRI might enable to easily distinguish tumor from the health tissue where no accumulation of the micelles was observed. Liposomes have proven to be highly suitable carriers for the delivery of several molecules with different physico-chemical properties allowing encapsulation either in the internal aqueous core (hydrophilic drugs) and/or intercalated in the lipid bilayer (lipophilic drugs). Therefore, these lipid carriers have been taken into consideration for the development of theranostic nanomedicines by carrying both therapeutic and contrast imaging agents [131,132]. Developing this concept, PEG liposomes encapsulating both doxorubicin as a therapeutic molecule and a gadolinium derivative (Gd-DOTA monoamide) as T1 MRI contrast agent have been proposed by Grange et al. [133]. Those liposomes were investigated in a severe immunodeficient mouse model of human Kaposi's sarcoma expressing neural cell adhesion molecules (NCAM). Active targeting toward these cells was achieved by decorating these liposomes with the C3d NCAM-binding peptide (C3d-lipo). Indeed, C3d-lipo showed an enhanced therapeutic efficacy and a lower toxicity compared to nontargeted control liposomes or to free doxorubicin. Moreover, the tumor accumulation of the liposomes and the therapeutic efficacy of the treatment were measured by MRI, clearly evidencing the reduction of the tumor mass after administration of C3d-lipo. A similar approach was followed by Cittadino et al. which investigated the theranostic properties of PEG liposomes, incorporating an amphiphilic Gd3+-based contrast agent (Gd-DOTAMAC(C18)2 and the glucocorticoid predinisolone phosphate, in a B16 melanoma mouse model [134]. Inhibition of the tumor growth was observed five days from the first treatment confirming that the release of the drug from the carrier was not significantly affected by the presence of the MRI contrast agent. Moreover, the Gd3+-based contrast agent allowed performing the non-invasive in vivo monitoring of

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the liposomes biodistribution and the simultaneous accurate assessment of the therapeutic response, highlighting the potential of this approach for in vivo follow up of disease progression. Doxorubicin and manganese loaded thermosensitive liposomes (Dox/Mn-LTSLs) were proposed by Ponce et al. [135] as multifunctional systems for drug delivery and MRI contrast enhancement. In this study, a novel MRI technique was used to monitor spatial and temporal tissue distribution of doxorubicin (i.e. drug dose painting) in a rat fibrosarcoma model allowing again to predict the therapeutic effect of the treatment. Different treatment protocols were applied to evaluate the influence of locally applied hyperthermia on the drug release and antitumor activity: (i) hyperthermia initiated 15 min before administration of Dox/MnLTSLs; (ii) administration of Dox/Mn-LTSLs 15 min before initiation of hyperthermia or (iii) half of the dose administered 15 min before the initiation of hyperthermia and the other half, 15 min after the initiation of hyperthermia. Saline and Dox/Mn-LTSLs, without hyperthermia, were used as controls. MR images of the animals were acquired before, during and after treatment with Dox/Mn-LTSLs plus hyperthermia. Signal enhancement was observed in all treated groups although the localization of the bright areas was dependent on the protocol followed. Uniform tumor enhancement was observed only following double administration of half of the dose of Dox/Mn-LTSLs, while unique administration before or after initiation of hyperthermia led to central or peripheric enhancement, respectively. Signal enhancement, due to shortening of T1 relaxivity was converted first to manganese and then to doxorubicin concentrations. This correlation allowed calculating the total dose of doxorubicin accumulated in the tumor, which was found significantly higher when hyperthermia was initiated 15 min before administration of Dox/Mn-LTSLs. This last protocol resulted in the highest therapeutic efficacy calculated as being the time needed (here 34 days) to increase the tumor volume by 5-times. On the contrary, a median time of 18.5 days was requested when Dox/Mn-LTSLs were administered before initiation of hyperthermia and 22.5 days were needed when the protocol using two injections was applied. Recently, comparable thermosensitive liposomes (HaT: Hyperthermia activated cytoToxic) [136], co-encapsulating Gd3+ and doxorubicin (HaTDOX-Gd) were tested in vivo, on a mouse model of mammary carcinoma (EMT-6) [137]. After i.v injection of HaT-DOX-Gd, animals were imaged by MR in order to follow the release and the uptake of doxorubicin in locally heated tumors. A perfect correlation was observed between the drug accumulation and the variation of the MR T1 relaxation time immediately after the treatment, thus confirming the possibility of using MRI to quantify the drug delivery and predict the therapeutic outcomes. Indeed, HaT-DOX-Gd+hyperthermia-treated mice showed a significant dosedependent inhibition of the tumor growth, while no reduction of tumor volume was observed in non heated mice. With the aim of a rapid translation of this strategy to clinical practice the same authors envisioned a protocol which would drive the evaluation of the best future treatment for each patient on the basis of the individual response, thus making possible to perform a real personalized therapy (Fig. 1). However, the methodology used in this study to rise tumor temperature (submersion in hot water of the tumor-bearing limb) is not clinically applicable. In clinical trials, local hyperthermia would be achieved with imaging-guided heating devices such as MR-focused ultrasounds. In a nutshell, multifunctional liposomes provide an excellent example of nanotheranostics which enable to monitor the effectiveness of therapeutic protocols and their optimization in real time, thus paving the road to the application of similar nanotechnologies for a personalized nanomedicine. 3.1.1.2. T2-weighted nanotheranostics. Multifunctional magnetic nanohybrids (MMPNs) have been prepared by Yang et al. [138] by combination of doxorubicin as anticancer drug, magnetic nanocrystals (MnFe2O4 or Fe3O4, mean diameter around 80 nm) as MRI contrast agent and biodegradable block copolymers, as stabilizers. The surface of the MMPNs was functionalized with the anti-HER antibody

Fig. 1. Flowchart of the proposed protocol for personalized therapy with the HaT-DOXGd formulation. Reprinted with permission from Elsevier Publisher: Biomaterials [137].

(HER, herceptin) (HER-MMPNs) in order to selectively target the human epidermal growth factor receptor 2 (HER2), a highly expressed tumor marker in metastatic breast cancer. The effectiveness of these nanohybrids has been evaluated for theranostic purpose in vivo on a subcutaneous model of breast cancer developed after injection of HER2/neu cancer marker-expressing NIH3T6.7 cells, in the proximal thigh region of mice. Animals were imaged by MR at different time points after injection of HER-MMPNs or NPs functionalized with an irrelevant antibody (IRR-MMPNs). With respect to images recorded pre-treatment, a clear and immediate color change to black associated to a ΔR2/R2pre (R2 = T2− 1) value increase of 50.5% was observed after injection of HER-MMPNs. At 12 h, the increase was of about 23.2%. By contrast, using IRR-MMPNs the signal enhancement was only of 14.4% immediately after treatment and of 6.2% at 12 h (Fig. 2a–h). Interestingly, HER-MMPNs not only increased the tumor contrast enhancement but also showed a significant therapeutic activity compared to IRRMMPNs, free doxorubicin, HER or DOX + HER control solutions. Indeed, HER-MMPNs caused a significant tumor growth inhibition (Fig. 2i), thus confirming active tumor targeting and important cell internalization by receptor mediated endocytosis which enabled both ultrasensitive tumor imaging (due to MnFe2O4 or Fe3O4) and therapeutic treatment (due to doxorubicin release). Doxorubicin-loaded thermally cross-linked superparamagnetic iron oxide NPs (Dox@TCL-SPION) are another promising nanoconstruction for combined cancer therapy and diagnosis [139]. Simultaneous contrast imaging enhancement and antitumor efficacy were evaluated in vivo on a subcutaneous model of Lewis lung carcinoma in mice. Before injection of Dox@TCL-SPION, the tumor appeared hyperintense in T2-weighted MR images, while post injection an intense darkening was observed, with a relative signal enhancement of 58%, suggesting large accumulation of the NPs. According to the relative signal enhancement, the maximal accumulation of Dox@TCL-SPION occurred between 4.5 and 12 h with a progressive elimination within the 24 h. As confirmed by MR imaging, the preferential accumulation of Dox@TCL-SPION in the tumor allowed achieving an excellent anticancer activity. Indeed, a significant tumor

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Fig. 2. MR images and color maps in tumor region of cancer targeting events of HERMMPNs (a–d) and IRR-MMPNs (e–h) in breast cancer bearing mice at various time intervals. (a,e) preinjection; (b,f) immediately after injection; (c,g) t = 1 h; (d,h) t = 12 h post injection of MMPNs. (i) Comparative therapeutic-efficacy study in breast cancerbearing mice. Adapted with permission from John Wiley and Sons Publishers: Angewandte Chemie International Edition [138].

growth inhibition (of 63%) was observed after injection of Dox@TCLSPION (0.64 mg Dox/kg) which was not observed with free doxorubicin at the same equivalent dose or even at a ten-times higher concentration

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(38% tumor growth inhibition for a dose of 5 mg/kg). The Dox@TCLSPION nanodevice was further modified by surface conjugation with prostate-specific membrane antigen (PSMA) aptamers for specific recognition of PSMA- over-expressing prostate cancer cells (Dox@Apt-hybrTCL-SPION) [140]. Imaging and therapeutic properties were evaluated in vivo in a LNCaP human prostate cancer mouse model. Animals were imaged by MR pre and post injection (t=2, 24, 48 h) of Apt-hybr-TCLSPION. After 2 h, an important darkening was observed in the tumor area with a relative signal enhancement (of 53%) in the region of interest. The signal drop was maintained up to 48 h, confirming the high accumulation of Apt-hybr-TCL-SPION. Contrariwise, at 2 h, scrambled aptamer conjugated TCL-SPION (scrApt-hybr-TCL-SPION) caused a lower relative signal enhancement (of 21%) and due to their progressive elimination, no signal drop was observed after 24 h (Fig. 3a). The therapeutic activity was assessed by evaluating the tumor growth rate in groups treated with Dox@Apt-hybr-TCL-SPION compared to free Dox, 5% glucose, Apthybr-TCL-SPION or scrambled Dox@scrApt-hybr-TCL-SPION treated control groups. Dox@Apt-hybr-TCL-SPION exhibited a superior inhibition of tumor growth and, at the end of the treatment, the tumor volume was around 54% of those in control groups (Fig. 3b). The reduction of the tumor volume was validated also by MR images recorded at the end of the treatment, which clearly confirmed the possibility of a non-invasive longitudinal monitoring of the effectiveness of the therapy. The association of iron oxide nanoparticles and doxorubicin has been investigated also by Quan et al. [141]. Human serum albumin, which has been shown to hold excellent ligand-binding properties [142,143], was used to coat the iron oxide NPs in order to achieve a high loading of the anticancer drug (D-HINPs). The potential as theranostic agents of D-HINPs was assessed on a xenograft 4T1 murine breast cancer model. D-HINPs were injected through the tail vein and three-dimensional gradient-echo scan MR images were acquired before injection, 1 h and 4 h post injection. Accumulation of D-HINPs in the tumor area was associated to a high hypointensity and an appreciable signal drop. In parallel to contrast enhancing properties, D-HINPs showed an interesting anticancer activity comparable to the commercial formulation Doxil. Nevertheless, despite this similar therapeutic efficacy, these NPs represent an interesting platform further exploitable for other therapeutic molecules. Another interesting example of theranostic nanomedicine, has been proposed very recently by Arias et al. [53]. In this construction, ultrasmall superparamagnetic iron oxide (USPIO) NPs (mean diameter and polydispersity index: 9±2 nm and 0.132, respectively) were incorporated into bigger NPs obtained by the self assemblage of a prodrug made of the anticancer drug gemcitabine conjugated to the squalene, a natural and biocompatible lipid (ie. the so-called “squalenoyl-gemcitabine conjugate” SQgem) [144,145]. The resulting USPIO/SQgem NPs (mean diameter around 270 nm) combine tumor therapy and MR imaging functionalities. Moreover, due to their magnetic susceptibility, these nanocomposites can be guided to tumors by application of an external magnetic field. The efficacy of this approach has been demonstrated in vivo on an experimental solid tumor model (L1210 murine leukemia). Under the influence of an external magnetic field, the intravenously injected USPIO/SQgem NPs, indeed, accumulated at the tumor site and could be visualized by MRI. T2-weighted images of the tumors post injection of USPIO/SQgem NPs showed hypo-intense areas (uniformly distributed in the entire tumor mass), due to a decrease of the T2 relaxivity, well correlated with the local concentration of NPs. After administration of USPIO/SQgem without the application of the magnetic field, a lower enhancement of the image contrast was observed (Fig. 4a–c). Moreover, the magnetically-guided USPIO/SQgem nanoparticles led to significantly higher anticancer activity compared to control groups, thus providing again the proof of concept that the simultaneous tumor visualization and treatment were possible (Fig. 4d). In addition, the same authors have already obtained promising results by enlarging this strategy to other contrast agents (Gd3+) and other anticancer drugs (paclitaxel, doxorubicin and cisplatin), thus confirming the versatility and the generic character of this nanoplatform.

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Fig. 3. (a) T2-weighted fast-spin echo images at the level of the LNCaP tumor on the right side of the mouse taken at 0, 2, 24, and 48 h after injection of Apt‐hybr‐TCL-SPIONs or scrApt–hybr–TCL-SPIONs. The dashed circle indicates the xenografted tumor region. (b) Antitumor activity of Dox@Apt‐hybr‐TCL-SPIONs in the LNCaP xenograft animal model (*P b 0.05, n = 6). Adapted with permission from John Wiley and Sons Publishers: Small [140].

Polymeric micelles for simultaneous in vivo monitoring by MR imaging and drug delivery were proposed by Blanco et al. [146]: PEGPLA (poly lactic acid) micelles loaded with doxorubicin and SPIO NPs were functionalized with the targeting ligand cRGD (cRGDDOX-SPIO micelles). The effectiveness of this nanoconstruction as theranostic agents has been assessed in vivo using an orthotopic H1299 non-small cell lung carcinoma model. T2 weighted images were accumulated before and 15 h after i.v. injection of cRGD-DOXSPIO micelles enabling to observe a significant darkening in the tumor areas which confirmed the accumulation of the micelles in this tissue. Antitumor efficacy was followed by bioluminescence imaging using the H1299 tumor model transfected with the firefly luciferase gene. The value of relative bioluminescence unit, measured immediately before injection of the micelles, was of 1.2 × 10 6. After two treatments, at d = 1 and d = 3, mice were imaged at d = 7, revealing a significant reduction of the relative bioluminescence unit to 5.6 × 10 5 which corresponded to a 50% reduction of the tumor volume. Similar results were also obtained on a subcutaneous model of lung carcinoma, thus confirming the anticancer activity of these multifunctional micelles. 3.1.2. MRI and chemo/photothermal therapy Phototermal therapy (PTT) applies Visible-NIR light to excite photoabsorber molecules that convert the energy of the incident light to heat thus inducing apoptotic cell death. The increase in temperature ranges between 45 °C and 300 °C and the effect can be obtained at sufficient depth with NIR radiation. PTT is highly specific and it allows irradiation with extreme precision only of the diseased tissue with minimal damage of the healthy surrounding one [147]. Important characteristic of the agents used for PTT is a significant optical absorbance in the NIR “optical window” of the spectrum in which the absorption of the endogenous chromophores is very low. However, these dyes show a significant photo-bleaching which represents the major limits to their wide application. To overcome this problem not-photo bleaching plasmonic metal NPs have been developed. An interesting multifunctional system for simultaneous imaging and bimodal therapy (i.e. PTT and chemotherapy) has been proposed through the construction of taxol-loaded PLGA (poly (lactide-co-glycolide)) NPs conjugated to iron oxide NPs and quantum dots (QDs) [148]. The surface of these NPs was further decorated with gold nanorods (Au NR/QD/Fe3O4/ Taxol-loaded PLGA NPs) with the aim to use the capacity of nanorods to convert NIR light to heat to achieve photothermal ablation of tumor tissue as well as the release of the entrapped drug. In vivo studies were performed on A549 lung carcinoma bearing mice. Intravenous injection of Au NR/QD/Fe3O4/Taxol-loaded PLGA NPs caused a significant darkening in the tumor area, thus revealing their potential as MRI contrast agents.

The therapeutic efficacy was evaluated following various protocols to investigate the effectiveness of photothermal and chemotherapeutic treatments alone or in combination. Laser irradiation of drug free Au NR/QD/ Fe3O4/PLGA NPs, intratumorally injected, caused a higher anticancer activity compared to the chemotherapy alone (i.e. injection of Au NR/ QD/Fe3O4/Taxol-loaded PLGA NPs without irradiation), although tumors started to grow few days after the treatment. As expected, the highest efficacy was obtained in mice treated with Au NR/QD/Fe3O4/Taxol-loaded PLGA NPs and then laser irradiated, which showed a progressive reduction of the tumor volume. Such a strategy offers the possibility of imaging and simultaneously applying a focused therapy, thus reducing the damage of the surround health tissue and improving the therapeutic outcomes. 3.1.3. MRI and pro-coagulant therapy There is a wide consensus to recognize that tumor growth and metastasis are significantly dependent on blood supply [149], and up to now, several strategies have been adopted using various therapeutic agents in order to inhibit tumor neo-angiogenesis [150–152]. Another interesting and alternative approach is represented by the occlusion of the tumor vasculature in order to induce tumor necrosis [153,154]. Beside their well known contrast imaging properties, iron oxide NPs can exert anticancer activity as a consequence of their pro-coagulant properties. In other words, due to the fundamental role of angiogenesis in tumor development, the ability of iron oxides to cause occlusion of tumor vessels could make these NPs a theranostic system by themselves, by combining imaging and therapeutic properties. The feasibility of this strategy has been exploited in a recent report, in which the procoagulant properties of iron oxides have been enhanced by coating these NPs with peptides able to recognize the fibrin–fibronectin complexes on the wall of tumor vessels [155]. Agemy and colleagues developed NPs with an elongated shape by coating elongated iron oxides (i.e. nanoworms, long dimension of ~70 nm and thickness of ~30 nm) [156], with the CREKA (Cys–Arg–Glu–Lys–Ala) tumorhoming peptide or analogs, directly or by introduction of a 5 kDa PEG spacer. The effectiveness of this approach as theranostic agent has been evaluated on nude mice bearing 22Rv1 orthotopic human prostate cancer xenografts. Peptide-coated nanoworms were injected at the dose of 5 mg iron/kg body weight and allowed to circulate for 7–8 h. Then, animals were subjected to T2-weighted MRI scans which revealed hypointense vascular signals through the tumors. For tumor treatment, injections were repeated for 14 days causing up to 70% of tumor flow blockade which led to a strong reduction in tumor size, as well as an extended survival of the animals. Interestingly, peptide coatednanoworms accumulated non-specifically also in liver and spleen but no signs of blood clotting were observed elsewhere in the healthy

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tissues. In fact, peptide coated-nanoworms recognize products of blood clotting in the tumor vessels and their accumulation enhances contrast of tumor vasculature allowing an improved visualization. Simultaneously, they cause further clotting thus producing new binding sites for nanoworm recognition. Taken together, these effects led to an effective blockage of the tumor vascularization leading to consequent tumor necrosis. Such a “self-amplifying” nanotheranostic combines imaging and therapeutic properties of iron oxide NPs and offers a new interesting and alternative approach for the treatment of tumor neo-angiogenesis.

Fig. 4. Examples of T2-weighted images of the tumors obtained at 2 h-postinjection of USPIO/SQgem NPs (a) in the absence of an external magnetic field and (b) guided by an external magnetic field (c) Percentage of the hypo-intensity tissues with T2 b 36 ms (white column), and with T2 b 20 ms (gray column). Mouse 2 was injected with nanocomposites without exposition to magnetic field. Mice 1, 3, and 4 were injected with USPIO/SQgem NPs with 2 h exposure to 1.1 T magnetic field. (d) In vivo anticancer activity of USPIO/SQgem NPs (with extracorporeal magnetic field) (5 mg/kg equivalent of gemcitabine) compared with placebo-treated group (drug unloaded USPIO/squalene nanocomposites), USPIO/SQgem NPs (no extracorporeal magnetic field applied), with SQgem NPs and with gemcitabine free in L1210 subcutaneous tumor bearing mice. Untreated (●), placebo USPIO/squalene NPs (○), gemcitabine (◊), SQgem NPs (▲), USPIO/SQgem composite NPs (no extracorporeal magnetic field) (Δ), USPIO/SQgem composite NPs (with extracorporeal magnetic field) (■). Statistical analysis was performed using Student's t-test to compare the statistical significance of USPIO/SQgem composite NPs independently with gemcitabine and SQgem NPs. Data with **p b 0.05 and ***p b 0.001 were considered as significant and very significant, respectively. Adapted with permission from Arias, J. L. et al. “Squalene Based Nanocomposites: A New Platform for the Design of Multifunctional Pharmaceutical Theragnostics.” ACS Nano 2011, 5, 1513–1521. Copyright © 2011 American Chemical Society [53].

3.1.4. MRI and photodynamic therapy The photodynamic therapy (PDT) is an emerging modality for the treatment of various diseases which uses a photosensitizer-based drug, oxygen and light of opportune wavelength to generate oxygen reactive species which cause photo-necrotic effect [157]. Due to the possibility to focalize the beam of radiation to a precise area of interest, there is a significant reduction of the risk of damaging nearby health tissues. Therefore, combination of a photosensitizer and an imaging agent would allow imaged guided treatment of disease and immediate monitoring of the efficiency of the therapy, thus orienting specific decisions related to treatment progression. Malignant gliomas represent the most common primary tumor of the central nervous system which poses several problems for early localization and chirurgical treatment due to the close vicinity to delicate anatomical structures of the brain. Iron oxide NPs have been shown to be useful to enhance MRI contrast and improve the differentiation of neoplastic from normal brain tissue, offering a prolonged delineation of tumor margins because of their high cellular internalization and their slow clearance from the tumor site [158]. In recent years, MRI contrast enhancement has been exploited in combination with PDT for the simultaneous imaging and treatment of this brain tumor [159,160]. Interesting results in this field were obtained first by Reddy et al., who proposed multifunctional polyacrylamidebased NPs encapsulating both iron oxide NPs and Photofrin (a photoactivable hemathophorphyrin agent) as imaging and therapeutic agent, respectively [160]. Specific targeting was achieved by using the F3 peptide that selectively binds the cell surface nucleolin, expressed on angiogenic endothelial cells within tumor vasculature. In vivo studies, carried out on intracranial 9L glioma-bearing rats, revealed that these multifunctional NPs could actively target gliomas cells and be detected using T2-weighted and diffusion MRI. After i.v. injection of the magnetic NPs, T2-weighted MR images showed significant contrast enhancement. F3-targeted NPs led to a longer and more intense signal enhancement compared to non-targeted one, thus confirming their higher accumulation and longer retention which could allow predicting a better therapeutic outcome. According to the possibility to correlate the magnitude of diffusion changes to animal survival [161], diffusion MRI was used to evaluate changes in tumor diffusion properties in each treatment group for up to two weeks after light activation of the Photofrin. The group treated with F3-targeted NPs showed the largest increase in mean tumor apparent diffusion coefficient values as well as the longest overall survival compared to control groups. Moreover, 40% of animals treated with F3-targeted NPs were found to be tumor-free at the end of the study (60 days after treatment) revealing a significant improvement of therapeutic outcome. This study clearly demonstrates the usefulness of such a vascular targeted nanotheranostic to identify the precise tumor localization, to monitor in real time both the immediate response to the treatment and the possible resurgence of tumor which could justify a further rapid intervention. 3.2. Optical imaging Biomedical optical imaging is a non-ionizing, non-invasive technique based on the specific optical properties of tissue constituents at different wavelengths [162]. Recent developments provide quantitative

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information, almost in real time, extending over a wide range in the resolution scale, therefore offering interesting possibilities for in vivo diagnosis and monitoring of treatment efficacy in cancer and other various diseases, often in combination with other imaging techniques [163]. The penetration depth represents the major limit of these techniques, due to the strong scattering properties of soft tissues [164]. Scattering is strong in the visible region of the spectrum (b700 nm), however it decreases at longer wavelengths, in the NIR region (700–900 nm), often called “biological window” for optical imaging. This region is, indeed, characterized by a low absorption and scattering in soft tissues. Recently developed fluorophores, which emit/absorb in this “optical window”, penetrate deeper in the tissues and represent interesting tools for clinical application of optical imaging techniques in early stage detection and characterization of pathological lesions which, however, must be located close to the radiation source, since penetration depth of NIR light in tissue is less than 1 cm. [165] Nanotheranostics can be developed by combination of NIR dyes and therapeutic molecules. This strategy has been explored by different groups which created multifunctional systems for diagnosis, treatment and, follow up of cancer. 3.2.1. Optical imaging and chemotherapy The NIR dye Cy5.5 was used by Kim et al. [166] to label paclitaxelloaded chitosan-based NPs (PXT-CNPs). Imaging and assessment of

the therapeutic efficacy were performed on mice bearing SCC7 murine squamous carcinoma tumors. In vivo NIR fluorescence (NIRF) images showed clearly delineated tumors due to the accumulation of NPs in this area. Fluorescence signal intensity was correlated to the concentration of the administered NPs and to the frequency of injection. Moreover, compared to the free dye, which was rapidly cleared, the fluorescence signal was stable up to 72 h (Fig. 5a,b). Not only NIRF imaging allowed to follow the biodistribution of the CNPs, but also enabled the non-invasive monitoring of the tumor growth rate in response to the treatment. NIRF images showed, indeed, a rapid increase of tumor size in control mice while a progressive inhibition of tumor growth was observed in treated mice. These results were confirmed by caliper measurements of tumors that reached, at the end of the study, volumes of 8000 mm 3 and 7900 mm 3 in control groups treated with saline or empty nanoparticles, respectively. Final mean volume was only 1000 mm 3 for mice treated with PXTCNPs, significantly smaller than in mice treated with free paclitaxel, thus clearly showing an improvement of the drug therapeutic activity. Moreover, PXT-CNPs enhanced mice survival rates allowing 8 mice out of 10 to survive until the end of the treatment while the high toxicity of the free paclitaxel treatment caused all mice died by day 21 (Fig. 5c,d). Deep tissue and organ imaging can be achieved also using NIR QDs. These semiconductor nanocrystals are extremely interesting as

Fig. 5. In vivo imaging of Cy5.5-labeled CNPs in SCC7 tumor-bearing mice. (a) The early-stage tumor models were generated by injecting subcutaneously SCC7 cells into the pectoral and dorsal sides of C3H-HeJ nude mice. After eight days, different size of tumors had grown to 2.6 ± 0.3 mm (solid arrow) and 6.2 ± 0.5 mm (dotted arrow). NIRF images were recorded 1-day post-injection of 3.3 μmol of Cy5.5-labeled CNPs (5 mg/kg), (b) Time-dependent tumor targeting specificity of free Cy5.5, Cy5.5-labeled GC polymers, and Cy5.5labeled CNPs, all with equimolar amounts of Cy5.5 (0.16 μmol), in SCC7 tumor-bearing mice. Comparative therapeutic efficacy studies of PTX-CNPs: (c) tumor size; (d) survival curves. Adapted with permission from Elsevier Publisher: Journal of Controlled Release [166].

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high sensitive fluorescent imaging agents, due to their large excitation spectrum and the possibility to modulate the emission, as a function of the nanocrystal size and chemical composition. However, due to the hydrophobic properties, surface modifications are required to allow their biological application. For example, Nurunnabi et al. [167] have encapsulated NIR QDs into micelles with the aim to achieve both the diagnostic and the therapeutic goal. Practically, QDs were encapsulated in a mixture of PEG–PCDA (10,12 pentacosadyinoic acid) and PCDA–herceptin conjugates, further stabilized by cross linking reaction. Herceptin is an IgG monoclonal antibody which targets the epidermal growth factor receptors present in breast and prostate cancer cells which highly express the HER-2 receptor. The resulting nanotheranostic could be used for targeted therapy and imaging due the combination of herceptin at the surface and QDs in the core of those micelles. The potential of the NIR QDs-loaded micelles to reduce tumor growth was investigated on HER-2 positive MDA-MB-231 human breast cancer-bearing mice. Specifically addressed to these cells, the QDs-loaded micelles were able to block the function of the overexpressed HER2, therefore causing a significant inhibition of tumor growth (i.e. by 77.3% compared to the control saline solution) (Fig. 6a-c). Moreover, the biodistribution was monitored in vivo by non-invasive fluorescence. Micelles were distributed rapidly through the animal, and a strong signal was observed at the tumor site due to accumulation of the NIR probe. Interestingly, this signal remained up to 5 days post injection (Fig. 6d). If the proof of concept has been done that such a construction could be used successfully at the preclinical stage for theranostic purpose, the application in clinical trials remains questionable, due to the toxicological issue of QDs. However, it has to be noted that the use of QDs in preclinical studies could represent an interesting advanced nanotechnology to help the pharmaceutical industry in the animal evaluation of their new discovered chemical entities. 3.2.2. Optical imaging and photodynamic therapy As already discussed before, PTD involves the administration of tumor-localizing photosensitizing agents, followed by their activation by photons of a specific wavelength. It results in a sequence of biological processes that cause irreversible photo-damage of tumor tissues [157]. However, irradiation can be used not only to activate the generation of reactive oxygen species but also to induce the emission of strong fluorescence signals, thus allowing the application of these molecules also as contrast agents for optical imaging. With the aim to develop theranostic nanomedicines which combine imaging contrast properties and therapeutic action in a single functional unit, various PDT sensitizers have been widely exploited. Indeed, in recent years, various porphyrin-loaded nanocarriers have been designed as multifunctional nanotheranostics whose application has already provided interesting and promising results in vivo. An example of such nanotheranostics has been proposed by Koo et al. [168]. In order to combine targeting, diagnosis and therapy, these authors have prepared pH-sensitive block copolymer micelles encapsulating the photosentitizer protophorphyrin IX (PpIX-pH-PMs). After intravenous injection, these polymeric micelles were able to accumulate at the tumor site, exploiting the EPR effect. Additionally, due to the tumoral acidic pH, a pH-responsive micellization/demicellization transition was expected, leading to a selective release of the entrapped drug. In vitro uptake studies confirmed the pH dependent release of the Pp-IX, that led to a higher fluorescent signal in the cell cytoplasm after incubation at pH 6.4 compared to pH 7.4. The in vivo effectiveness of this system has been evaluated on SCC-7 squamous cell carcinoma-bearing mice. 24 h after intravenous injection of PpIXpH-PMs, animals were irradiated with a 670 nm pulsed laser diode to excite PpIX molecules. A strong fluorescent signal was visible in the tumor (which could be distinguished from the surrounding health tissue) while in the case of the control groups (free PpIX) the

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fluorescence accumulated mainly in the liver without important signal enhancement in the tumor. To evaluate the in vivo antitumor activity, animals were irradiated twice for 30 min using a 633 nm laser, 24 h after injection of free PpIX or PpIX-pH-PMs (5 mg/kg PpIX). Laser irradiation led to production of the cytotoxic oxygen singlet which induced severe tissue damage with a more important reduction of the tumor volume in case of the PpIX-pH-PMs treatment. He et al. [169] have investigated the possibility to exploit the fluorescence and photosensitivity of methylene blue (MB), one of the most inexpensive of the commercially available NIR dyes, to develop a theranostic nanodevice, integrating imaging and therapeutic purposes. Practically, they prepared MB-encapsulated phoshonateterminated silica NPs (MB-encapsulated PSiNPs) demonstrating their effectiveness for real time in vivo NIR optical imaging and PDT. Intratumoral injection of the MB-encapsulated PSiNPs in mice bearing HeLA tumor allowed clear identification of the tumor margins and 12 h after injection, the induced fluorescence was used as guide to focus the laser light beam (635 nm, 5 min) for PDT. In vivo imaging was performed in the following days to assess the response to the treatment by measuring the necrotic area in the tumor. Gradual reduction of the tumor mass was observed in irradiated animals while no changes were observed in control tumors (Fig. 7). Although interesting, it has to be noted that in this study the nanotheranostic administration was performed intratumorally which limits the application to well accessible tumors. In the same direction, a polyacrilamide-based nanotheranostic was recently proposed for in vivo NIRF and PTD. A new synthetic strategy, the so called “post-loading approach” (i.e. drug loading into pre-formed NPs) enabled to achieve increased retention of both a sensitizer (HPPH) [3-(1′-17 hexyloxyethyl)pyropheophorbide-a] and a NIR cyanine dye. The tumor imaging properties and the phototherapeutic efficacy were evaluated on BALB/c mice bearing subcutaneous Colon 26 tumors confirming their applicability for delineating tumor margins and image guided treatment [170]. 3.2.3. Optical imaging and photothermal therapy So called “multifunctional nanoshells” (NSs) have emerged as promising tools to achieve both cancer therapy and imaging enhancement [44,100,171–175]. NSs consist of a spherical dielectric core coated with a layer of metal, typically gold, forming a thin shell. The possibility to use NS-based systems as therapeutic agents relies on their intrinsic property of absorbing NIR light resonant with the NS plasmon energy and convert it to heat which can be exploited for PTT. Therefore, following NSs irradiation, the local increase of temperature would enable minimally invasive photothermal ablation of tumor tissue. Multifunctional systems can be designed by modifications of the NS chemical structure with the introduction of NIR fluorophores at appropriate distance from the NS surface (~ 10 nm). The optical characteristics of the NSs can modify the emission properties of the fluorophore and enhance the fluorescence quantum yield. Additionally, iron oxide NPs can be incorporated in the spacer layer that separates the NIR dye from the metallic NP surface. The resulting NSs are extremely attractive to increase the sensitivity in optical fluorescence as well as in MR imaging, due to their specific relaxivity (Fig. 8) [176–178]. In practice, functionalized theranostic NSs would provide enhanced image contrast allowing to identify the precise localization of the tumor, to monitor the efficacy of the NIR thermal ablation (during the treatment or immediately after) and to detect an eventual resurgence of the tumor. By regulating the power of the laser, the same source could be used for imaging the tumor and for increasing the temperature of the NSs leading to cell death and tumor destruction. An important successful application of such theranostic strategy has been provided by Gobin et al. [179]. These authors have evaluated the potential of gold NSs (mean diameter around 120 nm) to enhance the contrast in tumors for optical coherence tomography imaging

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Fig. 6. Anti-tumor effects of the near-IR QD-loaded micelles. (a) Tumor volume on day 0, 7, 14 and 21 days; (b) Shrinkage in tumor volume and (c) changes in body weight after treatment with 5 mg/kg of near-IR QD-loaded micelles (■), and saline (●), respectively. (d) Non-invasive fluorescence imaging of tumor-bearing mice for 5 days after administration of near-IR QD-loaded micelles (10 mg/kg). Intensity bar shows the fluorescence intensity level of tumor side. The circle indicates tumor. Adapted with permission from Elservier Publisher: Biomaterials [167].

(OCT) and to induce photothermal cell death. Studies were carried out on a subcutaneously grafted mouse model of colon carcinoma (CT-26). Ten days after cell inoculation, gold NSs were injected intravenously and mice were imaged via OCT 20 h later. Images clearly demonstrated that gold NSs provided high contrast of tumor tissue compared to normal tissue in OCT, thus allowing a better definition

of the borders of the tumor. Noteworthy, no enhancement was observed in health tissue treated with NSs compared to untreated controls. Tumor imaging was followed by irradiation with a 808 nm laser for 3 min. Analysis of tumor regression, using the average measurement of the tumor size and the surviving populations, was carried out for 7 weeks post-treatment. Complete regression of the

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Fig. 7. In vivo imaging and PDT of subcutaneous-Hela-tumor-xenografted mice after different treatment: (a) 100 μl 44 mg/ml MB-encapsulated PSiNPs injection and 5 min light exposure (power intensity of 500 mw/cm2), the red circle indicates the region (i) injected the MB-encapsulated PSiNPs and (ii) exposed to light; (b) 100 μl 44 mg/ml MB-encapsulated PSiNPs injection; and (c) 5 min light exposure with power intensity of 500 mw/cm2. Reproduced with permission from Elsevier Publisher: Biomaterials [169].

tumor was observed in the majority of mice treated with gold NSs. At 21 days, the survival of NS-treated animals was significantly higher (long-term survival of 83%) than controls. In a similar approach, gold nanorods were PEG conjugated and complexed with a photosensitizers (Al(III) phthalocyanine chloride tetrasulfonic acid) (GNRs-AlPcS4) for fluorescence imaging and cancer treatment by combination of PTT and PDT [180]. These long circulating GNRs accumulated passively in tumors by EPR effect and could be used to induce hyperthermia following the absorption of an externally applied NIR light. The photosensitizer, irradiated at opportune wavelengths, emits strong NIR fluorescence and produces reactive oxygen species, thus inducing cell death by PDT. In GNRs-AlPcS4 the fluorescence emission and the production of reactive oxygen species by the photosensitizer could be strictly controlled, depending on the distance between the photosensitizer molecules and the gold nanorods. Indeed,

Fig. 8. Schematic representation of multifunctional gold nanoshells. Adapted with permission from John Wiley and Sons Publishers: Advanced Functional Materials [92].

due to the effective energy transfer between the photosensitizer molecules and the gold nanorod, the photosensitizers located close to the surface of the nanorod are non-fluorescent and non-toxic. However, after i.v. administration, GNRs-AlPcS4 passively accumulates in the tumor tissue where the photosensitizer is released and becomes highly fluorescent and phototoxic (Fig. 9). This strategy allows to noninvasively visualize the tumor by NIRF imaging and to apply localized PDT with minimal damage towards the healthy tissue. In addition, the simultaneous application of PTT may enhance the efficacy of the treatment. Indeed, mice bearing SSC7 squamous carcinoma tumor xenograft were imaged 1, 4 and 24 h post injection of GNRs-AlPcS4. NIRF images showed high fluorescence levels in the tumors which were clearly distinguished from the surrounding tissue already 1 h after injection. Same fluorescence levels were measured after injection of free AlPcS4. However, group treated with GNRs-AlPcS4, showed a significant increase of fluorescence intensity (1.5 fold) after the local application of hyperthermia up to 65 °C (irradiation with a 810 nm laser), which was not the case with AlPcS4 free. It was hypothesized that temperature induced the release of the phoptosensitizer still bound to the nanorod surface, leading to the subsequent augmentation of the fluorescence. PDT and PTT alone or in combined protocols were evaluated in the same tumor model, by measuring the tumor growth rates. A significant anticancer effect was obtained with application of PDT, however the highest improvement of the therapeutic activity was observed following the combined protocol (PTT plus by PDT 24 h after injection of GNRs-AlPcS4). On the contrary, PTT alone did not induce a sufficient therapeutic efficacy. 3.2.4. Optical imaging and gene therapy Gene therapy presents interesting potential for the treatment of severe diseases such as cancer and certain viral infections. The transfer of a therapeutic gene into a target cell would allow replacing a notworking gene, thus positively modifying the outcome of the disease. Together with genes, small interfering RNA (siRNA) molecules, which enable specific post-transcriptional silencing of target genes, have also

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Fig. 9. Schematic rapresentation of gold nanorod-photosensitizer complex. Adapted with permission from Jang, B. et al. “Gold Nanorod − Photosensitizer Complex for Near-Infrared Fluorescence Imaging and Photodynamic/Photothermal Therapy In Vivo.” ACS Nano 2011, 5, 1086–1094 Copyright © 2011 American Chemical Society [180].

attracted much interest. Indeed, the possibility to selectively knock out genes over expressed in tumors raised many hopes for their introduction in the therapeutic protocols for cancer [181,182]. With the aim of facilitating siRNA delivery to target cells, several nanocarriers have been developed [183]. Despite the promising results obtained in vitro, more work needs to be done to achieve a specific and adequate delivery to cancer cells in vivo [184]. Moreover, the translation to clinic of siRNA therapy requires not only the effective delivery to target cells but also the possibility to monitor their accumulation and therapeutic activity. Different groups have focused their attention on the development of multifunctional NPs which combine the delivery of siRNA molecules with imaging modalities for monitoring the delivery and to assess the efficacy of the treatment. For example, Kenny et al. [185] have developed PEGylated siRNA liposomes (LEsiRNA) in which Gd3+-conjugated lipids and AlexaFluor®-labeled siRNA molecules were introduced as MR and fluorescence imaging contrast agents, respectively. MR images of mice bearing OVCAR-3 human ovarian xenograft were registered before and 24, 48, 72 h post administration of LEsiRNA liposomes, the nonsilencing negative control liposomes or the clinical contrast agent Dotarem® (Guerbet, France). A significant increase of the MRI signal intensity was observed 24 h after administration of LEsiRNA suggesting their tumor accumulation. Moreover, 48 and 72 h post injection, the extent of liposome uptake correlated to a significant reduction of tumor growth, as compared to controls. These results show the high potential of these multifunctional liposomes combining two different imaging modalities: MRI for in vivo monitoring and fluorescence for the ex vivo evaluation of siRNA cell internalization (on tumor slice). Combining multimodal imaging and siRNA therapy has been successfully achieved by the development of magnetic NPs labeled with a NIR fluorophore (Cy5.5) and covalently linked through different linkers to: (i) siRNA molecules specific for a model (Green Fluorescent protein GFP) or a therapeutic (Survivin) gene and (ii) membrane translocation peptides (Fig. 10a) [186]. In vivo MRI and NIRF imaging (Fig. 10b,c) were performed after administration of these multifunctional nanoparticles to mice bearing 9L-GFP glioma or LS 174T human colon rectal carcinoma. In both cases, a significant reduction of the T2 relaxivity and a high fluorescent signal were observed within the tumors. No changes were observed in other tissues, thus

confirming the specific delivery of the NPs. The gene silencing was monitored by optical imaging (GFP inhibition). In the case of Survivin-targeting siRNA, the intense fluorescence signal, associated to siRNA delivery, correlated well with histological data, showing a high density of apoptotic and necrotic nuclei. 3.3. Combined MR/optical imaging and therapy Combination of MR, optical imaging and drug loading in a single nanoconstruct has been investigated by different groups. Thus, Giannella et al. [187] developed a theranostic nanodevice (mean diameter around 50 nm) composed of an oil in water nanoemulsion, loaded with iron oxide crystals and Cy7 dye (for MR and NIRF imaging respectively) as well as the glucocorticoid prednisolone acetate valeranate (PAV) as therapeutic molecule. The effectiveness of this nanotheranostic which combined the high spatial resolution of MR with the high sensitivity of optical fluorescence imaging has been evaluated on a colon cancer model. In vivo imaging by MR or NIRF demonstrated the preferential accumulation of the nanoemulsion in the tumors. Indeed, in the MR images of the mice injected with PAV nanoemulsions, tumors appeared bright compared to the surrounding tissue (Fig. 11a). The massive uptake of NPs in the tumor was also confirmed by NIRF imaging since the injection of Cy7-labeled nanoemulsions led to a strong fluorescent signal with a mean tumor/skin photon count ratio of 11.29±4.96 compared to only 0.83±0.013 for mice injected with Cy7-unlabeled nanoemulsions (Fig. 11b,c). To ascertain the possibility to use this multifunctional nanoemulsion for imaging guided treatment of experimental colon cancer, the therapeutic efficiency of various PAV loaded nanoemulsions was investigated. All animals treated with PAV nanoemulsion (independently of the presence of targeting molecules such as the RDG peptide) showed a significant inhibition of tumor growth compared to mice injected with the control nanoemulsion (i.e. drug unloaded) or saline solution. After three injections, over a period of eight days, the tumors in the group treated with PAV nanoemulsions were at least 50% smaller than in the control group. No significant differences were observed among simple PAV nanoemulsion, actively targeted nanoemulsions (containing the RGD peptide) and PAV+FeO loaded nanoemulsions (Fig. 11d,e). These results are really promising for personalized management of cancer. Indeed, it was

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Fig. 10. (a) Schematic representation of the MN-NIRF-siRNA probe; (b) In vivo MRI of mice bearing subcutaneous LS174T human colorectal adenocarcinoma (arrows) before and after administration of the nanoparticles; (c) a high-intensity NIRF signal on in vivo optical images confirming delivery of the nanoparticles to the target tissue (left, white light; middle, NIRF; right, color-coded overlay). Adapted with permission from Nature Publishing Group: Nature Medicine [186].

observed that the distribution and accumulation of this nanotheranostic in the various animal subjects may be monitored non-invasively and the optimization of the treatments may be guided by MR and NIRF images. Another extremely interesting theranostic concept arises from combined optical (upconversion luminescence (UCL)) and MR imagingguided tumor magnetically-targeted photothermal therapy [188]. For this, PEG-nanoparticles (MFNPs@–PEG) made of a core of upconversion NPs (i.e. lanthanide-doped rare-earth nanocrystals) [189], an intermediate layer of USPIO NPs and an external shell of gold were prepared (Fig. 12) and tested as theranostics in vivo on a 4T1 murine breast cancer-bearing mice model. After injection of MFNPs–PEG, the tumor guidance was achieved by applying a small magnet over the tumor of each mouse. In vivo UCL and MR images showed a bright UCL signal and a significant darkening (signal decrease 67%) in the tumor, 2 h after injection, thus suggesting a high accumulation of MFNPs–PEG under the magnetic field. On the contrary, control mice, not exposed to the magnetic field, showed a 7 times lower UCL signal and only a 18% decrease of the MRI signal. Then, magnetic-targeted non-invasive imaging was used as guide for PTT. Practically, 2 h after i.v. injection of MFNPs–PEG under magnetic field, mice were irradiated with a 808 nm laser for 5 min. Temperature at the surface of tumor reached 50 °C causing complete tumor ablation in all mice and no tumor re-growth was observed during 40 days follow up. Noteworthy, the absence of tumor growth inhibition was observed in control mice (untreated, irradiated only, injected with MFNPs–PEG without magnetic target and irradiated or, injected with MFNPs–PEG under magnetic field but not irradiated). These data clearly confirmed that the efficacy of the treatment was due to the combination of magnetic-targeted tumor uptake and laser irradiation. Indeed, when applied separately, the different treatments had no effect. Moreover, while for control mice, the average life span was of 14–18 days, combined treatment increased animal survival over 40 days without any sign of toxicity. This study clearly demonstrates the feasibility of the “see and treat approach”: MFNPs were found successful to monitor the disease status, predict the effectiveness of the treatment and validate the therapeutic outcome. Translation to clinic of this unique strategy offers again the possibility of personalization of therapies, thus improving the management of cancer.

3.4. Ultrasonography Ultrasonography is a common widely available, safe, non-invasive, non-ionizing, low cost and in real time clinical imaging modality. In ultrasonography, a transducer placed on the skin emits ultrasound waves which are partially backscattered by different structures of the body due to the impedance mismatch between different tissues. Backscattered waves return to the transducer and allow generating ultrasound images [190]. However, due to the weak difference of echogenicity between different soft tissues, ultrasound contrast agents are usually needed to improve imaging and to distinguish between diseased and healthy tissues. Air and biocompatible gaseous perfluorocarbons (PFCs), stabilized by a layer of phospholipids, proteins or polymers have been used to increase the contrast during ultrasound imaging because of the high impedance mismatch between gases and blood or soft tissues [62,191] Micro and nanosized ultrasound contrast agents encapsulating liquid PFCs, which show excellent mechanical and acoustic properties, have been recently introduced [192,193]. Moreover, along with the possibility to perform imaging, ultrasounds have a high potential for enhancing drug delivery. Indeed, the cavitation effect consequent to the application of ultrasounds can promote the release of encapsulated drugs and simultaneously increase the permeability of the cell membranes, thus enhancing drug specific uptake and therapeutic activity [194–196]. 3.4.1. Ultrasonograpy and chemotherapy After being entered in clinical phase 3 trials as blood substitutes, perfluorocarbon (PFC) NPs have attracted a wide interest for their application as theranostic system joining their above mentioned imaging properties in ultrasonography with the possibility to deliver and concentrate a pharmacologically active agent at the target site [197]. A promising theranostic approach using ultrasound as imaging modality is represented by the design of an oil in water emulsion made of liquid perfluoroctylbromide (PFOB) drops stabilized by a lipid layer in which the peptide melittin has been incorporated (melittinloaded NPs: mean diameter around 230 nm) [198]. Melittin is a non specific cytolitic peptide that permeabilizes membrane structure and induces cell lysis [199,200]. It has already been proposed as cytotoxic agent in the treatment of several cancers [201,202]. The effectiveness of the melittin-loaded NPs as drug delivery carrier has been

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Fig. 11. (a) (1, 2) Selected MR images of PAV nanoemulsion and CTRL nanoemulsion injected mice; (3, 4) MR images of PAV FeO nanoemulsion and FeO nanoemulsion injected mice. Red circles indicate the tumors. In (1) and (2), tumors appeared bright compared to surrounding muscle tissue. In (3) and (4), tumor areas appeared hypointense, indicative of FeO accumulation. (b) In vivo NIRF images of mouse injected with unlabeled nanoemulsions (left) and mice (two different sized tumors) injected with Cy7 nanoemulsion (middle and right) at the end of the study. (c) tumor/skin photon ratio at the same time point. Therapeutic effect of nanoemulsions: (d) photographs of typical tumors of mice injected with PAV nanoemulsion, RGD-PAV nanoemulsion, PAVþFeO nanoemulsion, and CTRL nanoemulsion; (e) tumor growth profiles. Adapted with permission from Gianella, A. et al. “Multifunctional Nanoemulsion Platform for Imaging Guided Therapy Evaluated in Experimental Cancer.” ACS Nano 2011 5, 4422–4433. Copyright © 2011 American Chemical Society [187].

Fig. 12. Schematic representation of multifunctional composite nanoparticles used for imaging guided cancer therapy. Adapted with permission from Elsevier Publisher: Biomaterials [188].

investigated in vivo on MDA-MB-435 xenograft models of breast cancer in nude mice. Ultrasound imaging was used to calculate the tumor volume at the beginning and at the end of the treatment. Intravenous administration of the mellitin-loaded NPs led to a significant inhibition of tumor growth (by 24.68±1.57% and 27.16±2.9% as compared with control saline solution or mellitin free emulsion, respectively). In this study, the phospholipid-stabilized PFOB nanoemulsion promoted the accumulation of the cytolitic peptide in the target cells by the “contact facilitated delivery” mechanism [72]. Simultaneously, they provided a significant contrast enhancement which enabled to monitor the therapeutic efficacy by ultrasound imaging. Another important application in cancer therapy which combines ultrasonic tumor imaging with ultrasound-enhanced targeted chemotherapy has been developed by Rapoport et al. [203]. This group proposed a nanodevice composed of (i) doxorubicin-loaded micelles made of biodegradable block copolymers (poly [ethylene glycol]‐block-poly[L-lactide] or poly [ethylene glycol]‐block-poly[caprolactone]) and (ii) nanodroplets of

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perfluoropentane stabilized by the same copolymers. After intravenous administration, micelles and nanodroples passively accumulated in the tumor due to EPR effect. At temperature higher than the boiling point of perfluoropentane, the nanodroplets started to coalesce, forming microbubbles. Further temperature increase with application of ultrasounds promoted the formation of bigger microbubbles in the tumor which provided a significant contrast enhancement for ultrasound imaging. B-mode ultrasound images, recorded after intravenous administration of these multifunctional micelles/ microbubbles, showed a large echogenic area in the tumor of MDA MB231 breast cancer-bearing mice, while no signal enhancement was observed in other organs, thus confirming passive tumor targeting. After intratumoral administration, a strong echo was generated within 1 min and persisted for several days showing the potential of these microbubbles as long lasting contrast agents. The efficacy of ultrasound mediated chemotherapy was evaluated on the same tumor model. It was observed that intravenous administration of the multifunctional micelles/microbubble formulation (0.75 mg/mL Dox/0.5% PEGPLLA/2% perfluoropentane) followed by treatment with 3 MHz ultrasound for 30 s inhibited tumor growth with a statistically significant difference compared to tumor group treated with the same formulation but without exposure to ultrasounds. In this latter case, the tumor growth profile was not significantly different from the saline-treated control group. These results show the pivotal role of ultrasounds which promoted drug release from the micelles and caused perturbation of the cell membrane, resulting in higher intracellular accumulation of the drug and better therapeutic outcome. In a successive study, the same group [204] designed theranostic paclitaxel-loaded block co-polymers stabilized PFC micelles which combined 19F MR and ultrasound imaging properties. The perfluoro-15-crown-5-ether (PFCE) was used as core forming material due to its excellent acoustic and resonance properties. The imaging potential of these micelles was investigated on the orthotopic human pancreatic MiaPaca-2 model in nude mice. Accumulation and distribution of the PFCE micelles were followed by ultrasonography and 19F MRI. An echogenic area was evident in the tumor as well as in the liver. The latter was due to the capture of the micelles by the RES cells. However, interpretation of the MR images was complex due to the strong dependence of the T2 relaxation time of the fluorine nucleus on the local oxygen concentration, which led to a decreased intensity of the signal produced in hypoxic areas. Since pancreatic cancer is characterized by a reduced vascularization and poor oxygenation, 19F MRI could underestimate the amount of micelles accumulated in the tumor. Therefore, this nanoconstruction is believed to be better adapted as imaging contrast agent for highly vascularized organs (such us liver, spleen heart and lung) and tumors. Interestingly, the ultrasound-mediated therapy associated with paclitaxel-loaded PFCE nanoemulsions showed excellent results in different cancer models treated with paclitaxel at a dose of 40 mg/kg, associated with a 1 MHz ultrasound treatment. For example, in MDA MB231 breast cancer tumor-bearing mice complete tumor regression was observed and no tumor relapse occurred during the following five months. In the above mentioned orthotopic human pancreatic MiaPaca-2 tumor model, the administration of paclitaxel-loaded PFCE nanoemulsions (without ultrasonography) caused only a stabilization of the tumor growth while a significant reduction of the tumor volume was observed in the group exposed to combined therapy, thus confirming the enhancement of the action of paclitaxel by ultrasounds. 3.5. Combined photoacoustic/optical imaging and photothermal therapy The combination of photoacoustic and optical imaging with PTT is another innovative approach which has been recently proposed by Lovell et al. [205] for theranostic application. In this study, the socalled porphysomes were prepared; they are spherical vesicles formed by the self assemblage of porphyrin–phospholipid conjugates.

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Porphysomes show high absorption of the NIR light and fluorescence self-quenching due to the confinement of the porphyrins in the lipid bilayer. Moreover, irradiated porphysomes convert laser energy to heat with a yield comparable to gold nanorods thus being attractive not only for imaging but also for photothermal treatments. The proof of concept has been done in vivo after intradermal administration to rats: porphysomes exhibited a strong photoacoustic signal and allowed to clearly detect the lymphatic network within 15 min. When injected intravenously in KB tumor bearing mice, porphysomes displayed high fluorescence signal in the tumor, not immediately but only 48 h after injection, thus confirming the initial self-quenched status of the porphyrins and the following reorganization of the porphysomes with dequenching of porphyrins in vivo into the tumor. The therapeutic efficacy of porphysomes has been demonstrated in mice irradiated with a 658 nm laser 24 h after injection. Monitored temperature reached 60 °C in tumors treated with porphysomes while it was only 40 °C in PBS-treated control mice. As a consequence, tumors were completely destroyed, whereas no inhibition of tumor growth was observed in mice which received only laser treatment or porphysomes alone (Fig. 13). 4. Nanotheranostic in cardiovascular diseases If the development of theranostic nanomedicines in oncology has received the majority of the attention, multifunctional nanomedicines may also have important impact in the treatment of cardiovascular diseases (CVDs). CVDs, which include various disorders of blood vasculature and the heart, are the main cause of death in the European Union accounting for over 2.0 million deaths each year. Nearly half of all deaths are from CVDs (45% deaths in women and 38% deaths in men). Despite significant clinical advancements, current methods for early detection and therapy of CDVs are limited and moreover their efficacy in preventing CVDs remains questionable. Given these facts, the development of novel techniques for improving imaging and treatment of CVDs represents a pressing challenge for nanomedicine. In particular, efforts should be directed to diagnosis and therapy of atherosclerosis, the principal cause of coronary heart disease whose growth rate is rising dramatically. In this context, the use of nanotheranostics which enable in vivo non-invasive imaging associated to the simultaneous evaluation of the therapeutic effect and the prediction of the treatment outcomes, gave rise to a wide interest. As it will be shown below, in cardiovascular diseases, imaging may reflect the treatment efficacy and become fundamental to optimize the further therapeutic protocol tuning it as function of each individual response. 4.1. MRI and anti-angiogenic therapy According to the fundamental role of neo-angiogenesis in the progression of the atherosclerotic lesions, various systems have been developed to normalize the extent of vascular development and to diminish the frequency of intraplaque hemorrhage, thus promoting plaque stabilization. Clearly, the neo vasculature proliferation is associated to high plaque instability and tendency to rupture with consequent myocardial infarction and stroke [206,207]. The intense neo-vessel formation within the atherosclerotic plaque is confirmed by a wide expression of αvβ3-integrin, an adhesion molecule marker of the vascular epithelial cells [208,209]. Among the various antiangiogenic drugs, the efficacy of the fumagillin and its water soluble functional analog, TNP-460, has been proven both in animal studies and in human clinical trials [210–212]. However, the high dose required to elite the therapeutic effect is associated to severe neurocognitive side effects [213,214]. In order to improve the treatment of the plaque progression, integrin-targeted PFC-loaded NPs [215,216] and liposomes [217] have been developed to explore the new proliferating vessels. Furthermore, αvβ3-targeted Gd3+-based NPs loaded with fumagillin have been proposed as theranostic systems to achieve a better management of the

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Fig. 13. (a) (1) Photoacoustic tomography imaging before and after intradermal injection of porphysomes; (2) fluorescence imaging after i.v. injection of porphysomes in a KB xenograft-bearing mouse. (b) Photothermal therapy set-up showing laser and tumor-bearing mouse; (c) representative thermal response in KB tumor-bearing mice injected intravenously 24 h before with 42 mg/kg porphysomes or PBS; (d) photographs showing therapeutic response to photothermal therapy using porphysomes; (e) survival plot of tumorbearing mice treated with the indicated conditions. Adapted with permission from Nature Publishing Group: Nature Materials [205].

atherosclerosis [218]. Actively targeted towards the neo-vasculature within the plaque, these NPs exert an antiangiogenic effect, which leads to a decreased plaque vulnerability and tendency to rupture because of the reduction of the neo-vessel extent. The presence of Gd3+ enabled to perform MRI in order to follow the distribution of the NPs and to detect the expression of integrins as marker of plaque angiogenesis. The level of plaque angiogenesis may be assessed and it would be possible to monitor non-invasively in real time the efficacy of the treatment and the patient outcome. The effectiveness as theranostic systems, of paramagnetic αvβ3-targeted Gd3+-based NPs loaded with fumagillin, was evaluated on hyperlipidemic New Zeland rabbits. In this animal model, the high cholesterol diet induced only a first stage atherosclerosis since only a minimal thickening of the aortic wall was observed after T1 weighted MRI. However, even at this early stage, the injection of αvβ3-targeted NPs caused a significant contrast enhancement (16.7 ± 1.1%) which allowed a clear visualization of the neovasculature in the plaque. Non-targeted NPs caused only a 10.8 ± 1.1% signal enhancement. The absence of signal enhancement in MR images recorded one week after the first injection, confirmed that the NPs previously injected were no more detectable. Following a second injection of αvβ3-targeted NPs, a reduction of signal intensity in the range 60– 80% was measured compared to the first injection. The decrease of MRI signal confirmed the efficacy of the treatment, which caused a reduction of the atherosclerosis related angiogenesis and of the expression of αvβ3 integrin as confirmed also by histological analysis. The high T1-weighted signal enhancement in the aorta wall at the day of initial treatment dramatically decreased after NP treatment (control group receiving unloaded NPs did not show any changes). Therefore, the spatial distribution and signal decrease after treatment can be used not only to detect the accumulation of the carrier and to evaluate the status of the disease but also to predict the efficacy of the treatment and further to monitor the effectiveness of the therapeutic protocol. These NPs offer, therefore, the opportunity for a rigorous follow up of the therapy. The same NPs were further tested for their efficacy after repeated treatment [219]. It was observed that if the signal enhancement progressively decreased (50–75% relative to control at week 2) after a

unique injection of fumagillin-loaded NPs, no differences could be observed in the MR signal between treated and control animals four weeks after injection. A second injection of NPs induced the same response, confirming the treatment was not affected by a previous drug administration (Fig. 14a). In these studies, fumagillin was used at a concentration up to 50,000 times lower than the cumulative oral dose of TNP6470 used in previous studies [213], and a single dose of this nanotheranostic was enough to induce a reduction of the neovasculature in the aortic wall. Therefore, this protocol is expected to be able to reduce the side effects associated with the traditional therapy with TNP-470. The translation of this approach in the clinical practice should allow an early detection of patients, often asymptomatic, which present a high risk of cardiovascular complications, due to plaques characterized by a high extent of new vessels formation. Moreover, the non-invasive monitoring of the progression of the disease should authorize the rapid identification of patients who are responding to the treatment and the assessment of the long-term antiangiogenic effect. 4.2. MRI and anti-restenotic therapy Restenosis is one of the complications of angioplasty which involves vascular remodeling mainly attributable to extracellular matrix production and activation, migration and proliferation of smooth muscle cells [220]. αvβ3 integrins highly expressed at the surface of stretch activated smooth muscle cells thus represent promising targets for the delivery of multifunctional NPs. For example, rapamycin-loaded αvβ3-targeted paramagnetic NPs were designed for vessels imaging after balloon angioplastly and simultaneous delivery of the anti-restenotic therapeutic agent whose effectiveness could be monitored [221]. The in vivo effectiveness of this concept has been investigated in New Zeland white rabbits. Animals were fed for 4 months with high fat diet to induce atherogenesis and then, exposed to balloon overstretch in the femoral arteries in order to cause vessel injury. After injury, rapamycin-loaded αvβ3-targeted paramagnetic NPs were perfused in the injured vessel lumen and allowed to stay for 5 min. Unbound NPs were then aspirated,

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Fig. 14. Theranostic nanomedicines for the treatment of cardiovascular diseases: (a) MR images of thoracic aorta (arrow) and vessel segmentation (outlined in yellow) showing false-colored overlay of percent signal enhancement as result of αvβ3-targeted paramagnetic nanoparticles at time of treatment and during the following weeks. On the 1 week image enhancement has clearly decreased due to the antiangiogenic effect of targeted fumagillin treatment. The level of signal enhacement decreses gradually at weeks 2 and 3 after treatment until week 4 when the level of enhancement is practically identical to the week 0 image. Reprinted with permission from Elsevier Publisher : JACC: Cardiovascular Imaging [219]. (b) In vivo localization of CLIO THPC nanoparticles in the carotid atheroma: (1) fluorescence imaging in the 750 channel demonstrating particle uptake by a carotid plaque; (2) fluorescence angiogram using fluorescein‐labeled dextran; (3) merged image of the two fluorescence channels. Adapted with permission from John Wiley and Sons publishers: Small [227]. (c) Hind limb skin tissue perfusion in a rat embolic model measured by a laser Doppler perfusion imager. After clot lodging into the left iliac artery, rtPA, rtPA covalently bound to PAA-coated MNP or equivalent MNP was administered from the right iliac arterial 5 min after introducing the clot. Data are representative of 8–10 experiments. Adapted with permission from Elsevier Publisher: Biomaterials [229].

the blood flow was reestablished and, after closure of excisions, animals were imaged by MR. MRI of femoral arteries was repeated two weeks after. An important signal enhancement was observed 30–40 min after the administration of αvβ3-targeted paramagnetic NPs and the reestablishment of blood flow which confirmed NPs accumulation and retention in the injured arterial wall segments. Contrariwise, no signal enhancement was detected after delivery of non-targeted paramagnetic NPs or saline solution. MR images recorded within 1 h from angioplasty and treatment did not show neither flow obstruction nor vascular wall dissections in all the treated groups. Two weeks later, vascular irregularities were observed in animals exposed to targeted or untargeted empty NPs or to saline solution. By contrast, only minimal irregularities were observed in animals treated with rapamicyn-loaded αvβ3-targeted paramagnetic NPs. These results clearly show the benefits of the local administration of rapamycin using targeted NPs which inhibited restenosis and lumen occlusion without delaying endothelial healing after balloon injury. Moreover, the non invasive monitoring allowed assessing the efficacy of the treatment. 4.3. Optical imaging and photodynamic therapy Macrophages are major components of the atherosclerotic lesions. They are responsible for the accumulation of oxidized lipoproteins, the release of inflammatory cytokines and extracellular proteinases [222] which play a key role in tissue remodeling processes thus promoting the degradation of plaque fibrous cap and the consequent plaque rupture. Due to the fundamental role of macrophages in the development and progression of atherosclerotic lesions, they represent another target for the detection and treatment of vulnerable plaques. Various studies have shown the high affinity of crosslinked dextran coated iron oxide (CLIO) NPs for inflammatory macrophages [223–226]. Thus, CLIO NPs may represent another interesting platform for the development of nanotheranostics in atherosclerotic lesions. Functionalized CLIO has been prepared by introduction of a NIR fluorophore and a potent chlorin-based photosensitizer (CLIO-THPC) [227]. The different spectral profiles of these molecules enable to monitor the distribution of the NPs and to exert PDT, respectively. The in vivo effectiveness has been evaluated on an Apo E −/− mice model of atherosclerosis. 24 h after CLIO-THPC injection, surgically exposed carotid arteries were imaged in the AF750 channel by intravital laser scanning fluorescence microscopy. Fluorescence images confirmed the localization of the NPs in the carotid atheroma lesion, in particular in the regions rich of

macrophages and foam cells (Fig. 14b). After the imaging session, mice where irradiated with a 650 nm laser light in order to activate the macrophage-targeted photosentitizer. Following treatment, mice were sutured and allowed to recover. One group of mice was sacrificed one day after the treatment and carotid arteries were subject to histological analysis which revealed a large number of apoptotic cells in the CLIO-THPC treated mice (55.5 ± 5.1% of the plaque area) compared to the control group (0.796 ± 0.10% of the plaque area). The other group of mice was imaged one week later, following a second injection of the nanotheranostic. Mice exposed PDT showed only a minimal uptake of CLIO-THPC, thus confirming the efficacy of the treatment. These findings demonstrate the promising approach of using such nanotheranostic for detection and treatment of atherosclerotic lesions.

4.4. Ultrasonography and thrombolytic therapy Thrombolitic agents are commonly used in the management of acute myocardial infarction and stroke [228]. Among the various thrombolytic agents, the recombinant tissue plasminogen activator (rtPA) is the most commonly used in clinical practice. It has been shown to benefit patients suffering thrombo-embolic diseases due to the induction of plasmin production, leading to lysis of fibrin clots. SPIO NPs have been developed also to deliver thrombolytic agents to the site of action by applying an external magnetic field. Again, the biodistribution of the NPs and the effectiveness of the therapy can be monitored in real time non-invasively. Ma et al. [229] have prepared poly acrylic acid-coated magnetic NPs (PAA-MNP), to which rtPA has been covalently linked (PAA‐MNP–rtPA). The effectiveness of these theranostic NPs has been evaluated in vivo in a rat embolic model, in which a clot was injected at the bifurcation of aorta and iliac arteries in order to reduce the left hind limb perfusion. After administration of PAA‐MNP–rtPA, free rtPA or PAA‐MNP, the perfusion of the hind limb skin tissue was monitored by a laser Doppler perfusion imager until 120 min after injection of the clot. Administration of PAA‐MNP–rtPA under magnetic guidance, led to a more important thrombolysis and restoration of the tissue perfusion compared to controls (Fig. 14c). Moreover, the highest thrombolysis induced by PAA‐MNP–rtPA was obtained at a rtPA dose b 20% of a regular dose. These results show interesting perspectives for achieving in vivo an effective thrombolysis and reperfusion. In addition, Doppler perfusion evaluation allows having an immediate response concerning the

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effectiveness of the treatment allowing to screen the patients for whom the thrombolytic treatment has to be eventually reevaluated. 5. Concluding remarks The emergence of the nanotheranostic concept and its further development illustrate the need for a pluridisciplinary approach (incl. physics, chemistry, material science, drug delivery and pharmacology) with the common objective of improving the management of cancer and other severe diseases. As detailed in the present review, interesting results have already been obtained and the proof of concept has been provided at the preclinical stage in cancer and cardiovascular disease treatment. The simultaneous delivery of imaging and therapeutic agents offers, indeed, the exciting possibility of early diagnostic and precocious feedbacks on the treatment effectiveness in real time, without detecting traditional end points (such as, for instance, the reduction of the tumor volume). If translated into the clinical stage (not done until now), this approach should enable the rapid adjustment of the therapy which would reflect the evolution of the disease in each patient. It is certain that a similar procedure, which takes into account disease and patients heterogeneity, paves to road to a personalized medicine. However, despite the wide enthusiasm associated with the presentation of every new and always more sophisticated theranostic nanomedicine, much work still needs to be done, before nanotheranostics could be effectively introduced into the medical practice. As pointed in this review, most of the currently available nanotheranostics have been investigated only in vitro, without providing a clear evidence of their feasibility as imaging and theranostic agents in vivo. In other studies, the in vivo investigation was performed, but only the imaging functionality was exploited: the therapeutic effectiveness of many of the theranostic nanomedicines still needs to be accurately verified. Nevertheless, even for the nanotheranostics which definitely demonstrated encouraging signs as detailed before, translation into clinical trials is a path strewn with obstacles. Several issues must be deeply investigated and clarified and certain answers are required [230]. It is especially essential to focus the attention on biocompatibility, drug/imaging agent loading capability, pharmacokinetic/pharmacodynamic parameters, and risk/benefit evaluation. A debated concern relies with the optimization of the dose required for achieving simultaneously the therapeutic and diagnostic purpose, which can be different by several orders of magnitude [231]. But the major limitation is related to the potential toxicity of the many imaging agents employed for the construction of theranostics nanomedicines. For instance, previously described nanotheranostics use iron oxide NPs in order to achieve a significant MRI contrast enhancement, however, without shedding lights on their toxicological profile. Although iron oxide NPs have been found to be relatively not toxic [232] and iron oxide NP-based formulations such as Feridex I.V.® and Sinerem® are currently clinically available, the safety of the single imaging agent is not a guaranty for the safety of multifunctional nanotheranostics in which diagnostic functionalities are combined to the therapeutic activity. Indeed, the specific physico-chemical characteristics of the nanotheranostics would strictly influence the biodistribution, cellular uptake and blood half-life of the iron oxide NPs [233]. Moreover, it must be considered that the in vivo fate of iron oxide NPs would be influenced by the dose and the frequency of administration of the nanotheranostics. Iron oxide NPs intended for diagnostic purpose are, indeed, supposed to be administered once or at least with a longer spacing time compared to nanotheranostics used in clinical protocols for simultaneous therapy and in real time monitoring of the diseases. Similarly, the safety profile the Gd 3+-based nanotheranostics has to be carefully investigated in order to assess any toxic accumulation of the contrast agent due to the potential slower clearance of the multifunctional NP. But still more importantly, the potential toxicity of gold NPs and QDs is a matter of concern. Several groups have investigated the potential toxicity

of QDs but currently available data are hardly comparable and sometimes controversial [234,235], because of the strictly dependence of the results on the synthetic method and the surface modifications of the QDs. The influence of these factors must be elucidated before QD-based nanotheranostics can move to clinics. Compared to QDs, gold NPs, might be considered safer due to their water solubility and the absence of heavy metals, but their biocompatibility remains questionable since their fate, after in vivo administration, is far to be fully established. Toxicological issues related to the use of all these contrast imaging agents represent therefore serious limitation for administration to humans. Thus, although the previously described nanotheranostics are highly promising for personalized nanomedicine, their translation into clinics would not be possible without a complete and detailed understanding of the potential toxicological risk associated to their use. Only facing all these challenges authorization from the Regulatory Agencies to start clinical trials and in patient assessment of their efficacy might be obtained. If these hurdles can be overcome, clinical application of theranostic nanomedicines would allow to detect, to investigate, and to understand the differences among patient, thus driving the clinical decisions and the elaboration of the therapeutic protocols and realizing the goal of the “personalized medicine”. Although it is evident that a great deal of effort is still required, the progress in the field of nanotheranostics is rapid and the nanotechnologies for personalized medicine will be a reality in a future not too far.

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