Nanoparticle-mediated delivery of suicide genes in cancer therapy

Accepted Manuscript Title: Nanoparticle-Mediated Delivery of Suicide Genes in Cancer Therapy Author: Riccardo Vago Veronica Collico Stefania Zuppone Davide Prosperi Miriam Colombo PII: DOI: Reference:

S1043-6618(16)30654-5 http://dx.doi.org/doi:10.1016/j.phrs.2016.07.007 YPHRS 3235

To appear in:

Pharmacological Research

Received date: Revised date: Accepted date:

15-4-2016 27-6-2016 5-7-2016

Please cite this article as: Vago Riccardo, Collico Veronica, Zuppone Stefania, Prosperi Davide, Colombo Miriam.Nanoparticle-Mediated Delivery of Suicide Genes in Cancer Therapy.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanoparticle-Mediated Delivery of Suicide Genes in Cancer Therapy Riccardo Vago2,3, Veronica Collico1, Stefania Zuppone1,3, Davide Prosperi1 and Miriam Colombo1,* 1

Università degli Studi di Milano-Bicocca, NanoBioLab, Dipartimento di Biotecnologie e Bioscienze,

piazza della Scienza 2, 20126 Milan, Italy. 2

Università Vita-Salute San Raffaele, Milano, I-20132, Italy

3

Istituto di Ricerca Urologica, Divisione di Oncologia Sperimentale, IRCCS Ospedale San Raffaele,

via Olgettina 60, 20132, Milan, Italy. *Miriam Colombo. Email: [email protected]; Phone (+39) 0264483302

Graphical Abstract

ABSTRACT Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy.

Keywords: non-viral gene delivery; nanovectors; suicide therapy; toxins; cancer treatment; active targeting.

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Contents: 1.

Introduction

1.1. State of the art therapies in cancer and their limitations 1.2. Nanoparticles as innovative carriers for anti-cancer drugs 2.

Colloidal Nanoparticles as Non-Viral Vectors for Gene Delivery

2.1. Gold nanovectors 2.2. Magnetic nanoparticles 2.3. Carbon nanovectors 2.3.1 Fullerene 2.3.2 Carbon nanotubes 2.4. Calcium phosphate nanovectors 2.5. Silica nanovectors 2.5.1 Mesoporous silica nanocontainers 2.5.2 Silica nanotubes 2.5.3 Dendrimer-like silica nanoparticles 3.

Therapeutic Agents

3.1. Directed Enzyme Prodrug Therapy 3.2. Bacterial and plant toxins: structure and mechanism of action 3.3. Toxin-based gene therapy 3.4. Tumor-specific expression of suicide genes 4.

Targeting Moieties

4.1. Antibodies and derivatives 4.2. Peptides and proteins 4.3. Aptamers 4.4. Other targeting moieties 5.

Conclusions and Perspectives

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1. Introduction Cancer figures among the leading causes of morbidity and mortality worldwide and the number of new cases is expected to rise by about 70% over the next 2 decades, as reported by the World Health Organization [1]. More than 100 tumor types exist, each with peculiar features requiring unique diagnosis and treatment. Lung, prostate, colorectal, stomach, and liver cancer are the most common types in men, while breast, colorectal, lung, uterine cervix, and stomach cancer are the most frequent among women. Important risk factors comprise smoking, virus infections, unhealthy diet, physical inactivity, UV radiations, alcohol and pollution and account for a substantial proportion of cancer mortality [2]. Nowadays, the management of cancer has significantly progressed and several tumors can be efficiently cured, by surgery, radiotherapy or chemotherapy, especially if they are diagnosed early. Cancer mortality can be reduced if cases are detected and treated early. Despite the important efforts and advancements over the past decades, cancer remains a big challenge to the scientific and medical community. A major issue is represented by the nature of therapeutic agents, most of which lack target selectivity causing devastating side effects to healthy tissues and promote the onset of chemoresistance after the first courses of administration. The development of drugs specifically exerting their activity on diseased cells without affecting healthy tissues is considered a critical step towards efficient and safe treatments.

1.1. State of the art therapies in cancer and their limitations Current clinical protocols are mostly based on the combination of surgical resection, radiation therapy and chemotherapy. Drugs used in classical chemotherapy are small molecules, like cisplatin, taxol, doxorubicin and other anthracyclines, used to treat the widest variety of tumor types. Cytotoxic chemotherapeutics commonly act disrupting the normal functions of a cell by inhibiting replication or inducing apoptosis. Thus, they preferentially kill rapidly proliferating cells and can effectively shrink solid tumors in some patients, though affecting also bone marrow, intestinal epithelial cells and hair follicles, causing unwanted toxicity. The restricted therapeutic index of classic antitumor drugs forces the clinicians to match the effectiveness of the drug with the ability of the patient’s organism to tolerate the associated side effects. Besides the absence of target selectivity, chemotherapy suffers from additional limitations, such as problems related to safe administration due to a lack of aqueous solubility [3]. Indeed, most chemotherapeutics either from natural source or deriving from synthetic processes are often strongly hydrophobic. For this reason, they mostly require oily moistures or lipophilic compositions to formulate the dosage, which contribute to the toxic effect of administrations. In addition, a major cause of failures of chemotherapy in human malignancies is due to a number of mechanisms including alterations in the drug target, activation of prosurvival 3

pathways and ineffective induction of cell death, that can be collectively defined as multidrug resistance [4]. For instance, an increased expression of efflux pumps such as P-glycoprotein (P-gp) in the cell membrane is responsible for transport of various anticancer drugs out of cells. Such overexpression is intrinsic in refractory cancer cell types, but in several instances basal expression of P-gp can turn into an overexpression after the first courses of treatment also in drug-sensitive tumors. Several alternative mechanisms operating at different steps of the cytotoxic action of the drug are responsible for the onset of the acquired tumor cell insensitivity, ranking from alterations of drug targets inside the cell, like the topoisomerases or amplification of dihydrofolate reductase, to the enhancement of the DNA repair machine; from the activation of the enzymes of the glutathione detoxification system to alterations of genes and proteins involved into the control of apoptosis (especially p53 and Bcl-2) [5]. Such an intricate biosignal network makes the identification of a conclusive treatment elusive. Therefore, as the majority of cancers is still resistant to the current therapeutic options, the development of alternative strategies remains urgent. Biotechnological drugs are a wide group of therapeutic agents obtained by genetic engineering methodologies that are increasingly produced and employed to improve the therapeutic solutions. Proteins, like antibodies and cytokines, as well as nucleic acids (both RNA and DNA) have been investigated as potential anti-tumor therapeutics. Over the past 15 years, RNA interference based technology have been widely employed to modulate gene expression at the post-transcriptional level. Small interfering RNA (siRNA) exogenously administrated to cells selectively anneal to mRNA and trigger its degradation [6]. One of the most innovative approaches to develop therapeutic agents with anti-tumor activity is the use of suicide genes. It does not consist in replacing not functional genes, but in expressing toxic products from various origins in target cells, causing their death. This strategy has been improved in the last three decades, since its efficacy was proven both in vitro and in vivo [7,8]. The selective delivery of suicide genes to the tumor tissue is the major challenge for this kind of therapy and several methods are currently under investigation [9-11]. Targeted therapies are emerging as specific approach in the treatment of cancer, based on their ability to preferentially or exclusively select and kill diseased cells, greatly limiting systemic side effects. The target specificity is addressed by the interaction of the drug with molecules, mainly protein receptors, expressed exclusively or over-expressed on the surface of tumor cells. Significant examples are represented by growth factor receptors, like the epidermal or the fibroblast growth factor receptor (EGFR and FGFR, respectively), induced by tumor cells to increase the proliferation rate and to trigger the process of invasion [12]. Hormone receptors represent another class of suitable targets, asin several organs, including breast, endometrium, ovary, prostate and testis, they control 4

when and how often cells multiply. Indeed, changes in hormone levels can interfere with this process leading to cancer development. Patients exhibiting advanced stage tumors often characterized by the spread of malignant cells in secondary organs through the bloodstream are not subjected to surgical resection, but hormone and biological therapies are the choice currently favored. Target-specific therapies are now rising as a valid contribution to anti-cancer therapies in combination with surgery or other treatments. They can contribute to decrease and confine the tumor mass allowing surgical resection or to determine a reduced administration of chemotherapeutic drugs, limiting the undesired effects of the latter.

1.2. Nanoparticles as innovative carriers for anti-cancer drugs The development of drug delivery systems that are able to modify the biodistribution, tissue uptake and pharmacokinetics of therapeutic agents is attracting increasing interest in biomedical research and in the pharmaceutical industry. Nanotechnology combined with advanced therapeutic agents offers great potential for addressing these challenges. In recent years, outstanding progress has been made in using nanovectors to direct drugs to tumor cells through surface ligands and to increase the targeted delivery by increasing serum residence time. Nanotherapeutic drug delivery systems showed important technological advantages providing increased circulation time of the drug, bypassing solubility limitations by incorporating hydrophobic substances, increasing the possible route of administration, and improving biodistribution of anticancer drugs. Furthermore, having a broad range of sizes, materials and surface characteristics, nanovectors provide control and sustain release of the drug both during the transportation and at the site of action [13]. Among the nonviral gene vectors, nanoparticles are one of the major challenging tools combining optimal characteristics in a single customized treatment [14]. Normally, they are carefully designed to offer several advantages addressing important issues, including 1) lack of immunogenicity and low systemic toxicity; 2) improvement ofthe genetic material stability, avoiding degradation during the blood circulation; 3) enhancement of the transfection efficacy, increasing the uptake of DNAs, RNAs, plasmids, etc.; 4) modulation of the therapeutic effect by changing the specific nanoparticle class (e.g., polymeric nanoparticles, liposomes and colloidal nanoparticles) and physical strategy; 5) targeting genes to a specific cell or tissue reducing non selective uptake and toxicity; 6) synergistic or cooperative effects by combined therapy (e.g., siRNA and antibodies); and 7) simultaneous association of diagnostic and therapeutic potential leading to the development of innovative “theranostic” tools for biomedicine [15]. The ideal nanoparticle for gene therapy should have optimal physico-chemical characteristics, includingsize, shape and surface charge, to bring agene to a specific cell or tissue overcoming biological barriers, such as the blood brain barrier (BBB). 5

Multifunctional nanoparticles for cancer gene therapy include polymeric, organic and inorganic nanoparticles. Currently cationic lipid and polymeric nanoparticles are the favorite formulation choicefor the systemic administration of siRNA in preclinical studies or clinical trials [16]. The standard approach to develop a gene loaded-multifunctional nanoparticle exploits the electrostatic interactions between the negatively-charged phosphate backbone of nucleic acids and a positivelycharged molecular layer bound to the nanoparticle surface (e.g., polyethylenimine (PEI) orpolylysine (PLL)). However, the interaction between nanoparticles and genes needs to be sufficiently tight to allow the gene delivery into the cell and the release of the complex inside the cytoplasm to be further translocated into the nucleus [17]. In addition, genes can be encapsulated in calcium phosphate or lipid based nanoparticles, or loaded onto the nanoparticlesurface by means ofcleavable linkers [18]. A combined therapy with chemotherapeutics and siRNAs has been investigated as an alternative strategy for increasingthe anticancer activity of the drug alone. Many groups have designed delivery systems based on nanoparticles for the co-delivery of anticancer chemotherapeutics and siRNAs [19,20]. For example, the suppression of genic expression of membrane proteins responsible for pumping out the drugs as resistance mechanism of cancer cells to chemotherapy, has been used as a strategy to reduce drug resistance upon treatment with cytotoxic agents, to increase intracellular drug concentration, and thereby to amplify the efficacy of the treatment [21]. Nanocarriers for gene therapy can be divided into 3 main groups: 1) polymeric compounds (mostly cationics); 2) inorganic nanoparticles; 3) lipid vectors and recombinant proteins, which can incorporate or form a complex with DNA. Recently, the DNA nanostructure itself has been used as a smart delivery vehicle in the form of oligonucleotide nanoparticles (ONPs) prepared through programmable self-assembly of short DNA fragments and therapeutic siRNAs (Figure 1) [22].

6

nanocapsules

plasmids

proteins siRNA

inorganic NPs liposomes

nanotubes

drugs

Targeted

Carrier

NPs

Therapeutic agent

Targeting moiety In vitro and in vivo models peptides

polypeptides

aptamers antibodies, Fab, ScFv

Clinical translation

Figure 1. Functional components for the design of nanoparticle-based delivery systems for effective treatment of cancer disease. 2. Colloidal Nanoparticles as Non-Viral Vectors for Gene Delivery The present review focuses on inorganic nanovectors that can be advantageous over non-viral bioorganic nanomaterials, such as liposomes, dendrimers, and biodegradable polymers, in terms of size and shape control and surface functionalization. (Figure 2) Inorganic nanoparticles can be designed to possess high colloidal stability and large surface area-to-volume ratio. A pool of functionalization strategies is presently available for preparing proper active drug and gene delivery vectors allowing easy drug loading and controlled release. Additionally, surface modifications with active or inert molecules can confer to inorganic nanoparticles the ability to cross the biological barriers (e.g., the BBB), to reach cancer cells, and also to reduce the nanoparticle cytotoxicity. Colloidal inorganic nanoparticles can be easily localized at tumor, inflammatory and infectious sites exploiting the enhanced permeability and retention effect (EPR) on the vasculature, in addition to the active targeting [23]. In particular, gene delivery by inorganic particles is processed for the majority by the amalgamation of cationic polymer complexes onto the surface of the nanoparticles. The stability corroborates towards the fact that the genes are protected from enzymatic and physical degradation via binding to cationic nanoparticles. The stability of the nanosystem also relies on the efficacy of gene delivered into the nucleus that purely depends upon the biologic mechanism of its delivery [23]. In order to optimize their use as nanovectors for gene therapy, inorganic nanoparticles have to be biocompatible and not cytotoxic, and to prevent antigenic activity. Importantly, due to the huge 7

surface area, they have excellent abilities to promote adsorption, concentration and protection of DNA [24]. Targeted nanoparticles are designed to confine the drug molecules in a therapeutic dosage at the tumor site. Additionally, they require a long circulation-time in plasma, thus increasing the bioavailability of the drug. The conceptual advance in using nanoparticles as gene vector is based on the ability to exit the blood stream to penetrate the target tissue, and once into, release the drug. In principle, these features allow to reduce the dose administered and increase the efficacy of the drug assumed by the patient, aiming to decrease side effects. Finally, they are intended to reduce the cytotoxicity of the drug loaded compared to the free one. Bearing in mind these perspectives, different classes of nanoparticles have been explored. Here we report some examples of most commonly used nanovectors. Metallic nanoparticles, including gold and iron oxide, mesoporous silica, carbon nanotubes and calcium phosphate nanoparticles, have been investigated to improve the efficacy of gene therapy.

Figure 2. Inorganic nanovectors used in gene therapy.

2.1. Gold nanovectors Gold nanoparticles (GNPs) have emerged has an attractive candidate for biomedical applications, including use as drug and gene delivery system. GNPs have unique optical properties, excellent stability, bioinertness, and biocompatibility in a broad range of administrations and do not exhibit significant cytotoxicity in vitro. In addition, GNP synthesis can be accomplished by straightforward and versatile approaches, they can be easily functionalized taking advantage of the gold affinity to 8

thiol/sulfide group and of their high surface-to-volume ratio [25]. However, despite these advantageous properties, the potential of GNPs for gene delivery remains limited, as a few studies have demonstrated that unmodified GNPs showed poor ability in carrying DNA [24]. Thus, surface modifications are necessary to increase the transfection efficiency of genes and also to confer biological affinity to GNPs [24]. Many groups developed GNPs functionalized with cationic polymers, such as polyethylene imine (PEI) of different molecular weights (from 800 Da to 25 kDa) and structures (e.g., linear or branched). This strategy exploits the electrostatic interactions between the positive charges of PEI amino groups and the negative charge of the genetic material. As an example, a GNP-PEI (800 Da) hybrid nanosystem exhibited transfection efficiency in COS-7 cells 3 or 4 orders more effective than unmodified PEI. However, PEI size seems to play a prominent role in affecting the transfection efficiency of gold nanoconjugates. Indeed, while higher molecular weight PEIs (i.e., 25 kDa PEI) in presence of serum medium results in lower efficiency, the efficiency of GNP with short PEI (i.e., 800 Da) can be retained or even enhanced (60-fold higher in 10% serum) [26]. Song et al. prepared PEI-capped GNPs for intracellular delivery of siRNA. Nanocarriers entered into cells and knocked down the target gene expression, namely the exogenous green fluorescent protein (GFP) and polo-like kinase I (PLK1) oncogenes, in MDA-MB-435s tumor cells. It might be speculated that GNPs-PEI could redirect the intracellular distributions of siRNA and therefore enhance its cytosolic availability leading to a better gene silencing efficiency compared to PEI alone [27]. An alternative strategy might exploit the strong affinity of gold for sulfur atoms, however it is not recommended to bind DNA directly to the gold surface through thiol groups, because the tight linkage makes the genetic material hard to release. In another work, a photothermal gene delivery system, based on a DNA plasmid electrostatically bound to GNPs, was developed. Niidome et al. prepared a polyethyleneglycol-orthopyridyl-disulfide (PEG-OPSS)-coated GNP complex that releases the plasmid under a pulse laser radiation without any fragmentation of the genetic material [28]. Similarly, Pawan et al. developed a gene release nanosystem based on an electric stimuli: the application of an electric field provides a net force that detaches the gene from GNP surface and pushes it into the nucleus of target cells [29]. Gold nanorods (GNRs) are lengthened nanoparticles with exceptional optical properties depending on their anisotropic shape, since they display a characteristic longitudinal localized surface plasmon resonance (LSPR) band. The plasmon signal is very sensitive to the surrounding local environment and, upon addition of target analytes, the interaction between the analytes and the surface of the GNRs leads to a detectable shift in the longitudinal band [30]. The shift in the longitudinal LSPR peak of GNRs provides a straightforward method to test their interaction with molecules, such as peptides, oligonucleotides and relevant macromolecules. Moreover, the ability of GNRs to bind nucleic acids 9

through electrostatic interactions under certain conditions makes these particles interesting vectors for the delivery of genetic material to target cells. For example, Prasad et al. synthesized an efficient GNRs-siRNA complex for gene silencing. The reduction of protein DARPP-32 (ERK and PP-1) expression confirmed the capability of GNRs-siRNA to suppress the DARPP-32 genes in dopaminergic neuronal cells. Upon GNR uptake, cell viability was still 98%, while the percentage knockdown of DARPP-32 gene expression increased of about 67%, markedly higher than the 30% knockdown obtained in cells treated with a commercial transfection agent. Notably, these nanoplexes were shown to be able to transmigrate across an in vitro model of BBB [31]. Another important feature of GNRs is their capability to yield local heating in nanoparticle-treated cells by exposing them to a laser radiation at a wavelength near their plasmon resonant absorption band, without harming surrounding healthy tissues. This feature is particularly useful for plasmonic photothermal therapy and can be additionally exploited to assist the release of the genetic material from the GNRs upon laser irradiation [32]. Bimetallic nanorods – i.e., an alloyed combination of gold and silver nanorods (Ag–GNRs) – acted as photothermal vectors for selective gene delivery, offering flexible optical properties with higher extinction efficiency in the near-infrared (NIR) range compared to pure GNRs. Their functionalization with specific aptamers conferred them the ability to selectively target mixed cancer cells and, combined with laser irradiation, gave higher relative dead cell percentage with respect to non-functionalized Ag-GNRs [33].

2.2. Magnetic nanoparticles Magnetic nanoparticles (MNPs) are broadly used for biological application thanks to their biocompatibility, as they degrade into the body into iron ions that enter the normal iron metabolic pathway. MNPs represent a very versatile system both for diagnosis and therapy. They are a versatile nanotool useful as contrast agents for magnetic resonance imaging (MRI) in the clinic, as drug delivery systems (DDS) and as mediators in cancer hyperthermic treatments. Moreover, MNPs have been used in gene therapy as transfection agents assisting the transport of nucleic acids or viral vectors into the cells [34]. In particular, gene delivery mediated by MNPs is commonly referred to as magnetofection [35-37]. A great advantage of magnetofection resides in that transfection efficiency can be improved applying an external magnetic field, which accelerates the gene-MNPs complex adsorption onto the cell surface promoting the phagocytosis of the genetic material [38,39]. Although this strategy proved to be successful with solid tumors, however it provided limited efficacy with highly invasive and infiltrating cancers (e.g., gliomas) so far, as these malignant cells are not easily accessible to a localized magnetic field. 10

MNPs are generally coated with different surfactants to prevent nanoparticle agglomeration, reduce the cytotoxicity, and to add functional groups useful for binding of the genetic material [35,37, 40]. PEI is the most used between a wide range of coating polymers. However, as already mentioned, PEI-based gene therapy is limited by toxicity caused by the high positive charge density of PEI, which causes disruption of cell membrane, detrimental intracellular “proton sponge” effects and impairment of mitochondrial activity resulting in reactive oxygen species (ROS) release [41]. In an attempt to overcome this problem, lipid-modified magnetoplexes were developed by coating MNPs with quaternary ammonium ion-based aminooxy and oxime groups, further functionalized with lipid side chains for generating safe magnetofection nanovectors. The authors found that magnetoplexes showed higher transfection efficiency in MCF-7 breast cancer cells compared to available cationic liposome formulations, both with or without magnetic assistance [42]. In vivo, these gene delivery vehicles can be addressed to their target site principally exploiting the EPR effect to passively accumulate at the tumor tissue. However, it is possible to improve the MNP ability to target cancer cells by functionalizing them with active targeting molecules that can promote a receptor-mediated endocytosis, resulting in increased transfection efficiency. Following this suggestion, Kievit et al. proposed magnetovectors stabilized with copolymers of chitosan, PEG and PEI for active gene delivery. pGFP was delivered to C6 glioma cells in a xenograft mouse model using magnetic carriers activated with chlorotoxin (CTX), a promising targeting agent able to specifically recognize a wide variety of cancers, including brain tumors, prostate, skin, and colorectal cancers. Interestingly, CTX does not affect the accumulation of nanoparticles at the tumor site, but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression [36,43]. It is assumed that the unique magnetic properties of MNPs can be exploited to further enhance the targeting efficiency of magnetofection. In vitro data on Saos-2 osteoblasts confirmed that the transfection efficiency of magnetic nanovectors is strongly dependent on the presence of an applied magnetic field, [36,39] while in vivo experiments showed that targeted MNPs are able to enhance the targeting efficiency under the force of the magnetic field [44]. Finally, the superparamagnetic character of nanoscale magnetite allows for the localization of gene nanovectors both in vitro and in vivo, as it can be favorably utilized as contrast agent in magnetic resonance imaging (MRI).

2.3. Carbon nanovectors 2.3.1. Fullerene The term “Fullerfection” has been used to underline the ability of modified fullerenes to efficiently deliver genes to cells. Fullerenes are water-insoluble allotropes of carbon having a unique sphericalshaped structure bearing several intriguing properties, including photosensitivity, redox activity and 11

high chemical reactivity [45,46]. The low solubility of fullerene can be improved by surface modification, for example introducing amino groups useful for the subsequent immobilization of genetic material. Maeda-Mamiya et al. synthesized an amphiphilic tetra-amino fullerene (TPFE) forming small aggregates with double-stranded DNA that protected nucleic acid from nucleases. The DNA/TPFE complex was stable in serum conditions and had size (50-100 nm) suitable for being internalized into the cells by endocytosis. The nanocomplex could penetrate into the nucleus where the DNA was released by two different mechanisms, either by removing the amino groups or by neutralizing them with an amide reaction. TPFE could target liver and spleen in vivo more efficiently than commercial Lipofectin and avoided acute toxicity in liver and kidneys [46]. 2.3.2. Carbon nanotubes Carbon nanotubes (CNTs) are one dimensional seamless cylindrical graphene sheets, tunable in dimensions (length and diameter) and composition (material and number of walls). Both single- and multi-walled carbon nanotubes were found to form stable complexes with DNA plasmids, thus allowing for the successful delivery of genes [47]. Moreover, CNTs are adopted in drug delivery and gene therapy thanks to their easy translocation across the plasma membranes of cells with an endocytosis-independent mechanism. Surprisingly, this unusual pathway of entrance did not induce cell death and exhibited low general cytotoxicity in vitro, although concerns about the ultimate biocompatibility and poor solubility have limited their widespread use in biomedical applications [37,48]. However, CNT solubility can be increased by chemical surface modification, which allows also the easy functionalization with biomolecules, imaging agents, drugs or genes. In principle, CNT cavity should allow loading large amounts of drugs or genes, and their open ends could act as a gate for drug loading or release and exhibit also very advantageous intrinsic properties such as strong optical absorbance in the NIR region leading to photoluminescence, strong Raman scattering and photoacoustic signals that could be exploited to track gene delivery. These properties can be used to trigger drug or gene release from CNT by illumination with NIR light: the absorbance of NIR waves produces a localized heat stimulating the release of drugs or genes from carbon surface [49].

2.4. Calcium phosphate nanovectors Calcium phosphate nanoparticles (CPNPs) are intensively employed in gene delivery thanks to their high transfection efficiency, either alone or in combination with viral and non-viral vectors. CPNPs show low immunogenicity and cytotoxicity and are retained to be highly biocompatible. Indeed, they can be found throughout the body in the form of amorphous calcium phosphate and crystalline hydroxyapatite, which is the major natural inorganic component of bones and tooth 12

enamel. Their easy degradation in a biological environment has the advantage to make CPNPs nontoxic, but on the other hand limits the possibility of their long-term storage [50-52]. CPNP synthesis is straightforward, easy to scale-up and very cheap. Moreover, calcium, as well as many other bivalent cations, can form ionic complexes with nucleic acids resulting in stable structures with high oligonucleotide payload [53]. It has been demonstrated that CPNPs are able to transport nucleic acids across the cell membrane via ion channel-mediated endocytosis. Despite the advantages of CPNPs in gene delivery, there are some issues that limit their potential at present, such as 1) poor reproducibility in synthetic protocols due to various experimental variables, including pH, temperature, DNA concentration, cell type, etc.; 2) unsatisfactory colloidal stability, which causes aggregation affecting the transfection efficiency. For the same reason, CPNPs suspension storage cannot be protracted for longtime [54]. A PEG shell with the function of stabilizing and making the nanoparticles stealth, or a lipidic shell stabilizing and helping the particles to penetrate into the cellular membrane, were proposed as possible solutions to overcome this limitations [55,56].

2.5. Silica nanovectors 2.5.1. Mesoporous silica nanocontainers Among the various kinds of nanoparticles, mesoporous silica nanoparticles (MSNs) are attractive for their porous nature, the easy surface manipulation, the long-term stability, the ease of preparation in large quantities at low cost, and, importantly, for their postulated biocompatibility. MSNs have been recently applied for controlled release of drugs and genes thanks to their controllable size between 50 and 300 nm, which allows a facile cellular endocytosis without significant cytotoxicity, and to the uniform and tunable pore size (usually in the 2 to 6 nm range). MSN porosity provides high surface area and large pores volume useful for functionalizing both the inner and the outer surfaces with different moieties. All these features provide a set of control parameters that allow to adjust the loading of different molecules and to modulate the release kinetic profile. Moreover, compared to other polymer-based carriers, MSNs are more resistant to heating, pH variations, mechanical stress, and hydrolysis-induced degradation due to their stable and rigid framework. The lack of interconnectivity between individual porous channels prevents premature release of drugs/genes and makes the individual cylindrical pores independent reservoirs for drug encapsulation and release, as long as both ends of a given channel are capped [57]. Kim et al. synthesized amine-modified monodisperse MSNs with cationic pores of >15 nm, thus large enough to encapsulate plasmids, at least partially, without using potentially toxic cationic polymers for DNA immobilization. MSNs with 23 nm pores showed efficient cellular uptake, high 13

plasmid-DNA (pDNA) loading capacity, remarkable protection against DNase Ι, and low cytotoxicity [58]. In a different approach, Hartono et al. developed large pore MSNs (LPMSNs) functionalized with degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) (PDMAEA–LPMSNs) as nanocarriers for gene delivery. The unique design of PDMAEA–LPMSNs endowed this system with multiple functions derived from both the organic and inorganic moieties. PDMAEA is a cationic polymer covalently attached to the MSN surface that binds negatively charged siRNAs. It undergoes selfcatalyzed hydrolysis in water, generating a nontoxic anionic polymer poly(acrylic acid) (PAA), and controls the release of siRNA in cells, thus PDMAEA degraded only after being internalized by target cells, independent of external degradation triggers [59,60] In order to improve the therapeutic efficacy of silica nanovectors, Lin’s group developed MSN-based stimuli-responsive systems, based on a variety of chemical entities as gatekeepers (nanoparticles, organic molecules, or supramolecular assemblies) to regulate the encapsulation and release of drug molecules [61]. The stimuli-responsive vectors are designed to release the cargo in a proper concentration at the desired target in a determined time [62]. The zero premature release make them particularly useful when the delivered cargo is toxic (e.g., anti-cancer drugs). Mesoporous silica nanoparticles are also used as multitasking vectors both for drug and gene delivery. Meng et al. demonstrated that MSNPs can restore doxorubicin (Dox) sensitivity in a drug resistant squamous carcinoma cell line by co-delivery of siRNAs that knockdown the drug resistance gene. The phosphonate-coated particle pores allowed the electrostatic Dox interior attachment and the subsequent release by protons in an acidifying endosomal environment of a MCF7 multidrug resistance (MDR) xenograft model in nude mice. Moreover, the phosphonate modification ensured an exterior cationic PEI-PEG coating, used for the electrostatic attachment of siRNAs. The systemic administration of Dox and siRNA dual delivery nanoparticle resulted in synergistic inhibition of tumor growth in a MDR tumor xenograft model in vivo [63,64]. 2.5.2. Silica nanotubes Wu et al. reported fluorescent silica nanotubes (SNTs) internally modified with 3-aminopropyl silane to generate a polycationic surface adjusted to hold CdSe/ZnS core-shell quantum dots (QDs) or DNA molecules through electrostatic forces. Gene delivery experiments were performed inserting a plasmid DNA-SNT complex in COS-7 cells. Although the efficiency of SNT-mediated DNA transfection is about four times lower than that of conventional calcium phosphate, the advantage of this strategy is the broad range of cargo biomolecules carried by SNTs [65,66]. Son et al. developed magnetic silica nanotubes (MNTs), in which magnetic nanoparticles are embedded in silica nanotubes, to exploit the advantage of MNPs to track the drug delivery through MRI analysis and to

14

control the drug carrier by an external magnetic field, and hence to address it to specific sites in vivo. [67] 2.5.3. Dendrimer-like silica nanoparticles Dendrimer-like silica nanoparticles (HPSNs) are peculiar silica nanoparticles preserving the skeleton of a dendrimer that could be designed with articulated branches developing from a centralradial core. HPSNs with hierarchical pores are supposed to possess more benefits than conventional MSNs with uniform mesoporous or even dendrimer-like silica nanoparticles without uniform mesopores. One advantage of HPSNs derives from their structure: center-radial open pores have much higher accessibility for various sized molecules to the internal surface of the particles, which is favorable for the loading and the mass transport of large biomolecules. Importantly, the synergistic combination of multiple-scale pores can balance the diffusion of guest molecules with different sizes through the porous matrices. Du et al. exploited these platforms to gain a codelivery synergistic effect: they prepared HPSNs-PEI to deliver into KHOS osteosarcoma cells the anticancer drug TPT, mostly accommodated inside the mesopores, together with PLK1 siRNA, in turn tightly bound to the cationic PEI [68]. MATERIAL

VECTORS

ADVANTAGES

LIMITATIONS

FIELD OF RESEARCH

REF.

fibroblast-like cells from monkey kidney tissue: COS-1 cells and COS-7 cells;

[26, 187, 190]

Human cervical cells: Hela;

optical properties

cancer

[187, 188, 189, 191]

biocompatibility

gold

Gold nanoparticles (GNPs)

non-cytotoxic versatile synthesis

Human breast cancer: MDA-MB-435s cells

[25, 27]

human lung carcinoma: A549

[27]

human and murine prostate cancer cells: PC-3 cells in vivo invertebrate

[17]

In vivo study on freshwater polyp: Hydra vulgaris

[191]

in vivo mouse model: C57BL/6J mice

[191]

easy functionalization gold affinity sulphur atoms

to

high surface volume ratio

to

breast carcinoma BT474;

15

cells:

[33, 192]

Gold nanorods (GNRs)

low citotoxicity

iron oxide

Magnetic Nanoparticles (MNPs)

easy surface functionalization

surface functionalization necessary

answer to an external magnetic field

Human cervical cancer cells : HeLa cells (CCL-2)

[33]

human lung carcinoma: A549

[31]

Human breast cancer cells: Hela cells

[29]

acute lymphoblastic leukemia: CCRF-CEM cells (CCL-119 T-cell)

[33]

Human choriocarcinoma: JEG-3, JAR, 1. human endometrial:RL95-2;

[44]

Human cervical cancer cells: Hela and HeLaS3;

[34] [44]

Colon from human (supraclavicular lymph node metastasis): Lovo cell lines

[44]

human liver cancer cells: Hep-G2

[44] [193]

Human breast cancer cells: MDA-MB-435-GFP ( αvβ3 integrin +) and MCF-7;

[42] [194]

Adenocarcinomic human alveolar basal epithelial cells: A549-GFP (αvβ3 integrin -)

[194]

human osteosarcama cells: Saos-2 cells

[38]

mouse embryonic fibroblast cells: NIH3T3

[34]

In vivo study on Flank xenograft tumor of C6 glioma cells in mice

[43]

decree transfection time

16

answer to an external magnetic field imaging application Magnetic Nanotubes (MNTs)

Carbon Nanohorn (CNH)

encapsulation drugs modulated by inner surface functionalization

necessary surface functionalization [67] cytotoxicity modulation

low solubility easy surface functionalization

prostate cancer cells: androgen independent

functionalization necessary

[47]

prostate cancer PC-3;

easy surface functionalization high chemicalactivity high

Fullerene

photosensitivity

hydrophobicity

redox property

low solubility

no acute toxicity

functionalization

In vivo study on female C57/BL6 mice

[46]

necessary DNA protection

carbon

penetration into the nucleus Human peripheral blood

Carbon Nanotube (CNT)

easy surface

mononuclear

functionalization

(PBMCs): T cells;

inner void to encapsulate molecules

cells

[195]

low solubility HeLa-derived MAGI cells

[195]

functionalization is necessary

pancreatic

cell

line:

inner and outer

MiaPaCa2-HRE cells with

functionalization

an reporter

17

HIF-1α/luciferase

[199]

tip as gate

[49, 197,

no damaging

cervical

cancer

internalization

(Hela): (CCL-2.2);

cells

198, 199]

NIR absorption Raman scattering

human

lung

carcinoma

cells: A549 (CCL-185)

[196]

photoacustic signal low cytotoxicity Human lung cancer line: H460 cells in vivo study on female nude mice

easy production and

[18, 55]

[18]

scale up low cost production lower immunogenicity

calcium phosphate

Calcium phosphate nanoparticles (CPNPs)

stabilized nucleic acid

DNA degradation poor reproducibility in

targetability

precipitation and transfection

membrane permeability

mesenchymal stem cells

[50]

pancreas human PanC-1 cells

cells:

[201]

hepato cellular carcinoma cells: Huh-7 cells

[52]

human lung cancer cells: NCI-H-460 cells

[18]

long-time stored cervical

cancer

cells:

HeLa;

biodegradable

[55, 201]

osteoblast precursor cell

biocompatible

line derived from high DNA loading

musculus calvaria:

[200]

MC3T3-E1; in vivo study on female athymic nude mice

18

[18]

high loading/encapsulation of molecules zero

premature

release cell type or tissue human cervical cancer cell

specificity and site

line: Hela

directing ability.

[20, 58, 68, 202, 206]

controlled release of molecules with a easy surface functionalization stable

and

rigid

the modification of the inner voids

framework

for pore caps can

silica

Mesoporous Silica Nanoparticles (MSNs)

long-term stability

limit

the

drug

squamous carcinoma cells:

loading capacity

KB-31 (drug sensitive) and

tuneable particles and

and/or

KB-V1 (doxo resistant)

volume pores size

diversity

the

[63]

of

compatible drugs

gate control

that can be loaded

human osteosarcoma cells: KHOS cells

high surface area and

[59, 204]

large pore volume inner

and

outer

breast

functionalization

cancer

MCF7/MDR

cells: xenograft

[62]

model in nude mice multifunctional platform

astrocyte cells

[202]

low citotoxicity murine melanoma cells: B16F10

biocompatibility

[205]

protect DNA biodegradability

Human breast cancer cells: MDA-MB-231 cells

19

[206]

penetration into the cell membrane

primary astrocyte cultures from neonatal rat cerebral

facile synthesis

[62]

cortex: P0 to P3 large

quantity

production competitive cost inner Silica Nanotubes (SNTs)

and

outer

functionalization

[66]

low citotoxicity human liver cancer cells:

silica Starlike Hollow Silica Nanoparticles (SHNPs)

advantage of silica

HepG2

NPs

[207] fibroblast-like cells from

stimuli responsive

monkey

kidney

tissue:

COS-7 cells human glioblastoma cancer tuneable optoelectronic property quantum dots

delivery and imaging tracking

cells: U87 potential in vivo

mouse fibroblasts: NIH-

toxicity

3T3

[208]

human Mesenchymal Stem

stability

Cells (hMSCs)

Table 1. Inorganic nanoparticles for gene therapy

3. Therapeutic Agents As mentioned in the above section, nanoparticles have been shown to deliver a huge amount of therapeutic agents to provide stability, to withstand the aggressive environment in the blood stream, prolonging their circulation half-life, to increase the solubility of hydrophobic drugs, to improve biodistribution, to reduce immunogenicity and confer target specificity. Nucleic acids are especially sensitive to degradation within the circulation by nucleases and prone to trigger activation of the innate immunity. For this reason, they have taken great advantage from nanoparticle-mediated delivery. This is particularly evident in RNA-based therapeutics, such as small interfering RNA 20

(siRNA), that have been demonstrated to be active as anti-tumor effectors in various animal models and are currently under investigation in clinical trials [69] Although to a lesser extent compared to RNA, DNA-based drugs have also been explored. A few notable studies reported the employment of nanoparticle-delivered therapeutic genes in oncological field, opening new routes toward the application of gene therapy in cancer research and treatment. The transfection of tumor suppressor genes, including p53 [70], Bik [71], FUS1 and LKB1 [72] in different cancer cells resulted in significant inhibition of tumor growth due to decreased cell proliferation and apoptosis induction. P53 gene expression in tumors led to sustained anti-angiogenic effect as well, thereby contributing to slow down the disease progression [73]. Another approach is represented by Rexin-G, a liposome containing a non replicative, pathology-targeted retroviral vector, bearing a cytocidal “dominant negative” cyclin G1 construct as a genetic payload, which has the ability to retard tumor growth by disruption of cyclin G1 activity, thus inducing apoptosis of tumor cells [74]. Caspases [75] and DNAses [76] encoding genes have been demonstrated to be effective in killing tumor cells in vitro and their potential has been considered for streamlining into in vivo trials. Cancer cell death and tumor growth arrest can be obtained directly by using suicide genes coding for bacterial or plant toxin or upon activation of an administered prodrug by enzyme coding genes. The selectivity of the suicide cancer gene therapy is defined by how accurately the vector can deliver its cargo to the tumor environment and/or how restrictedly the gene can be expressed in tumor cells. To date, two major gene delivery systems have been developed: recombinant disarmed viruses and non-viral vectors. Replication incompetent viruses have the advantages of high-efficiency infection of dividing and non-dividing cells, long-term stable expression, and the ability to accommodate large transgenes. Their use is limited owing to their side effects such as toxicity, immunogenicity and costs of large scale formulations. The drawbacks of viral vectors stimulated the search for non-viral gene delivery methods, which offer improved safety profiles and ease and less expensive production, but suffer from significantly lower transfection efficiency. The latter issue has been in part counterpoised by effective and specific targeting. Up to now, cationic liposomes and cationic polymers are the non-viral gene delivery methods most commonly used in transfection protocols from cell lines to clinical trials [77].

3.1. Directed Enzyme Prodrug Therapy Also known as gene/prodrug activation therapy, the directed enzyme prodrug therapeutic approach allows the combination of passive, active, and transcriptional targeting strategies to maximize anticancer activity at the tumor site, while minimizing the impact on normal tissues. It is a two-step process, where the selective delivery of the enzyme-coding genes to tumors is followed by the 21

systemic or intratumoral administration of a non toxic prodrug in a subtherapeutic dose [78,79]. Therefore, transduced cells, which actively express the enzyme, accumulate prodrug’s toxic metabolites, that lead them to death. Every year, a few new or modified enzyme/prodrug systems are proposed and some of them find their way towards preclinical and clinical studies. As of January 2015, 157 out of 2076 clinical trials (7.7%) conducted worldwide focused on suicide gene therapy, indicating that it is now assumed to be a major approach to cancer gene therapy [80]. The most widely used enzyme/prodrug systems that underwent clinical trials usually originated from viruses or bacteria [81]: examples include herpes simplex virus thymidine kinase/ganciclovir (HSVTK/GCV), cytosine

deaminase/5-fluorocytosine

(CD/5FC),

nitroreductase/CB1954

(NTR/CB1954),

carboxypeptidase G2/nitrogen mustard (CPG2/NM), purine nucleoside phsphorylase/6-methylpurine deoxyriboside (PNP/MEP), and cytochrome-P450/oxazaphosphorine (CYP450/Oxp). A commonly described method using herpes simplex virus thymidine kinase (HSV-tk) consists in the expression of the HSV-tk gene leading to the production of viral thymidine kinase that metabolizes ganciclovir (GCV) to ganciclovir monophosphate, which in turn is converted by the cellular kinases into ganciclovir triphosphate, an analog of deoxyguanosine triphosphate. As a consequence, DNA polymerase is inhibited and the incorporation of the compound into DNA results in chain termination and tumor cell death [82]. Several animal cancer models have been established in order to demonstrate the anti-tumor activity of the TK/GCV system, among which leukemia [83], glioma [84,85], bladder carcinoma [86], intrahepatic metastasis of liver cancer [87], colon adenocarcinoma [88], and oral cancer, [89,90]. Similar to HSV-TK/GCV system, apoptosis is also involved in the mechanism of cytotoxicity induced by the cytosine deaminase/5-fluorocytosine suicide system (CD/5-FC) [91]. The CD enzyme catalyzes the hydrolytic deamination of cytosine into uracil, converting the non-toxic prodrug 5-FC to a highly toxic chemotherapeutic agent, 5-FU, which is then transformed by cellular enzymes into potent pyrimidine antimetabolites (5-FdUMP, 5-FdUTP, 5FUTP). In vivo anti-tumor activity of the CD/5-FC combination has been demonstrated in several animal models, including fibrosarcomas [92], carcinomas [93], gliomas [94] and peripheral metastases deriving from different origins [95,96]. The successful results achieved in vivo led to the application of enzyme/prodrug systems in clinical trials as an additional therapy with promising results reported in patients with chronic lymphocytic leukemia, acute lymphocytic leukemia, brain tumors, prostate tumor [97].

3.2. Bacterial and plant toxins: structure and mechanism of action Toxins from bacterial or plant origins are dangerous molecules produced as defensive strategy to ensure the survival of such organisms. They have been structurally and functionally optimized under 22

evolutionary pressure to effectively harm and kill intoxicated cells and these remarkable features have been exploited for therapeutic purposes. Among bacterial toxins, Diphtheria toxin (DT) and Pseudomonas Exotoxin A (PEA) are the most studied for the anti-cancer treatment. DT is produced and secreted by a pathogenic strain of bacterium Corynebacterium diphtheria. It consists of two subunits linked to each other by disulfide bridges. Subunit A contains the catalytic (C) domain and subunit B consists of the translocation (T) and receptor-binding (R) domains. Upon binding to the heparin binding epidermal growth factor precursor on the cell membrane, DT is taken up in endocytic vesicles and a pH-induced conformational change in the T domain triggers its insertion into the endosomal membrane and makes possible the transfer of the C domain to the cytoplasm, where it exerts its lethal effect. PEA is an amino acid polypeptide secreted by the bacterium Pseudomonas aeruginosa as one of its virulence factors. It is produced as a single polypeptide chain composed of three structural and functional domains (domains I, II, and III). The intoxication process starts with the binding of domain I to the CD91, also called alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (2MR/LRP), on the surface of the cell. Once internalized, exposure to a low endosomal pH triggers major conformational changes within its structure and to the generation of an enzymatically active 37 kDa fragment, that is retrogradely transported through the Golgi apparatus to the endoplasmic reticulum, where the domain II mediates its translocation to the cytosol. DT and PE belong to the mono-ADP-ribosyl transferase family and exert their function by blocking the protein translation via catalyzing adenosine ADP ribosylation of the elongation factor 2 and thus killing cells through an apoptosis pathway [98]. Other well known toxins with great therapeutic potential are ribosome inactivating proteins (RIPs). They belong to the N-glycosidase family of toxins, widely distributed in nature, but predominantly found in plants and bacteria. RIPs irreversibly block protein translation as well, even though they differ from the above mentioned bacterial toxins since they depurinate a specific adenine base in the universally conserved GAGA-tetraloop located in 23/26/28S rRNA, also known as the -sarcin/ricin loop. As a consequence, the ribosome is unable to bind the elongation factor 2, rsulting in the irreversibe arrest of the protein synthesis and apoptotic cell death. Plant RIPs have been mainly classified into two groups: type I RIPs, composed by a single polypeptide chain with catalytic activity, and type II RIPs, heterodimers consisting of an A chain, functionally equivalent to the type I, linked to a B subunit, a binding domain, which is endowed with lectin-binding properties. Ricin, produced by Ricinus communis, is the prototype plant type II RIP, an exceptionally potent poison, thanks to its galactose-B-lectin domain that binds to glycans exposed on the cell membrane and mediates toxin entry into most mammalian cells. After internalization, ricin is retrogradely transported along the secretory pathway to the endoplasmic reticulum, where the disulfide bonds between the two chains 23

is reduced and the catalytic A chain is translocated into the cytosol and there it promptly inactivates target ribosomes [99]. Type I RIPs, like saporin from Saponaria officinalis, lack binding domain and have been demonstrated to follow a different intoxication route [100]. Both plant and bacteria toxins have been extensively studied to construct anti-cancer conjugates, that, by acting in a cell cycle independent manner, can target both quiescent and rapidly dividing cells, resulting suitable to fight both aggressive cancers and tumors with slower progression. To confer target specificity, catalytic domains have been bound to antibodies, providing antigen-specific binding affinity, or fused to ligands, usually a growth factor domain, directed to molecules overexpressed on the surface of malignant cells. The most successful toxin-based drug is Denileukindiftitox, the first recombinant fusion formed by a truncated form of DT and human cytokine interleukin-2 which the US Federal Drug Administration (FDA), approved for the treatment of cutaneous T-cell lymphomas. Numerous other toxin-based conjugates are under investigations in pre-clinical and clinical trials against hematological as well as solid tumors with encouraging results [101]. The cytotoxicity of these compounds depends on multiple factors that can seriously limit their efficacy. Solid tumor core is not easily accessible and the drug effect is often confined to the surface of the tumor mass. Toxin conjugates have to efficiently interact with their targets on the cell surface, be internalized and released in the cytosol to exert their inhibitory activity. Cancer cells can acquire resistance caused by loss or down-regulation of target receptors by autocrine ligands, resulting in reduced binding and internalization of the recombinant toxin. In addition, protein toxins can be subjected to lysosomal and proteasomal degradation, once penetrated into the tumor cells. All these concerns explain why a relevant number of toxin-derived drugs did not confirm their efficacy under clinical trials.

A

Type I RIPs

Type II RIPs G

G _ G A

G A

eEF2

B

eEF2

DT Protein synthesis inhibition

PEA

24

Figure 3. Toxin mechanisms of action. (A) Type I (saporin, gelonin, etc.) or type II (ricin) ribosome inactivating proteins (RIPs) act on 23/26/28S rRNA, by depurinating a specific adenine base in the conserved GAGA-tetraloop. (B) The catalytic domain of bacterial toxins such as Diphteria toxin (DT) and Pseudomonas Exotoxin A (PEA) inactivates the eukaryotic elongation factor 2 (eEF2) by ADP ribosylation. Both activities cause the inhibition of protein translation, promptly leading to cell death.

3.3. Toxin-based gene therapy Gene therapy has been investigated as an alternative or complementary strategy to overcome the limitations of conventional anti-cancer treatments. Plasmid DNA shows several advantages compared to proteins: its production and purification is less expensive and time-consuming even at large scale; it does not trigger immune response, even after multiple administrations; very low amount of protein directly synthesized inside the target cells is sufficient to induce apoptotic cell death; eventually, tumor cells do not develop resistance to the drug (Figure 3). To date, a few toxins have been employed in suicide cancer gene therapy and DT can be considered the prototype of toxins since it was the first and the best-studied one for this purpose. DT gene has been delivered through viral and non-viral vectors, expressed under constitutive or tumor-specific promoters and demonstrated to be effective in many cellular and animal models [102], so that it is currently under investigation in clinical trials. Two phase I/II clinical trials involving DT gene under regulation of the H19 promoter sequence, complexed with the cationic polymer polyethylenimine (PEI), were completed. In the first study, the complex was administrated in patients with unresectable, locally advanced, non-metastatic pancreatic cancer and the safety, tolerability, pharmacokinetics and preliminary efficacy in a dose-escalation trial were evaluated. H19/DT was safely administered by intratumoral injection under computerized tomography or endoscopic ultrasound guidance. A gradual arrest of tumor growth was observed in 4 weeks after initiating the treatment. Furthermore, when the treatment was completed, two patients were administered with a subsequent chemotherapy or chemoradiation therapy. After two weeks, down-staged pancreatic tumors were considered surgically resectable. Partial responses were also reported [103]. In the second, patients with superficial bladder cancer who have failed previous therapy with the Bacille Calmette-Guérin were enrolled and the efficacy and toxicity of intravesical instillations of H19/DT were assessed. H19/DT prevented new tumor growth in two-thirds of the patients and ablated a third of the marker lesions. Prolonged time to recurrence was observed in responding patients, with mild adverse events [104]. Overall, these studies showed positive results, though they were limited by the small number of patients and need to be extended. DT is a rare case of toxin gene delivered by nanoparticles. A cationic biodegradable poly(B-amino ester) polymer has been developed and shown to deliver the DT gene to the tumor site 25

in animal models. The tumor-restricted expression of DT was obtained by employing promoter sequences of two genes that are highly active in ovarian tumor cells, MSLN and HE4. The administration of DT nanoparticles directly to subcutaneous xenograft tumors and to the peritoneal cavity of mice bearing primary and metastatic ovarian tumors resulted in a significant reduction in tumor mass and a prolonged life span compared to control mice. Importantly, the growth of ovarian tumors in mice treated with DT nanoparticles was suppressed more effectively, and with minimal nonspecific cytotoxicity, compared to clinically relevant doses of cisplatin and paclitaxel [105], indicating this approach as suitable for clinical application. PEA gene is also a good candidate for cancer therapy, since it is efficient in inhibiting protein synthesis in a DT similar extent. A plasmid encoding a truncated but active form of PEA complexed with cationic lipids as transfection agents has been proven to block protein synthesis and induce apoptosis in breast carcinoma cell lines. PE/cationic lipid complexes were injected into tumor xenografts in athymic nude mice and attenuated the tumor growth [106]. PEA was delivered into various human cancer cells using SV40 vectors packaged in vitro (pseudovirions) and the number of viable cells was reduced significantly in all the PEA-transduced cells with similar toxicity. Human adenocarcinoma cells growing in mice were treated with intratumoral injection of PEA packaged in vitro and tumor size decreased significantly. Notably, the combined treatment of Dox with in vitropackaged PEA reduced tumor size slightly more than each of the treatments separately [107]. Retroviral vectors, in which either PEA or ricin were placed under the control of the thyroid hormone (T3) tunable promoter of the rat myelin basic protein, were highly toxic towards rat (C6) and human (U-373-MG) glioblastoma cells and were capable of eradicating experimentally induced brain tumors in Wistar rats [108]. Constructs expressing the type I plant RIP saporin (SAP) gene was prepared by placing the region encoding the toxin under the control of the CMV or SV40 promoters and their ability to block protein synthesis was successfully tested in melanoma cell lines, with the CMV driven expression of SAP resulted the most effective. Few nanograms of plasmids complexed with cationic lipids were sufficient to irreversibly inhibit the expression of luciferase reporter gene and kill transfected cancer cells. Saporin gene DNA, complexed with a cationic lipid, was able to significantly reduce tumor growth when injected intratumorally and its effect was increased upon repeated administrations. The antitumor effect was specifically due to the intrinsic RIP activity of the toxin, since a plasmid encoding the catalytic inactive mutant SAP-KQ did not kill cultured cells and did not delay tumor growth in mice [109,110]. In the context of ligand targeted delivery, SAP gene was complexed with basic fibroblast growth factor 2 (FGF2) carrying a polylysine tail and demonstrated to significantly decrease the number of viable FGF-2 receptor-bearing cells. Interestingly, the activity of SAP activity 26

was comparable to that of herpes simplex virus thymidine kinase gene upon activation of the ganciclovir prodrug. The specificity of targeted gene delivery approach was demonstrated in competition assays with free FGF2 [111]. The activity of a recombinant plasmid expressing another plant type I RIP, gelonin, was recently assessed on human ovarian cancer. Gelonin gene transfected by using biodegradable cationic heparinpolyethyleneimine (HPEI) nanogels resulted in the suppression of cell growth in vitro. Gelonin/HPEI complexes were then tested in an ovarian carcinomatosis model, showing a tumor growth inhibition following intraperitoneal administration [112]. The potential of plant RIPs in suicide cancer gene therapy remains partially unexpressed and further development of constructs and delivery systems are strongly encouraged.

3.4. Tumor-specific expression of suicide genes In case of systemic delivery of suicide genes, the detrimental expression of the toxin in inappropriate tissues has to be absolutely prevented. The safety of the toxin based suicide gene therapy method has been developed by two distinct strategies that can be applied individually or in combination. The first one is based on the possibility to deliver in vivo the toxin encoding vector by conjugation of its carrier with an appropriate targeting moiety, usually an antibody or a growth factor, interacting with a molecule over-expressed on the surface of malignant cells. The second takes advantage of the restricted expression of the therapeutic gene in a given tissue and has been used more frequently, due to its simplicity. The gene is placed under the control of a cancer specific promoter and is expressed only in the tumor cells, reducing the off-target toxicity. To identify a promoter that exclusively directs the expression of the gene of interest in the tumor cells is of great importance. In the last decades, several cancer/tissue promoters have been tested and valuable examples are the H19, the human telomerase reverse transcriptase (hTERT), the carcinoembryonic antigen (CEA), the osteocalcin (OC), the urokinase-type plasminogen activator receptor (uPAR) and the ERBB2 promoters [84, 113]. hTERT encodes for the catalytic protein subunit of telomerase, a polymerase that acts to stabilize telomere lengths and is highly expressed in tumors, but not in normal, differentiated adult cells. It successfully underwent phase I clinical trial, providing promising results in patients with advanced solid tumors. [114]. H19 is a paternally-imprinted, oncofetal gene that encodes a long non coding RNA (lncRNA) acting as a “riboregulator”, which is expressed at substantial levels in embryonic tissues, in different human tumor types, and marginally expressed or completely unexpressed in the corresponding adult tissues. Its promoter has been widely used in gene therapy because of its specificity to cancerous cells and, at the same time, demonstrates strong activity similar to the Simian virus 40 (SV40) promoter [115]. Mucin 1 (MUC1) is a member of the mucin 27

family which encodes a membrane bound glycosylated phosphoprotein. Its expression has been associated to tumor aggressiveness in pancreatic ductal adenocarcinoma (PDA) and other cancers [116]. Similar to H19, MUC1 has been employed as a therapeutic target for promoter-driven DT in in vitro model of heterogeneous PDA. It has been shown that the transfection with MUC1 promoterdriven DT construct induced an increased cell death, amplified in combination with drugs that enhance MUC1 expression in PDA cells [117]. Another relevant example is represented by Frizzled7 (Fzd7), whose expression correlates with the activation of the Wnt/β-catenin pathway, resulting in the nuclear accumulation of wild-type β-catenin. Recent studies indicate Fzd7 as the Wnt receptor most commonly up-regulated in a wide variety of cancers, such as colorectal cancer, triple negative breast cancer, pancreatic cancer and hepatocellular carcinoma [118]. Xu et al. demonstrated the ability of Frz1 promoter to direct Shiga-like toxin1 (Slt1) expression in vitro in different cancer cell lines, while leaving normal cells unaffected. In addition, in vivo studies of mice bearing liver tumor showed a decrease in the cell proliferation and tumor growth after transfection with Frz1 driven, Slt1 encoding plasmid [119]. The main safety advantage of specific promoter therapy is often accompanied by some limitation, like the weak activity, resulting in a decrease of therapeutic efficacy. To overcome these challenges, promoters can be modified in order to improve their activity. This is the case of chimeric and artificial promoters, currently under investigation, which consist in the introduction of enhancers or in the fusion of two different promoters, proven to increase their transcriptional activity [120]. In 2012, Amit et al. selected two different regulatory sequences from the human cancer-specific promoters H19 and IGF2-P4 to create a double promoter vector expressing the diphtheria toxin A-fragment (DTA). They demonstrated that the double promoter vector exhibited higher transcription activity compared to each single promoter, in a broad spectrum of malignant cell lines resembling bladder, pancreas, ovary cancer, glioblastoma and hepatocarcinoma. To expand the applicability of the tumor-specific promoters, more universal promoters capable of acting in a wide range of tumors, but not in normal cells, can be employed. Although this approach slightly increases the risk of affecting normal tissues, it is considered to be more economically viable, since the same constructs can be used for the therapy of a wide range of tumors. Additionally,

the

specificity of promoter-targeted therapy can derive

from tumor

microenvironment peculiarities, such as hypoxia. In fact, as tumor vasculature is often disorganized and inadequate, hypoxic areas are common in the malignant tumor mass. In response to this condition, cells up-regulate the transcription of genes involved in angiogenesis, anaerobic metabolism, vascular permeability and inflammation. In most cases, this process is initiated by the transcription factor hypoxia-inducible factor (HIF), which binds to hypoxia responsive elements (HREs). Hypoxiatargeting for gene therapy has been achieved using promoters containing hypoxia responsive elements 28

(HREs), in order to drive transgene selective and efficient activation at the tumor site. Even if the use of tissue/tumor-specific promoters results in a constitutive expression of the transgene in the target tissue, in some circumstances it is preferable to exogenously regulate the duration and level of expression through exogenously controlled inducible promoters such as radiation-, heat- or druginducible promoters [121].

4. Targeting Moieties In order to be effective, a targeted drug delivery system must respect four requirements: retain, evade, target and release [122]. Once drug has been loaded into a certain delivery vehicle, it has to reside sufficient time in blood circulation to reach a specific area of the body, be retained through specific properties within it (i.e., targeting), and, eventually, released within a time that allows drug to display its function. Two types of tumor targeting strategies have been reported so far, the “passive” and the “active” targeting routes. The so-called “passive targeting” mostly associates with the targeted drug delivery and refers to the passive drug accumulation in the tumor site due to tumor anatomical and pathophysiological differences from normal tissues, such as leaky vasculature and decreased rate of clearance. This phenomenon is commonly referred to as “enhanced permeability and retention” (EPR) effect. Most drugs and nano-sized agents accumulate within tumor tissues due to the EPR effect and release there their therapeutic payloads. However, the major limitation of EPR effect is that it provides relatively modest specificity, offering varying response rates, likely related to its broad heterogeneity observed among tumor types and within individual tumor. On the other hand, “active targeting” may represent a complementary way to overcome these limitations. It is inspired on the concept of the Paul Ehrlich’s “magic bullet”, with the aim to provide effective site-specific drug delivery. Active targeting is also useful to monitor tumor growth and burden through in vivo diagnostic imaging. In fact, the direct interaction between a ligand and its specific receptor or epitope expressed on cell surface allows the payload uptake by the targeted cell. These cell surface molecules display important functions in different biological processes, such as signal transduction, cell adhesion and migration, cell-cell and intra- and extracellular environment interactions and their abnormal expression is often related to tumorigenesis, making them of great interest for cancer diagnosis and therapy. Classical approaches to actively target over-expressed surface molecules have provided a broad spectrum of ligands, selectively directed against these tumor-associated antigens, such as peptide hormones, growth factors and mainly monoclonal antibodies. Agent providing active targeting have to accumulate to a significant degree in the pathologic tissue, exhibiting high binding affinity and specificity for their targets. In addition, they should not reside persistently in the bloodstream or in the organs of metabolism: to this aim, the small size may facilitate a rapid blood 29

clearance and tissue uptake. These characteristics lead to an improved therapeutic potential with reduced side effects. (Figure 4)

Proteins and peptides Aptamers Tf

half-chain of mAb

TfR

Antibodies and derivatives PSMA

Scfv/Fab

Folate receptor

Folate

EGFR Leptin

Carbohydrate

HER2

Monoclonal Ab

Figure 4. Active targeting approaches for nanovectors delivery; peptides, monoclonal antibodies and derivatives and aptamers can be linked to the nanovectors to target over-expressed surface molecules.

4.1. Antibodies and derivatives Nowadays, monoclonal antibody (mAb)-based therapy is one of the most successful and important strategies for the treatment of patients with hematological malignancies and solid tumors. The main type of therapeutic mAbs is represented by IgG, an Y-shaped protein (approximately 150 kDa) composed by two identical antigen binding sites (Fab), with a variable sequence, and a conserved Fc domain, which plays a role in modulating the immune cell activity, including complement activation, and regulating serum half-life [123,124]. The presence of two epitope binding sites in a single molecule offers an exceedingly high selectivity and binding affinity for the target of interest; for this reason, antibodies have become the first major class of biological drugs that could serve as ‘magic bullets’ in the diagnosis and therapy of cancer. Depending on the antibody, in fact, it is possible to target a broad variety of surface antigens involved in the pathophysiology of the disease, which are found mutated or even selectively expressed in diseased cells and tissues compared to the normal counterparts. Therefore, a key challenge over the past 20 years has been to identify antigens that are suitable for antibody-based therapeutics. Since 1997, FDA approved 12 antibodies for their application in oncology, and a large number of additional therapeutic antibodies are currently under investigation in Phase II and III clinical trials [125]. Rituximab, a humanized anti-cluster of differentiation (CD) 20 antibody used for treatment against B-cell lymphoma, was the first therapeutic anti-cancer antibody to be approved. Validation of rituximab was quickly followed by trastuzumab, a humanized anti-HER2 mAb used for treatment of HER2 positive breast carcinoma, and cetuximab, 30

a chimeric anti-HER1 mAb for treatment of colorectal cancer. Other therapeutic antibodies include alemtuzumab, a humanized anti-CD52 mAb approved in 2001 to treat chronic lymphocytic leukemia, and bevacizumab, a humanized anti-VEGF mAb designed to inhibit angiogenesis and used for treating colorectal and lung cancer [126]. Typically, mAbs are combined with conventional chemotherapy and their introduction into cancer treatment regimens has led to ameliorate the course of the disease and improve the clinical outcome. As a result, survival rates have raised up to 5 years for a number of cancer types. Although the impact of mAbs on cancer therapy is plainly evident, current therapeutic mAbs are associated with several drawbacks that hinder widespread use of these molecules, such as huge monetary costs of production, that involves relatively complex processes in mammalian cells; large size, around 150 kDa, which generally causes an inadequate pharmacokinetics and tissue accessibility. Furthermore, high dosage administrations are required to achieve clinical efficacy, due to the limited duration of action, contributing to undesired immunogenicity and toxicity [127]. In an effort to overcome these limitations, current developments in the field of recombinant technology have yielded alternative mAb structures, among which are humanized, chimeric, and/or fragmented antibodies. Single-chain antibody fragments (ScFv), for example, consisting of the smallest functional fragments of antibodies that can bind to the antigen (the variable regions Fv) linked with a flexible peptide, provide specific therapeutic benefits in the treatment of solid tumors, due to a reduction in size (approximately 27  kDa) thereby favoring improved tissue penetration and biodistribution. Moreover, they do not contain the Fc region of the antibody which can induce immunogenicity and antigenicity [128]. Nanomedicine supplies an additional strategy to circumvent mAb-based therapy limitations. MAbs and their derivatives have been extensively examined as escort molecules to confer target selectivity to nanoparticles. Such conjugates have the potential to elicit effective targeting and release of therapeutic cargoes at the disease site, while minimizing off-target side effects caused by dosing. The human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of invasive breast cancers and is associated with a more aggressive tumor phenotype, worse prognosis and reduced survival rate. Several antibodies have been developed against HER2 and employed as targeting moiety for nanoparticles. A PEGylated hybrid magnetic nanocrystal system conjugated to fluorescently labeled anti-HER2 antibody was demonstrated to prevalently accumulate at the tumor site by using a multifaceted bioanalytical approach, combining fluorescence, magnetic relaxivity, transmission electron microscopy and histological experiments in vivo and ex vivo [129]. Nanoparticles consisting of a mucic acid polymer conjugate of camptothecin and containing on average a single anti-HER2 antibody molecule have been developed and tested in nude mice bearing HER2 over-expressing BT-474 human breast cancer 31

tumors. As a result, after intravenous tail vein injections in mice, nanoparticles displayed prolonged in vivo circulation and showed complete tumor regression [130]. A comparative study of internalization, trafficking, and metabolism in breast cancer MCF7 cells of multifunctional nanoparticles formed by iron oxide spherical nanocrystal coated with an amphiphilic polymer shell and functionalized with anti-HER2 antibody or with alternative lower molecular weight variants such as the half-chain and scFv fragments has been performed. In vitro, in vivo, and ex vivo analyses of the targeting efficiency and of the intracellular fate of targeting moieties suggested that the highly stable half chain is the best candidate for application in breast cancer detection [131]. PEG-poly(D, Llactide) (PLA)-based nanoparticles functionalized with anti-HER2 ScFv have been developed to deliver a small interfering RNA (siRNA) against the gene coding for polo-like kinase 1 (Plk1). Improved cellular uptake, effective Plk1 silencing and enhanced tumor cell apoptosis in HER2+ breast cancer cells were observed. More importantly, nanoparticles markedly enhanced the accumulation of siRNA in HER2+ breast tumor tissue and remarkably improved the efficacy of tumor suppression [132]. Iron oxide nanoparticles conjugated to an antibody against the epidermal growth factor receptor deletion mutant (EGFRvIII), expressed on the surface of human glioblastoma multiforme cells, were exploited for therapeutic targeting and MRI after convection enhanced delivery. Nanoparticles were delivered to the intratumoral and peritumoral regions in the brain of mice implanted with highly tumorigenic glioblastoma xenografts showing a significant increase in animal survival [133]. Prostate specific membrane antigen (PSMA) is highly expressed in almost all prostate cancers and its expression progressively increases in higher Gleason grade and hormone refractory tumors. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles bearing 131I labeled anti-PSMA antibody and curcumin as an anticancer agent were evaluated in C4-2 xenograft mouse model, exhibiting a specific accumulation at the tumor site in a dose dependent manner and a significant anti-cancer activity [134]. A preclinical study that employed a polymeric nanoparticle targeting PSMA containing the chemotherapeutic agent taxol showed enhanced tumor accumulation and prolonged tumor growth delay in tumor-bearing mice. Preliminary data of a phase I study in patients with advanced or metastatic solid cancers displayed cases of tumor shrinkage [135]. The single chain antiprostate stem cell antigen (PSCA) is a prostate-specific glycosylphosphatidyl-inositol-anchored glycoprotein that is marginally expressed in normal prostate and overexpressed in prostate cancer tissues. Theranostic polymer nanoparticles conjugated with anti-PSCA scFv antibody and loaded simultaneously with anti-cancer drug taxol and superparamagnetic iron oxide nanocrystals were developed for both cancer therapy and ultrasensitive MRI. Enhanced cellular uptake ability and antiproliferative effect of the targeted nanoparticles in prostate cancer PC3 cell line were observed [136]. The receptor for transferrin (Tf), referred to as TfR1, is ubiquitously expressed at low levels in most 32

normal human tissues, while is up-regulated in metastatic and drug-resistant malignant cells and its expression can be correlated with tumor stage or cancer progression. Anti-TfR specific antibodies can be attached to the surface of liposomes loaded with chemotherapeutic drugs. Immunoliposomes delivering oxaliplatin has completed a Phase I clinical trial and is in a Phase Ib/II in second line patients with gastric, gastroesophageal, or esophageal adenocarcinoma [137]. Another system under clinical evaluation is represented by immunoliposomes encapsulating plasmid DNA coding for wild type p53 in phase I clinical trial. Minimal side effects were observed in patients with advanced solid tumors and the majority of them demonstrated stable disease. The accumulation of the transgene in metastatic tumors, but not in normal skin tissue, in a dose-related manner was observed [138]. Up to date no antibody-nanoparticle conjugates have been approved by FDA, although the encouraging preclinical results in vitro and in vivo in cancer treatment via both passive and active targeting show that this combination is promising for cancer treatment. However, several limitations, including large size and difficulty in conjugation to nanoparticles, have hampered the use of monoclonal antibodies (mAbs) as “magic bullets” for the targeted delivery of nanoparticles. Thus, at present, other smaller-sized ligands including peptides have attracted greater attention.

4.2. Peptides and proteins Peptides are attractive targeting molecules due to their small size, low immunogenicity, ease of manufacture at small costs and of conjugation to the nanoparticles. Commonly, they are obtained from the binding regions of the protein of interest and phage display approach is mainly used to identify appropriate peptide-targeting ligands by selection of huge libraries (~1011 different sequences). Although peptides can have drawbacks, such as low target affinity and susceptibility to proteolytic cleavage, these issues could be addressed by introducing chemical modifications or displaying the peptides multivalently [139]. Conjugation may yield stochastic ligand densities and spatial orientations and a few different approaches have been studied for the development of peptidefunctionalized nanoparticles with the principal aim to bind the targeting moiety without losing the functionality, The most relevant ones are represented by the direct conjugation involving nanoparticle surface functional groups (e.g., amine, aldehyde, or active hydrogen groups); the employment of chemical linkers that can control the binding orientations of ligands and act as spacers (e.g., PEG derivatives,

N-succinimidyl-3-(2-pyridyldithio)-propionate

(SPDP),

succinimidyl-4-(N-

maleimidomethyl) cyclo-hexane-1-carboxylate (SMCC), and the conjugation mediated by affinity tags inserted into the protein primary sequence or recombinant protein linkers [139]. 33

Natural ligands and short peptides derived from them have been used as targeting moieties to specifically deliver therapeutic agents to TfR expressing cancer cells. Bacterial and plant toxins such as diphtheria toxin, ricin A chain, saporin and artemisinin has been conjugated to Tf and shown to be active in a variety of cancer cells upon TfR-mediated internalization and to significantly induce tumor regression in xenograft mouse models [137]. Tf has been conjugated to the surface of nanoparticles for the direct targeting of these structures as carriers of anti-neoplastic drugs or genetic material to tumors. For instance, the synthetic miR-29b has been selectively delivered to acute myeloid leukemia cells by Tf-conjugated anionic lipopolyplex nanoparticles, resulting in a significant decrease if the cell growth and longer survival of mice engrafted with tumor cells and treated with nanoparticles. Furthermore, priming cancer cell with nanoparticlesb efore the anti-neoplastic decitabine resulted in a improved anti-leukemic activity compared with the drug alone in vivo [140]. Self-assembling PEGylated nanoparticles based on calcium/phosphate and cationic liposomes and containing the zoledronic acid, a potent aminobisphosphonate inducing cell growth inhibition and apoptosis, have been functionalized with Tf to target glioblastoma multiforme cells. The treatment with nanoparticles resulted in higher in vitro cytotoxic activity with respect to the free drug and showed a higher antitumor efficacy in immunosuppressed mice bearing intracranial human tumor xenografts, inducing a significant tumor weight inhibition and an increased life survival [141,142]. Hormone receptors are good candidates for targeted therapy since they are known to be upregulated in several types of tumors like breast, ovary. prostate, lung and colon cancer. A synthetic analog of Luteinizing Hormone-Releasing Hormone (LHRH) peptide was conjugated to the distal end of PEG polymer to direct the BCL2 targeted siRNA nanoparticles specifically to the cancer cells. Nanoparticles were stable in plasma and were specifically taken up by tumor cells, accumulating siRNA in the cytoplasm of cancer cells and providing gene silencing efficiently. In vivo body distribution data confirmed that targeting by LHRH peptide significantly changed body distribution of dendrimers, since they were capable to preferentially accumulate in the tumor, while non-targeted dendrimers were found mainly in the liver and kidney [143]. LHRH peptide conjugated to cisplatinbearing polyelectrolyte nanogels has been demonstrated to mediate the selective accumulation in ovarian cancer cells. The LHRH-nanogel cisplatin formulation was more effective in reducing the tumor volume and less toxic than equimolar doses of free cisplatin or untargeted nanogels in the treatment of receptor-positive ovarian cancer xenografts in mice and resulted in a prolonged survival time. The analysis of tumor and organ accumulation revealed that only a trace amount of cisplatin was detected in the organs of reticuloendothelial system such as liver and spleen, confirming the target specificity [144]. Very recently, the HSYWLRS peptide sequence has been identified as a specific ligand for aggressive neuroblastoma, a childhood tumor mostly refractory to current 34

therapies. Such heptapeptide has been exploited to specifically deliver doxorubicin-loaded stealth liposomes to the tumor, resulting in increased tumor vascular permeability and enhanced tumor penetration of the drug. This formulation proved to exert a potent antitumor efficacy leading to delay of tumor growth and abrogation of metastatic spreading, accompanied by absence of systemic toxicity and significant increase in the animal life span [145]. Chlorotoxin, a peptide reported to bind selectively to glioma cells was linked to PLGA nanoparticles with a silver core and conferred target selectivity to different human glioblastoma cell lines. A single whole brain X-ray irradiation, performed before nanoparticle injection, enhanced the expression of the chlorotoxin targets, MMP-2 and ClC-3, and, through blood-brain barrier (BBB) permeabilization, potently increased the amount of internalized nanovertors even in metastatic dispersed cells, generating an efficient antitumor synergistic effect able to inhibit in vivo tumor growth in a glioblastoma multiforme mouse model [146]. A platform for the efficient image-guided delivery of siRNA to breast tumors and the mediation of a robust therapeutic effect has been developed by conjugating superparamagnetic iron oxide nanoparticles to the EPPT peptide, which targets the tumor-specific antigen uMUC-1 found in more than 90% of human breast adenocarcinomas, near-IR dye Cy5.5 for optical imaging and a siRNA against the antiapoptotic gene birc5. The nanodrug bioavailability in the tumor tissue throughout the course of treatment was monitored and a preferential tumor uptake determined the induction of considerable levels of necrosis and apoptosis in the tumor, translating into a significant decrease in tumor growth rate. [147]. The ability to combine different approaches often offer the possibility to improve the rate of success. Doxorubicin-loaded hollow gold nanospheres targeting EphB4, a tyrosine kinases over-expressed on the cell membrane of multiple tumors and angiogenic blood vessels have been developed and tested in combination with photothermal ablation. As targeting moiety, a 14-mer peptide identified by phage display technology was used as a reference to develop a cyclic peptide as a second-generation EphB4-binding antagonist with improved plasma stability and high-receptor binding affinity. Increased uptake of targeted nanoparticles was observed in EphB4-positive tumors and the treatment resulted more cytotoxic upon near-infrared laser irradiation. Significantly decreased tumor growth was observed when compared to treatments with nontargeted nanoparticles and no systemic toxicity evaluated as body weight loss was detected [148]. It is well-known that tumors can not grow without an adequate vascular supply. By releasing growth factors, cancer cells stimulate the neoangiogenesis. Newly formed blood vessels are characterized by specific integrin receptors, absent in normal cells, representing an attractive target to develop novel therapeutic strategies. In the last decades, many efforts have been spent to identify ligands specific for receptors on neo-formed vessels, in order to design new anti-angiogenic drugs or to selectively deliver different therapeutic agents to tumors [149]. An example is cilengitide, a cyclic 35

RGD-based pentapeptide which shows nanomolar affinities for the integrins αvβ3, αvβ5 and α5β1, and high selectivity towards the platelet receptor IIb3. Its anti-angiogenic and, thus, anti-tumor activity resulted in a significant reduction of vessel density and delay of tumor growth and metastasis. Cilengitide is currently under investigation in phase I/II and III clinical trials in the treatment of several tumors, like non small cell lung cancer, glioblastoma, melanoma and breast cancer in combination with classical chemotherapeutic agents [150]. To tackle classical drawbacks of drugs upon systemic administration, such as fast blood clearance, high kidney and liver uptake, poor BBB penetration, low tumor specificity and rapid washout from tumors, nanoparticles carrying cilengitide were prepared using gelatin and Poloxamer 188-grafted heparin copolymer. The tumor level of cilengitide in a rat model of glioblastoma was increased over 3-fold, tumor retention prolonged and renal clearance significantly reduced when compared with the free drug. Additionally, the combination with the ultrasound-targeted microbubble destruction resulted in a significantly extended median survival period [151]. Likewise, the head-to-tail-cyclized hexapeptide containing the iso Asp-Gly-Arg (iso DGR), a mimetic of Arg-Gly-Asp (RGD) motif, has been developed and proved to recognize RGD-dependent integrins (such as vβ3, vβ5, vβ6, vβ8 and 5β1) with different affinity and selectivity, depending on iso DGR conformation and molecular scaffold. Gold nanoparticles bearing tumor necrosis factor alpha (TNF-), a well know tumor vessel damaging agent, and iso DGR as tumor targeting moiety, have been injected in mice bearing WEHI fibrosarcomas showing a significantly delayed tumor growth [152]. Although successful studies have been documented, problems associated with their cost, size, long blood residence and immunogenicity led to the search of new targeting agents and clinical protocols. In the last decade, many evolving treatments based on molecular targeting have been developed to effectively deliver therapeutic cargoes toward and into cancer cells.

4.3. Aptamers Another important category of targeting agents is represented by aptamers, a class of ligands which exhibit a great potential for therapeutic and diagnostic applications in various disease, such as cancer. The term “aptamer” refers to short single-stranded (ss) RNA or DNA oligonucleotide molecules, typically generated through Systematic Evolution of Ligands by EXponential enrichment (SELEX) [153,154], by which they are selected from random sequences directed to the target cells. They can fold into 3D well-defined structures that show a high binding affinity and specificity towards their targets via non covalent bonds. This particular recognition process aligns with the antigen-antibody reaction and ensures a broader range of possible molecules to interact with, when compared to antibodies, such as ions, drugs, toxins, phospholipids, sugars, peptides, proteins, viruses, bacteria, 36

whole cells, and even tissues. In contrast to other targeting molecules like antibodies or derivatives, DNA and RNA aptamers bind to their target by using their entire sequence. Aptamers exhibit several advantages over other ligands typically used in targeted therapy. First of all, the small size allows the penetration of tissues barriers to reach target sites in vivo more efficiently than the larger-sized protein antibodies. Second, due to their nucleic acids nature, aptamers are thermally stable at high temperatures, denaturing and refolding into their 3D conformation multiple times without undergoing lack of activity. Being oligonucleotides, they should not be recognized by the immune system and do not stimulate any immunogenic response. Furthermore, aptamers wellestablished synthesis protocol allows for a rapid, large-scale and low cost production. However, as the reverse side to the coin, the small size could also represent a disadvantage, exhibited by a high susceptibility to renal filtration which, together with serum nuclease degradation, causes a decrease of effectiveness [155]. To overcome these obstacles, chemical modification technology has been adopted as a strategy to enhance aptamers stability [156]. Conjugation of aptamers to bioconjugates, such as polyethylene glycol (PEG) polymers, has been employed to modify aptamer pharmacokinetics [157] in order to increase the molecular weight and prolong its circulating half-life, enhancing its stability and decreasing its toxic accumulation in off-target tissues. Furthermore, they can be coupled to diagnostic or therapeutic agents through covalent or not covalent bonds, functioning as drug delivery systems (chemotherapy drugs, among which doxorubicin is the most widely used, [158,159], toxins and small interfering RNAs. Recently, aptamers have been utilized in nanomedicine to prepare ligand-directed “active targeting” delivery systems. The conjugation of aptamers to nanomaterials is allowed by the latter large surface area/loading capacity and versatile chemistry. This combination has great potential not only in the targeted delivery of drugs [160], but also in improving the therapeutic index of the treatment. One comprehensive study reported a formulation of AS1411-functionalized liposomes encapsulating DOX [161]. AS1411 is a 26-nucleotide guanosine-rich DNA aptamer with potential apoptotic induction activity, which binds surfaceexpressed as well as translocated nucleolin (NCL) in many types of cancer cells [162]. It has been demonstrated that these targeted liposomes exhibit a high targeting efficiency and enhanced cytotoxicity in MCF-7 breast cancer cells; moreover, in vivo results indicate an improved anti-tumor activity against xenografted MCF-7 breast tumors in nude mice, due to enhanced tumor tissue penetration and the early onset of tumor suppression [161]. Many other formulations of AS1411 conjugated to nanoparticles were investigated in preclinical studies for cancer therapy and diagnosis, showing promising preclinical results owing to high tumor targeting. Early-stage data from a phase I clinical trial, in which a maximum tolerated dose therapy was tested, confirmed its safety in high grade solid tumors. In addition, phase II trials have been performed 37

to assess the antitumor effect on acute myeloid leukemia (AML) and renal cell carcinoma (RC) [163]. To date, no aptamer-driven nanoparticles have been applied in clinical trials. DOX-encapsulated DOTAP:DOPE liposomes were used in combination with a cancer cell-specific ssDNA aptamer (SRZ1), whose anti-cancer efficacy have been demonstrated both in vitro and in vivo [164]. The presence of the SRZ1 aptamer on the nanoparticles surface was found to significantly enhance the uptake efficiency of DOTAP:DOPE-DOX NPs. As a consequence, the accumulation of DOX within the tumor (determined by body imaging in vivo and biodistribution analysis), caused suppression of tumor growth and increased survival rate of 4T1 tumor-bearing mice. Another example is represented by aptamer A10, an RNA aptamer whose use has been focused on prostate cancer research for decades. In vitro studies demonstrated Its effectiveness in actively binding to the extracellular domain of the prostate-specific membrane antigen (PSMA), expressed on prostate cancer cell surface, and its subsequent uptake by targeted cancer cells. New generation A10 aptamers have been chemically synthesized to improve chemical synthesis, stability, and activity in vivo). Tang et al. prepared an A10 aptamer functionalized doxorubicin–polylactide (DOX-PLA) nanoconjugate (DPN) to target PSMA expressing cells in canine hemangiosarcoma (cHSA) [165]. They demonstrated that A10 DPNs conjugate bound to PSMA expressing cancer cells, mediated the drug internalization and exerted greater cytotoxic effects compared to non functionalized DOX-PLA DPNs and free DOX. In addition, therapeutic safety and the absence of organ toxicity were observed in vivo, where two consecutive intravenous infusions of A10 DOX-PLA DPNs led to significant anti-tumor effect. An interesting approach is represented by the dual-aptamer system used to target both PSMApositive and PSMA-negative cells in prostate cancer. To achieve this goal, they immobilized A10 RNA aptamer and DUP-1 peptide aptamer, specific to PSMA negative cells, on the surface area of superparamagnetic iron oxide nanoparticles (TCL-SPION), enabling the targeted delivery of DOX in both types of prostate cancer cells. This proof-of-principle study demonstrated that dual conjugates can act as specific drug delivery vehicles against the tumor heterogeneity and anti-cancer drug resistance, increasing the bioavailability of DOX to target cells, minimizing the side effects [166].

4.4. Other targeting moieties Not only antibodies, proteins and aptamers can confer target selectivity to nanoparticles, but also other moieties ensure specific delivery to the tumor site. Representative examples are small molecules like folate, flavin mononucleotide (FMN), anisamide or carbohydrates (galactose and hyaluronic acid). Small molecules are a heterogeneous class of compounds with infinitely diverse structures and properties, characterized by minute size and inexpensive to produce. Among them, folate is one of 38

the most extensively utilized ligands for targeted drug delivery devices. Its receptor is frequently upregulated in many types of human cancers and nanoparticles conjugated with folate are internalized by cells, then released in the cytoplasm and proceed to interact with intracellular components [167]. Folate receptor targeted delivery of DOX has been achieved by biodegradable polymeric micelles, self-assembled from a di-block copolymer of PLGA and PEG. They exhibited increased cellular uptake, circulation time, and decreased cardiotoxicity with respect to the chemotherapeutic alone, the latter effect indicating that folate was able to differentiate between healthy and tumor tissue with greater specificity [168]. Folate has been also utilized to direct lipid-coated cisplatin nanoparticles co-loaded with microRNA-375 to chemotherapy resistant hepatocellular carcinoma cells. Such therapeutic enhanced apoptosis and induced cell cycle arrest in tumor cells in vitro and significantly inhibited tumor growth and delayed the tumor relapse in a primary carcinoma mouse model [169]. Sigma receptor, a membrane bound protein that binds haloperidol and various other neuroleptics with high affinity are an interesting target since they are over-expressed in certain human malignancies including prostate cancer. An anisamide derivatized ligand with high affinity for sigma receptors has been incorporated into liposomes encapsulating DOX and demonstrated to specifically deliver the drug to prostate cancer cells. Studies after intravenous administration showed that incorporation of anisamide into liposomes significantly improved their accumulation into the tumor and led to a significant growth inhibition of established DU-145 tumor in nude mice with minimal toxicity [170]. The vitamin riboflavin is fundamental for cellular metabolism, therefore it is strongly up-regulated in cells with a high metabolic activity, including cancerous cells. For this reason, the riboflavin carrier protein (RCP) constitutes a suitable target for cancer and activated endothelial cells. In this effort, the coating of utrasmall superparamagnetic Iron oxide nanoparticles (USPIO) with FMN, the endogenous RCP ligand, made them target-specific. In vitro studies showed the biocompatibility, high labeling efficiency and RCP-specific uptake of USPIO from prostate cancer and activated endothelial cells [171]. Carbohydrates form another class of small molecule targeting ligands that selectively recognize cell surface receptors, such as lectins. The asialoglycoprotein receptor resides only on the surface of hepatocytes at a high density and it binds carbohydrates, such as galactose, mannose, and arabinose, which can thereby be exploited as effective liver targeted drug delivery systems in vivo. Poly(2-(2aminoethyoxy)ethoxy)phosphazene nanoparticles bearing a galactose residue as targeting moiety have been demonstrated to increase the transfection efficiency of plasmid DNA. Even if the conjugation with galactose significantly decreased the cytotoxicity of nanoparticles, displayed the selective gene expression in the tumor and liver with relatively low gene expression in the lung or 39

other organs [172]. Hyaluronic acid is a copolymer of N-acetyl D-glucosamine and D-glucuronic acid, widely used as targeting macromolecule. It can bind to CD44, a cell-surface glycoprotein over-expressed in various tumors, and involved in cell proliferation, differentiation and migration, as well as in signaling for cell survival. Therefore, hyaluronic acid has been utilized as an efficient cancer targeting moiety for tumor targeted diagnostic and therapeutic applications. A multifunctional nanoagent formed by hollow Prussian blue nanoparticles as photothermal agent and drug carrier, camptothecin as chemotherapeutic agent, and hyaluronic acid grafting PEG as capping agent for prolonged blood circulation time and CD44 receptor mediated tumor targeting has been developed. This nanoconstruct showed excellent biocompatibility, efficient internalization by cells through the CD44 receptor mediated active targeting and could significantly improve the therapeutic efficacy compared with either therapies alone because of a good synergetic effect [173]. Attempts to improve target selectivity and cell uptake have prompted researchers to develop nanoparticles conjugated with various ligands with different individual targets. Recent examples of multifunctionalized nanoparticles include the concurrent presence of anti-EGFR and MOV18 antifolate receptor antibodies on gold nanoparticles [174]; the dual-targeting of v3 and galectin-1 of paramagnetic liposomes [175] and nanoparticles simultaneously targeting the nucleolin, integrin αvβ3 and Tnc proteins [176]. Multiple targeting proved to significantly enhance specificity and signal intensity compared to each one individually [177].

5. Conclusions and Perspectives Albeit important advances in cancer chemotherapy have been achieved in the last decades, the need for better therapies for advanced stage solid tumors remains urgent. Most therapeutic approaches are still routinely based on surgical debulking followed by chemotherapy using cytotoxic agents, mainly involving anthracyclines, taxanes and cisplatin, or biologicals, including peptides or monoclonal antibodies, which seldom result in complete remission in compromised patients and often lead to poor quality of life. In addition, current clinical approaches fail in advanced cancers, including for example breast, ovarian, pancreatic, lung and liver cancers. For this reason, much effort has been spent in the direction of developing alternative therapeutic strategies based on targeted gene therapy in the attempt to exert a cytotoxic action both on the primary tumor and on the peripheral metastases. Gene therapy looks particularly promising in those diseases in which metastatic tumors are prevalently confined to the peritoneal cavity (e.g., ovarian cancer), so that tumor treatment can be directly accessed by intraperitoneal delivery of therapeutic genetic material. In theory, this paradigm provides the additional advantage of improving the therapeutic efficiency and selectivity compared 40

to conventional chemotherapy. In practice, however, these advantages are far from being fulfilled at the preclinical and clinical levels, as very low targeting efficiency and poor DNA stability could be achieved in living organisms by means of currently available protocols. Among the therapeutic DNA-based approaches, suicide gene therapy has great potential because encoding genes are designed to induce programmed cell death as soon as they reach cancer cells. Tumor growth arrest is expected to be achieved selectively upon activation of an administered prodrug by the enzyme coding genes. However, due to the strong effectiveness of suicide therapy in cell killing, this strategy could be highly detrimental for normal tissues unless optimal control on tumor targeting can be efficiently attained. The efficacy and safety of this strategy is intimately related to the possibility to precisely transfer the therapeutic genes to the specific target and to act selectively therein. Indeed, the therapeutic index strictly depends on the selectivity of the suicide cancer gene therapy, which in turn is defined by the accurate choice of the proper vector. Hence, one of the main challenges that gene therapy urgently needs to face resides in developing effective and safe targeting vehicles for nucleic acids. For long time, with few exceptions, most efforts have been focused on the use of viral vectors to deliver the DNA to the cells. However, few attempts to translate gene therapy to the clinics invariably resulted in serious consequences for the patients associated to the use of viral vectors [177]. Nowadays, the advent of the era of nanotechnology provides renewed spur to the research in this field and offers unprecedented solutions for the identification of appropriate vectors for nonviral DNA delivery [178]. For example, a phase I clinical trial using cationic liposomes as vectors for gene delivery in patients with both HER2/neu-overexpressing and low-expressing HER2/neu breast and ovarian cancers was reported in 2001 [179]. Although the antitumor efficacy in this study was limited and associated with adverse side effects, this first attempt provided room to open new possibilities in the development of alternative nonviral carriers for targeted gene transfer. More recently, several studies have been conducted to explore more efficient and sophisticated nanoparticle-based gene delivery systems to improve the effectiveness of suicide therapy, obtaining promising results in vivo [95, 180-184]. It is now generally assumed that targeted therapy can reduce the severe side effects associated with conventional chemotherapy and radiotherapy. In addition, targeted therapy can push the boundaries of the therapeutic indices by confining the levels of cytotoxic agents to the primary tumor and metastases. A wide variety of nanoparticle systems and targeting ligands are now available and offer a plethora of possible solutions to improve the targeting efficiency in a broad range of malignancies. The improved knowledge of tumor environment deriving from recent discoveries in cancer physiology and the advanced tools available for drug delivery allows researchers to design tunable multifunctional nanovectors capable of long lasting circulation in the bloodstream preventing 41

rapid clearance from the reticuloendothelial system [185], until they reach the loose tumor vasculature where they can easily extravasate accumulating selectively at the tumor by EPR effect (Figure. 5a,b) [186]. Then, accurate surface decoration of nanoparticles promotes ligand binding to specific molecular receptors in cancer cells triggering nanoparticle uptake vesicular compartmentalization followed by endosomal escape and gene release into the cytoplasm (Figure 5c,d). The DNA translocates into the nucleus where it can activate the mRNA transcription (Figure 5e), which in turn translates into the cytosolic toxin that results in cell death (Figure 5f). Increasing the specificity of the nanocarrier and optimizing the drug loading and release are essential tasks to improve the quality of vectors. In conclusion, as mentioned previously, we are still far from clinical translation of gene therapy exploiting nanostructured nonviral vectors. However, we can anticipate that two main approaches proved to have concrete chances to be developed toward preclinical and clinical assessment: colloidal nanoparticles (e.g., gold and iron oxide nanoparticles) can bring the nucleic acids by tight adsorption on their surface mediated by interaction with a cationic polymer coating, while calcium phosphate nanoparticles can incorporated the genetic material inside the CaP inorganic matrix, eventually releasing the DNA/RNA cargo once they have been internalized into the tumor cytoplasm. Hence, recent advances in nanotechnology hold great promise in combining the potential of suicide gene therapy with the targeting efficiency of the last generation nonviral nanovehicles to revolutionize cancer therapy and improve the quality and duration of a patient’s life.

42

a.Cationic polymer-coated nanoparticle +

+

+ +

Toxin encoding plasmid

+

+ +

Targeting moiety

+

+

+ Complexation

b.

Blood circulation Tumor tissue leaky vasculature

EPR effect Cell death

c.

Uptake by receptor mediated endocytosis Cytoplasm

Y

Ribosome inactivation

toxin

f.

Translation mRNA

Y

d.

Endosomal escape Nucleus

+ + + + + + + +

Transcription

pre-mRNA

e.

Figure 5. Schematic representation of nanoparticle-mediated gene delivery in vivo. a. Nanoparticle preparation. The nanoparticles are functionalized with targeting agents and loaded with a toxin encoding plasmid complexed with cationic polymers. b. Passive tumor targeting. Circulating nanoparticles passively extravasate in the tumor environment through increased permeability of the leaky vasculature and ineffective lymphatic drainage (EPR effect). c. Active tumor targeting. The functionalization of nanoparticles with targeting ligands promotes cell-specific recognition and binding to receptors that are overexpressed on tumor cells, resulting in enhanced uptake and internalization. d. Endosomal escape. The proton sponge effect causes the Internalized nanoparticles and the associated plasmid escape from the endosome into the cytoplasm. e. Toxin expression and activity. Once the toxin-encoding gene is expressed in the cytosol the resulting protein inhibits the ribosomal activity, blocking protein synthesis and causing cell death (f).

43

ACKNOWLEDGMENTS This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) “My First AIRC Grant” (MFAG 2014 Ref. 16144).

References [1] World Health Organization, Cancer http://www.who.int/cancer [2]

World

Health

Organization,

Media

Center,

Cancer

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