Alfonso Garcia-Bennett

Matthias Nees

Bengt Fadeel
email: bengt.fadeel@ki.se

In Search of the Holy Grail: Folate-Targeted Nanoparticles for Cancer Therapy

Keywords: nanoparticle, folic acid, targeted delivery, toxicity NPs-nanoparticles, QD-quantum dot, MRI-magnetic resonance imaging, SPIONs-superparamagnetic iron oxide nanoparticles, FOLR-folate receptor, EPR-enhanced permeability and retention, PEG-polyethylene glycol

Targeted drug therapy or "smart" drug delivery, potentially combined with simultaneous imaging modalities to monitor the delivery of drugs to specific tissues, is arguably the "holy grail" of pharmacology. Therapeutic approaches that exploit nanoparticles to deliver drugs selectively to cancer cells are currently considered one of most promising avenues in the area of cancer therapeutics and imaging. The potential to deliver active chemotherapeutic drugs in the vicinity or directly within specific tumors via receptor mediated pathways, and to image tumors through the use of nanoparticles has been conceptually and experimentally shown for several classes of nanoparticles. Nanoparticles functionalized with the vitamin folic acid are of particular interest as a variety of malignant tumors are known to overexpress the folate receptor(s). Indeed, several nanoparticle architectures with improved retention time, administration route, biocompatibility, absorption, and clearance are being proposed and are in late stage clinical development. This commentary highlights some of the most important concepts related to nanoparticles and folate-mediated drug delivery and imaging in cancer research.

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Introduction

The use of nanoparticles in therapeutics, medical imaging and diagnostics is receiving considerable attention, as interdisciplinary research teams involving physicists and material chemists are increasingly combining their technologies and know-how with the biomedical community. [START_REF] Fadeel | Nanomedicine: reshaping clinical practice[END_REF] Numerous publications attest to the potential use of nanoparticles (whether organic, inorganic or metallic) in biomedical applications such as magnetic resonance imaging (MRI), [START_REF] Nan | Inorganic nanoparticles for MRI contrast agents[END_REF] tissue repair and bone regeneration, [START_REF] Vallet-Regi | Nanostructured hybrid materials for bone tissue regeneration[END_REF] immunoassays, [START_REF] Kim | Biomedical applications of superparamagnetic iron oxide nanoparticles encapsulated within chitosan[END_REF] detoxification of biological fluids, [START_REF] Gupta | Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications[END_REF] hyperthermia, [START_REF] Kim | Surface-modified magnetite nanoparticles for hyperthermia: Preparation, characterization, and cytotoxicity studies[END_REF] and as drug delivery vehicles, [START_REF] Slowing | Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers[END_REF] to name a few. This comprises the use of nanoparticles for theranostics, a novel field that combines diagnostic and therapeutic applications suitable for imaging and monitoring of individual patient's responses to therapy, paving the road towards personalized medicine. The concerns regarding the toxicological properties of nanoparticles are being addressed by an emerging community of nanotoxicologists, devoted to the thorough understanding of hazardous properties of nanoparticles (NPs) both in vitro and in vivo. [START_REF] Fadeel | Better safe than sorry: Understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications[END_REF] Procedures to mitigate toxicity, wherever it is found, are being applied. In addition, the environmental effect of NP production, their longterm impact in the environment, and regulatory issues are being tackled. Importantly, it will be necessary to categorize NPs and to assess each NP on a case-by-case basis in order to avoid inadequate generalizations that may hinder their commercial use including the application of NPs in medicine.

Among the biomedical applications of nanoparticles, their use in cancer therapeutics is the most anticipated. Irrespective of their composition, NPs easily penetrate cellular membranes through active and passive mechanisms due to their small size, allowing them to act as drug carriers with tunable pharmacokinetic properties, enabling slow or sustained release of their A c c e p t e d M a n u s c r i p t 4 payload. Of particular value to the oncology field is the potential use of NPs for targeted drug delivery, thus minimizing unwanted effects on bystander cells and tissues. This promises to reduce the toxicity of chemotherapeutic treatments by reducing the doses that are administered.

The enhanced permeability and retention (EPR) effect which has been widely observed for liposomes and other macromolecules appears to be also applicable to NPs of various forms.

This passive form of cell targeting relies on the diffusion limitation encountered by tumor growth of above a certain size (around 2 mm), which affects the intake of vital nutrients and oxygen. [START_REF] Rubin | Microcirculation of tumors. II. The supervascularized state of irradiated regressing tumors[END_REF] In order to overcome this respiratory limitation, neo-angiogenesis occurs, resulting in an increased but usually immature tumor vasculature and lymph vasculature, which allows the penetration of nanoparticles below a certain size (approximately 2 µm), depending on the tumor type. Particles capable of penetrating through the interstitial tumor vasculature have considerably higher retention times than those in normal tissues, resulting in the EPR effect. [START_REF] Haley | Nanoparticles for drug delivery in cancer treatment[END_REF][START_REF] Byrne | Active targeting schemes for nanoparticle systems in cancer therapeutics[END_REF] The EPR effect is an important phenomenon that has been widely observed for all categories of NPs. In addition, active site-specific targeting and effective uptake of NPs has also been achieved via receptors on the cell membrane, which may mediate uptake through various phagocytic or endocytic mechanisms. To activate such receptors, a recognizable ligand needs to be tethered to the NP surface. Careful and efficient functionalization as well as encapsulating technologies for NPs has been developed over recent years, including biocompatible shells such as lipids, polymers, micelles or silica coatings of different kinds. In this context, folate mediated targeting holds substantial promise and has been one of the major strategies assimilated by the nanomedical field for targeted drug-delivery and imaging of tumors. [START_REF] Low | Folate-targeted therapeutic and imaging agents for cancer[END_REF] It is a daunting task to provide a comprehensive summary of the wide variety of NP architectures that have been investigated in recent years. Therefore, the aim of the current A c c e p t e d M a n u s c r i p t 5 commentary is merely to highlight some of the key aspects of folic acid mediated uptake, nanoparticle functionalization with folate linkers, and the current state-of-the-art for targeted imaging and drug delivery. We will also discuss nanoparticle-biomolecule interactions and whether the "holy grail" of targeted drug delivery based on the use of nanoparticles is, in fact, achievable.

Nanoparticles for biomedical applications

The European Union, a strong advocate for tighter control of chemical manufacturing, has initiated a serious discussion on the regulation of nanomaterials, and the following definition of nanomaterials from a regulatory perspective was recently proposed; 13 hence, nanomaterials:

(i) consist of particles, with one or more external dimensions in the size range 1 nm -100 nm for more than 1 % of their number size distribution;

(ii) have internal or surface structures in one or more dimensions in the size range 1 nm -100 nm;

(iii) have a specific surface area by volume greater than 60 m 2 /cm 3 , excluding materials consisting of particles with a size lower than 1 nm.

A particle is here defined as a minute piece of matter with defined physical boundaries as described by the International Standard Organization. 14 This definition is attractive, although it places several materials within the nanoparticle description which have not been classified as such previously, eg. the zeolite family of crystalline aluminosilicates. Further classification with respect to composition would be useful, in light of the many types of nanomaterial compositions that have been reported and its direct effect on the functions and behavior of the NPs.

A c c e p t e d M a n u s c r i p t 6 Organic, lipid or polymeric based nanomaterials encompass one of the largest groups of NPs.

It is important to note, however, that all of these different sub-groups have their individual properties, surface reactivity, toxicological behavior and functional applications. Similarly, their method of preparation, scalability and purity is different. In general, organic based NPs are easily functionalized as their terminal surface groups lend themselves to conventional organic chemical modification, as well as possessing numerous terminal groups on their external surface available for conjugation. Pegylation i.e. the attachment of polymeric chains of ethylene glycol covalently to the external surface of the particle or reaction with fluorochromes prior to conjugation with folates is also easily achieved. [START_REF] Tallurya | Ultra-small waterdispersible fluorescent chitosan nanoparticles: synthesis, characterization and specific targeting[END_REF] The latter is often necessary as organic NPs do not inherently offer any optical or detection properties.

Polymeric and lipid based NPs such as dendrimers or liposomes, offer the advantage of a relatively straightforward incorporation of drugs within their internal void or cavities, and the potential specific release in the affected area. [START_REF] Patri | Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex[END_REF] Micellar derived nanoparticles allow the encapsulation of hydrophobic drugs, since these rely on the amphiphilic behavior of surfactant molecules. This is of particular interest in cancer therapy as the formulation and delivery of poorly soluble drug lead compounds is a major hurdle in assessing their potential. Polymeric, non-ionic surfactants are already in wide use in the pharmaceutical industry as adjuvants.

Surfactants containing poly(ethylene oxide) chains have been found to prevent the recognition of the micelle by the reticuloendothelial system thus allowing for longer systemic circulation. [START_REF] Francis | Polymeric micelles for oral drug delivery: why and how[END_REF] Zheng et al. [START_REF] Zheng | Rerouting lipoprotein nanoparticles to selected alternate receptors for the targeted delivery of cancer diagnostic and therapeutic agents[END_REF] generated a "natural" nanoplatform by conjugating tumor-homing molecules to the protein components of lipoproteins, resulting in the rerouting of the nanoparticles from their normal lipoprotein receptors on scavenger cells to selected cancerassociated receptors.

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The large compositional variety of available inorganic (metal) oxide based nanomaterials makes them the most versatile family of NPs. The development of synthesis and characterization procedures for inorganic oxide NPs is fueled by their wide use as catalyst or catalyst supports in the petrochemical and fine chemical industry as well as in other bulk material industries. Sol-gel synthesis routes are significantly advanced to allow sequential layers of oxides (for example silica) to be deposited over inorganic oxide NPs. This method has been successfully employed to improve the dispersability under physiologic conditions and/or mitigate the toxicity of superparamagnetic iron oxide nanoparticles (SPIONs), which offer the possibility to physically (magnetically) direct particles to the affected tissue. [START_REF] Vogt | High quality and tuneable silica shell-magnetic core nanoparticles[END_REF] Silica surfaces are characterized by surface hydroxyl groups, which are easily functionalized through hydrolysis and condensation reactions with widely available siloxane-based functional groups, which may then be conjugated to, or instance, folates. Direct conjugation to SPIONs has been recently achieved via catechol-derivative anchor groups which possess irreversible binding affinity to iron oxide. [START_REF] Amstad | Ultrastable iron oxide nanoparticle colloidal suspensions using dispersants with catechol-derived anchor groups[END_REF] Applications of SPIONs investigated to date include in vitro magnetic separation and in vivo applications such as cancer treatment using hyperthermia, in magnetic drug targeting, MRI and gene delivery. [START_REF] Lin | Development of superparamagnetic iron oxide nanoparticles (SPIONs) for translation to clinical applications[END_REF][START_REF] Zhao | Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent[END_REF][START_REF] Zhang | Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake[END_REF][START_REF] Nahrendorf | Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis[END_REF][START_REF] Zhang | Self-assembled coatings on individual monodisperse magnetite nanoparticles for efficient intracellular uptake[END_REF] In a recent study, SPIONs were coated with polymer fibers to create magnetic, thermosensitive folateconjugated NPs, that were loaded with the anti-cancer drug doxorubicin, thereby achieving thermoresponsive drug release in L929 fibroblast cells in vitro with low toxicity.

Unfortunately, in this study, the targeting properties of the particles were not evaluated. [START_REF] Rastogi | Evaluation of folate conjugated pegylated thermosensitive magnetic nanocomposites for tumor imaging and therapy[END_REF] The loading capacity of inorganic oxide nanoparticles can be improved through the introduction of pores within the particle. There are various methods of achieving this, but the most useful is to employ pore templating agents during the preparation of the NPs. If the templates are themselves ordered or if they self-assemble into ordered structures, highly

A c c e p t e d M a n u s c r i p t ordered porosity can be achieved once the material has been formed and the template has been removed. This is the case for mesoporous oxide nanoparticles, exemplified by mesoporous silica compositions which are widely studied for medical applications due to the low toxicity of amorphous silica, its biocompatibility, tunable surface chemistry, and high surface areas and pore volumes. Hence, we have found that mesoporous silica particles of different sizes are non-toxic to primary human immune-competent cells. [START_REF] Vallhov | Mesoporous silica particles induce size dependent effects on human dendritic cells[END_REF][START_REF] Witasp | Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibodyopsonized target cells[END_REF] Moreover, recent studies have shown that mesoporous silica nanoparticles are biocompatible in vivo and preferentially accumulate in tumors. [START_REF] Lu | Biocompatibility, biodistribution, and drugdelivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals[END_REF] The preparation of ordered mesoporous silica relies on the use of surfactant and non-surfactant templates. [START_REF] Kresge | Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism[END_REF][START_REF] Che | A novel anionic surfactant templating route for synthesizing mesoporous silica with unique structure[END_REF][START_REF] Atluri | Nonsurfactant supramolecular synthesis of ordered mesoporous silica[END_REF] This method has been widely applied to prepare materials with varying porous structures, from 2-dimensional (2D)-cylindrical ordered pores to 3D cage type structures, with large surface areas (over 1000m 2 /g) and large pore volumes available for conjugation and loading of fluorophores, drugs, proteins, or folates. [START_REF] Wang | On the controllable soft-templating approach to mesoporous silicates[END_REF] Mesoporous materials have been the subject of numerous studies as drug delivery vehicles,

and it has been shown that structural parameters such as pore connectivity, pore size, surface functionalization (hydrophobicity), and particle size can be altered in order to control the pharmacokinetic profile of drugs loaded within the pores. [START_REF] Slowing | Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers[END_REF][START_REF] Vallet-Regi | Mesoporous materials for drug delivery[END_REF] Metallic NPs such as quantum dots (QDs) offer the potential to act as imaging agents due to their controllable light emission as a result of their tunable crystal size and semi-conductor properties. The same applies for gold NPs, where detection is based on their excellent light scattering and light absorption properties. Hence, such metallic NPs offer the advantage that no further detection molecule needs to be conjugated to the particle surface. However, it is difficult to conjugate folates (see following section) directly to the surface of metallic nanoparticles, as this often implies oxidation of the surface with a possible change in their optical properties. Therefore, amine and thiol terminated polyethylene glycol (PEG) groups

A c c e p t e d M a n u s c r i p t can be covalently reacted to metallic NP surfaces, thus offering an organic moiety capable of conjugation to folic acid, without altering the particle properties. The use of PEG chains on the surface of NPs have additional advantages as their presence reduces the amount of unspecific protein adsorption (opsonization) and has been found to drastically increase the blood circulation time as well as biodistribution of the particles. [START_REF] Alexis | Factors affecting the clearance and biodistribution of polymeric nanoparticles[END_REF] Finally, carbon nanotubes have also been investigated as drug or siRNA delivery vehicles in preclinical models, but it remains to be understood whether the benefits outweigh the potential risks. 36

Exploiting folate receptors on cancer cells

The fundamental role of folates (folic acid) in nucleotide and DNA synthesis was established in the 1970s. The primary form of folates entering human circulation via intestinal cells is 5methyltetrahydrofolate monoglutamate. The conversion of deoxyuridylate (a precursor to RNA) into thymidylate (a precursor to DNA) is catalysed by thymidylate synthase, which requires 5,10-methylenetetrahydrolofate as a cofactor. Folates and the folate cycle are therefore essential for rapidly proliferating cells and tissues, which includes advanced stages of cancer, and critical for the "1-carbon pathway", a key initiating step in the synthesis of nucleotide precursors as building blocks for DNA synthesis in the S-phase of the cell cycle (Figure 1). In many cells, the intracellular acquisition of folates is primarily mediated through folate receptors alpha, beta and gamma (FOLR1-3), which bind to folates with a very high affinity of 10 -9 M. Hence, in particular folate receptor alpha or FOLR1, and many of the folate-cycle related genes, are highly expressed in rapidly proliferating cells. FOLR1 and FOLR3 are considerably overexpressed in a subset of cancers of the breast, pleural

A c c e p t e d M a n u s c r i p t 10 mesothelioma, [START_REF] Bueno | The alpha folate receptor is highly activated in malignant pleural mesothelioma[END_REF] endometrium/uterine [START_REF] Dainty | Overexpression of folate binding protein and mesothelin are associated with uterine serous carcinoma[END_REF] and particularly the ovary, while folate receptor beta or FOLR2 is a neutrophilic lineage marker and may be a promising target in myeloid leukaemia. [START_REF] Ross | Folate receptor type beta is a neutrophilic lineage marker and is differentially expressed in myeloid leukemia[END_REF] Expression of FOLR2 can be effectively induced by the differentiationpromoting effects of all-trans retinoic acid (ATRA) [START_REF] Li | Targeting human clonogenic acute myelogenous leukemia cells via folate conjugated liposomes combined with receptor modulation by all-trans retinoic acid[END_REF] . Thus, ATRA treatment of acute myelogenous leukaemia (AML) has been successfully combined with the simultaneous delivery of folate-receptor beta targeted nanoparticles. Expression of FOLRs often correlates with poor patient outcome [START_REF] Hartmann | Folate receptor overexpression is associated with poor outcome in breast cancer[END_REF] and has been deemed predictive for the therapeutic response to anti-cancer drugs such as cisplatin, [START_REF] Ottone | Relationship between folate-binding protein expression and cisplatin sensitivity in ovarian carcinoma cell lines[END_REF] whilst their levels remain usually low in most normal cells and tissues (see Figure 2). The high expression of FOLR1 particularly in ovarian cancers led to its cloning from such tumors twenty years ago. [START_REF] Campbell | Folate-binding protein is a marker for ovarian cancer[END_REF] All three folate receptors are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) tail, although soluble variants also exist. In fact, soluble folate receptors have been detected in blood, serum and ascites of cancer patients [START_REF] Basal | Functional folate receptor alpha is elevated in the blood of ovarian cancer patients[END_REF] and may thus be useful as biomarkers of disease in cancer patients.

Expression of FOLR1 and FOLR3 often correlate with the malignancy of different female genital tract malignancies [START_REF] Wu | Expression of folate receptor type alpha in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix[END_REF] and have been useful as markers to distinguish ovarian from breast cancer metastases. [START_REF] Yuan | Expression of the folate receptor genes FOLR1 and FOLR3 differentiates ovarian carcinoma from breast carcinoma and malignant mesothelioma in serous effusions[END_REF] However, expression of FOLR1 is also high in normal kidney and placenta, which reflects the body's requirement to actively retain important vitamin-like cofactors such as folates, and to prevent their excretion. Furthermore, FOLR1 expression is high in normal lung tissue, which could relate to the rapid turnover of lung epithelial cells and/or tissue-specific protective effects exerted by folate-requiring mechanisms. Thus, lung, kidney and to some extent the placenta represent potential targets for therapy-related toxicity in any therapies that exploit folic acid for drug transport.

The ideal approach for the therapeutic exploitation of folate receptors in cancer treatment is the Trojan horse approach, whereby the FOLRs and the cellular receptor-mediated delivery can be combined with the use of membrane-translocating peptides such as the TAT protein of the human immunodeficiency virus [START_REF] Zhao | Paclitaxel-loaded, folic-acidtargeted and TAT-peptide-conjugated polymeric liposomes: in vitro and in vivo evaluation[END_REF] , which increases the likelihood to release the cargo by membrane disruption into the cytoplasm. FOLR1 expression often correlates directly with the activity of the intracellular folate cycle/1-carbon pathway and thymidine kinase, [START_REF] Sun | Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity[END_REF] and is co-expressed with metabolic genes required for oxidative respiration as a result of enhanced growth. Therefore, receptor-mediated endocytosis of folates/folate receptor conjugates could be combined with drugs that inhibit folate-related pathways (eg. Candidates such as the epidermal growth factor (EGF)/EGF receptor, Her-2/ERBB2, integrins, transferrin receptor, somatostatin/growth hormone receptor (GH/GHR), and glucose transporter (GLUT1) have been suggested. Some of these molecules are currently being targeted in clinical trials (reviewed in [START_REF] Ledermann | Targeted trials in ovarian cancer[END_REF] ), although none of these trials utilize NPs. EGFR may be a particularly useful target, since many tumors such as head and neck cancers, glioblastomas, and lung adenocarcinomas strongly overexpress EGFR, but rarely show overexpression of FOLR1. [START_REF] Wheeler | Understanding resistance to EGFR inhibitors-impact on future treatment strategies[END_REF] The utilization of cellular drug-entry ports into the cell provides unprecedented specificity of drug delivery that typically exceeds the uptake of free drugs by at least an order of magnitude. [START_REF] Moghimi | Nanomedicine: current status and future prospects[END_REF] This process significantly improves the direct bioavailability and reduces the toxicity of folate-conjugated drugs or NPs compared to free drugs, in particular for lipophilic drugs such as taxanes, doxorubicin, or even extremely hydrophobic derivatives such as SN-38, the active metabolite of irinotecan. [START_REF] Ebrahimnejad | Preparation and in vitro evaluation of actively targetable nanoparticles for SN-38 delivery against HT-29 cell lines[END_REF] The delivery of siRNAs is another field of applications that would greatly profit from specificity. [START_REF] Thomas | Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues[END_REF] Furthermore, the specificity of drug uptake can be easily monitored and quantitatively measured by competition with free folates/folic acid, a concept that can become very useful for mechanistic studies and optimization purposes. An alternative mechanism of active cellular uptake, independent of clathrin-coated pits, is mediated through the solute carrier protein SLC19A1 (RFC-1), which actively transports folates across a gradient into the cellular interior, in conjunction with a proton pump. The SLC19A1 carrier protein is occasionally upregulated in some cancers.

These two unrelated mechanisms, receptor-versus carrier-mediated import, may compete with each other on certain cell types, and this could explain the failure of folate uptake via A c c e p t e d M a n u s c r i p t 13 FOLR1 eg. in mesothelioma cells. [START_REF] Khokhar | Despite some expression of folate receptor alpha in human mesothelioma cells, internalization of methotrexate is predominantly carrier mediated[END_REF] Considering the increasingly widespread experimental use of folates as bait for cancer drug delivery, the relative activity of these mechanisms has to be carefully elucidated.

Of course, it remains to be understood whether chronic treatment with folate-targeted NPs may lead to the downregulation of folate receptor expression and whether this will impact upon the efficacy of the approach. Experimental data addressing this topic are not available; however, it is conceivable that resistance mechanisms related to the loss of FOLR1 surface expression could arise in patients undergoing NP-based therapies. The "escape" of cancer cells from FOLR1-targeted particles might be related to adaptation to folate deficiency, which is observed for instance under methotrexate treatment. Careful phenotyping of cancer cells from patients, to select those patients that are most likely to benefit from novel, targeted approaches will be needed.

Examples of folate-conjugated nanoparticles

There is no "one-size-fits-all" conjugation method for folates. The choice of strategy depends largely on the type of NP and on the specific function required. Broadly speaking, the preparation of folate-conjugated nanoparticles involves the following steps: (i) preparation of the folate linker (and/or detection group); (ii) preparation of the nanoparticle surface; (iii) reaction and purification. Two common folate linkers are shown in Figure 3. Low and colleagues have described the preparation of folate linkers and the versatility of folic acid to accommodate varying tethering groups, without disturbing cellular uptake. [START_REF] Low | Chapter 17[END_REF] For example, the folate-ethylenediamine linker is easily conjugated with carboxyl groups on a NP surface.

Linkers may need further reaction or to be activated by N-hydroxysulfosuccinimide (NHS) or molecules. Folic acid-PEG-thioctic acid conjugates bonded to 10 nm gold NPs showed higher uptake by KB cells that overexpressed folate receptors than naked gold NPs. [START_REF] Dixit | Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting off receptor-positive tumor cells[END_REF] The authors suggest the increased solubility (strictly speaking colloidal stability) over a pH range from 2 to 12 and electrolyte concentrations of up to 0.5 M NaCl as a possible reason for enhanced uptake.

There are several advantages of using NPs conjugated to folates, rather than using the therapeutic drugs directly conjugated to folate. First, the potential to deliver large quantities of drugs through individual receptor molecules and the utilization of the very effective receptormediated endocytosis pathway is especially feasible when combined with porous NPs or such particles that contain internal voids allowing large amounts of cargo to be transported. This is the case, for instance, in mesoporous silica nanoparticles, liposomes, polymeric spheres or dendrimers. [START_REF] Patri | Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex[END_REF][START_REF] Francis | Polymeric micelles for oral drug delivery: why and how[END_REF] This facilitates the use of drugs with less potency, but with higher efficacies due to improved pharmacokinetic behavior. It also allows the incorporation of lipophilic drugs that are difficult to deliver to the target tissue by standard intravenous or oral routes. [START_REF] Mellaerts | Increasing the oral bioavailability of the poorly water soluble drug itraconazole with ordered mesoporous silica[END_REF] Additionally, NPs can be very useful to ascertain tight packaging of hydrophilic drug molecules which otherwise have a tendency to cause off-target effects due to percolation into irrelevant tissues. Such hydrophilic compounds may also have poor cell-and membrane penetrating properties. Moreover, the use of NPs in drug formulation may allow drug candidates that do not possess optimal solubility properties, to be encapsulated into dispersible matrices, avoiding modifications to the chemical structure of the drug. Specific drug release of NPs incorporated by receptor-mediated transport mechanisms may then be triggered within the endosomal compartments of cells, as multivalent folate conjugates are A c c e p t e d M a n u s c r i p t processed differently compared to monovalent conjugates. [START_REF] Low | Folate-targeted therapeutic and imaging agents for cancer[END_REF] NPs may thus act as multifunctional devices, not only allowing to image and diagnose tumors but also to monitor therapeutic responses. This is the case of NPs used as theranostics, an approach suitable for the concomitant treatment and monitoring of responses to therapy. [START_REF] Yang | Multifunctional SPIO/DOX-loaded wormlike polymer vesicles for cancer therapy and MR imaging[END_REF] Ideally, a functional therapeutic target (such as doxorubicin or methotrexate) is combined with a fluorescent tracking device, such as near-infrared dyes or QDs. [START_REF] Santra | Cytochrome C encapsulating theranostic nanoparticles: a novel bifunctional system for targeted delivery of therapeutic membrane-impermeable proteins to tumors and imaging of cancer therapy[END_REF] The control of drug pharmacokinetic properties though drug loading into NPs may ensure the maintenance of an adequate systemic therapeutic dose for prolonged periods of time which additionally improves patient compliance.

Several research groups have developed mesoporous particles and nanoparticles conjugated or loaded with folate, and combined folate functionalization with therapeutic drug schemes with the purpose to use them as targeted delivery agents. [START_REF] Lu | Biocompatibility, biodistribution, and drugdelivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals[END_REF][START_REF] Wang | Mesoporous materials: Why PMO? Towards functionality and utility of periodic mesoporous organosilicas[END_REF] Linden et al. have shown how multifunctional mesoporous silica functionalized on their external surface with fluorescent molecules and folate targeting groups via hyper branching polymers are actively taken up in higher concentrations by cells expressing the folate receptor, in comparison to (normal) cells expressing low levels of folate receptors. [START_REF] Rosenholm | Targeting of porous hybrid silica nanoparticles to cancer cells[END_REF] We have recently reported on a novel nonsurfactant route for the synthesis of highly ordered mesoporous materials, based on the supramolecular templating of stacked arrays of the tetramer-forming pterin groups of folic acid. [START_REF] Che | A novel anionic surfactant templating route for synthesizing mesoporous silica with unique structure[END_REF][START_REF] Zhou | Release of folic acid in mesoporous NFM-1 silica[END_REF] This method leads to hexagonally ordered mesoporous structures with gyroid, spherical, and chiral morphologies with pores on the order of 25-30 Å in diameter and surface areas above 1000 m 2 /g. In this approach folates are pre-loaded into the mesopores during the synthesis enabling the efficient co-encapsulation of drug targets (antifolates, cisplatin and others) within the pores directly within the synthesis of the inorganic particles, and relying on their interaction with folate tetramers through π-π interactions. Studies are currently ongoing demonstrated that mesoporous silica nanoparticles materials are well tolerated by mice. [START_REF] Lu | Biocompatibility, biodistribution, and drugdelivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals[END_REF] The maximum tolerated dose of the NPs was determined to be 100 mg/kg following intravenous administration. Biodistribution studies using human cancer xenografts revealed that the folateconjugated particles were taken up preferentially in tumors. In addition, the drug delivery properties of mesoporous particles were demonstrated in vivo by delivery of camptothecin released from loaded mesoporous particles, resulting in a considerable decrease of tumor volume. [START_REF] Lu | Biocompatibility, biodistribution, and drugdelivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals[END_REF] Mansoori et al. [START_REF] Mansoori | Comparative study of two folateconjugated gold nanoparticles for cancer nanotechnology applications[END_REF] have reported a comparative study of two folate-conjugated gold NPs using a variety of linkers (4-aminothiophenol and 6-mercapto-1-hexanol). The study provides an interesting analysis of folate receptor tissue distribution and particle endocytosis in cells with both high and low folate receptor expression (HeLa and MCF-7, respectively). The authors conclude that the type of conjugation has a direct effect on the cell death associated with the particles, with the 4-aminothiphenol linker being superior to the mercapto-1-hexanol linker.

Furthermore, this in vitro study clearly demonstrates the selectivity of some folate linker conjugates towards cells with high folate expression, and shows that 98% of the HeLa cells were killed after stimulating the particles with intense pulsed light after only four hours of incubation.

In sum, the abovementioned NPs appear to be the most promising candidates for future increased efficacy and effect of the NPs in comparison to the free drugs which is not merely attributable to an EPR effect. We, therefore, conclude that these NPs have reached sufficient critical mass in terms of their development to move towards more serious determination of their applicability in a clinical context. Table 1 summarizes some of the important aspects of these NPs.

Other receptor-mediated pathways of uptake

As discussed above, the identification of additional ligand/receptor pairs that show significant tumor-specific expression could be highly rewarding. Byrne et al. [START_REF] Byrne | Active targeting schemes for nanoparticle systems in cancer therapeutics[END_REF] discussed two such pathways involving: (i) the human epidermal receptor (HER) family of tyrosine kinases which are overexpressed in one-third of all solid tumors and mediate cell signaling pathways for growth after binding of the growth factor ligands, [START_REF] Radinsky | Level and function of epidermal growth factor receptor predict the metastatic potential of human colon carcinoma cells[END_REF] and (ii) transferrin receptor which is overexpressed in malignant cells due to the increased requirement of iron. Both of these receptors pathways have been exploited already by the pharmaceutical industry or are in advanced clinical stages of development, for active products without including NPs. [START_REF] Mendelsohn | Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer[END_REF] The endothelin (ET) family of receptors participates in the growth and progression of a variety of tumors such as prostatic, ovarian, renal, pulmonary, colorectal, cervical, breast carcinoma, Kaposi's sarcoma, brain tumors, melanoma, and bone metastases, and offers a further route for targeting. [START_REF] Bagnato | Endothelin receptors as novel targets in tumor therapy[END_REF] A number of tumor signaling pathways, and nuclear and apoptotic receptors are reported as overexpressed to significant levels by tumor cells; [START_REF] Conzen | Nuclear receptors and breast cancer[END_REF][START_REF] Blázquez | Cannabinoid receptors as novel targets for the treatment of melanoma[END_REF][START_REF] Fulda | Signaling through death receptors in cancer therapy[END_REF] however, in many of these receptor targeting approaches the development of suitable antagonists is less developed than for those mentioned above and further research is needed in order to exploit their potential clinical use.

A c c e p t e d M a n u s c r i p t

Receptor-mediated endocytosis mostly occurs via clathrin-coated pits and vesicles, and requires a dedicated intracellular machinery of transport molecules such as arrestins, dynamins, and stabilins. An alternative system is represented by caveolin-dependent endocytosis, which utilizes lipid raft domains in the plasma membrane for forming caveolae, non-clathrin coated vesicles that nevertheless fuse with early endosomes and serve similar purposes. Internalization of particles may occur through additional, poorly understood mechanisms that utilize neither caveolae nor coated pits/vesicles. The ubiquity of the endocytosis processes, and the spectrum of diverse receptor/ligand pairs being transported and recycled via this regulated turnover, provides ideal mechanisms to be exploited or hijacked, eg. by naturally occurring viruses or by complex NP-based drug delivery systems.

Considerable efforts have been focused in recent years on the mode of uptake of NPs lacking specific ligands, such as folic acid. For instance, Zhang and Monteiro-Riviere 71 utilized 24 different endocytosis-interfering agents to investigate the mechanism by which QDs enter cells, and found that QD internalization is primarily regulated by the G-protein-coupled receptor-associated pathway and low density lipoprotein receptor/scavenger receptor pathways, whereas other endocytic pathways may play a minor role in the uptake of QDs.

Other investigators have shown that non-functionalized QDs may enter the nucleus in a sizeand surface charge-dependent manner. [START_REF] Nabiev | Nonfunctionalized nanocrystals can exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments[END_REF][START_REF] Conroy | CdTe nanoparticles display tropism to core histones and histone-rich cell organelles[END_REF] Chen and von Mikecz reported that silica nanoparticles translocate into the nucleus. [START_REF] Chen | Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO 2 nanoparticles[END_REF] More recent spatio-temporal mapping of the uptake of silica particles of different sizes have disclosed that these NPs enter one by one engulfed in a vesicle, without evident involvement of classical-clathrin-coated pits; endosomal structures were first occupied, and later lysosomes were populated, while no particles were found to reside in the nucleus or in other organelles. [START_REF] Shapero | Time and space resolved uptake study of silica nanoparticles by human cells[END_REF] Understanding how cells handle pristine NPs is of importance when specific pathways of uptake are exploited; It is also worth considering the possibility of targeting tumoral endothelial cells for therapeutic gain. In an elegant proof-of-principle study, Hood et al. [START_REF] Hood | Tumor regression by targeted gene delivery to the neovasculature[END_REF] demonstrated that systemic administration of lipid-based NPs coupled to an integrin-targeting ligand can deliver genes selectively to blood vessels in tumor-bearing mice, resulting in death of the tumorassociated endothelium, ultimately leading to tumor cell death and sustained regression of established tumors. In more recent studies, integrin-targeted NPs encapsulating the cytotoxic drug doxorubicin were shown to home to tumor vasculature, with a 15-fold increase in antimetastatic activity in mice as observed with systemic administration of the free drug. [START_REF] Murphy | Nanoparticle-mediated drug delivery to tumor vasculature suppresses metastasis[END_REF] Ruoslahti and colleagues have recently identified a cyclic peptide (iRGD), that, when chemically conjugated to a drug, can carry the drug deep into extravascular tumor tissue. [START_REF] Sugahara | Tissuepenetrating delivery of compounds and nanoparticles into tumors[END_REF] The peptide targets tumors by binding integrins that are specifically expressed on the tumor vessel endothelium. They also showed that co-administration of iRGD enhances drug delivery and activity of drugs that are not chemically conjugated to it, including liposomes carrying doxorubicin. [START_REF] Sugahara | Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs[END_REF] Wong et al. [START_REF] Wong | Multistage nanoparticle delivery system for deep penetration into tumor tissue[END_REF] propose a multi-stage nanoparticle delivery system in which 100 nm nanoparticles "shrink" to 10 nm nanoparticles after they extravasate from the tumor

The biomolecular corona on nanoparticles

When NPs are presented to a physiological environment such as human plasma, they selectively absorb biomolecules (i.e. proteins, lipids) to form a so-called biomolecular corona on the surface of the NP. [START_REF] Cedervall | Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles[END_REF][START_REF] Lundqvist | Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts[END_REF][START_REF] Röcker | A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles[END_REF][START_REF] Dell'orco | Modeling the time evolution of the nanoparticle-protein corona in a body fluid[END_REF][START_REF] Casals | Time evolution of the nanoparticle protein corona[END_REF] It has been argued that this corona of biomolecules determines what cells "see"in other words, cells do not respond to the naked NP surface but rather to the proteins and/or lipids immobilized on the NP surface. [START_REF] Walczyk | What the cell "sees" in bionanoscience[END_REF] If this were the case, then this would appear to challenge the very concept of targeted delivery of NPs based on attachment of specific targeting ligands such as folic acid onto the surface of the NP, as such ligands would potentially become inaccessible (or "invisible") to cellular receptors on cancer cells due to the corona of plasma proteins (in the case of intravenous delivery of NPs). One wellknown strategy to reduce the (non-specific) binding of proteins to NPs is to attach PEG chains; this is also thought to prevent NP uptake by cells of the reticuloendothelial system. [START_REF] Kunzmann | Toxicology of engineered nanomaterials: Focus on biocompatibility, biodistribution and biodegradation[END_REF] In a recent study, the full repertoire of SPION-binding blood proteins was determined using a novel two-dimensional differential mass spectrometry approach. [START_REF] Simberg | Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance[END_REF] The identified proteins showed specificity for surface domains of the NPs: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and other proteins were shown to be secondary binders. Interestingly, transferrin and albumin, the most abundant plasma proteins, showed no significant enrichment on the SPIONs. However, subsequent in vivo studies in mice suggested that the identified proteins did not play a significant role in the hepatic clearance of these NPs. [START_REF] Simberg | Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance[END_REF] Importantly, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIONs in plasma, suggesting that the NP surface could be "seen" by liver macrophages or by other cells, regardless of the protein coating. Notwithstanding, a better grasp of the nature of the biomolecule corona of NPs remains an important goal, as the adsorbed plasma proteins and

Conclusions and future perspectives

Nanoparticles are commonly defined as particles in the range of 1-100 nm. However, the small size per se is not necessarily of interest, and a more appropriate definition should therefore take into consideration the novel properties that arise when materials are produced at the nano-scale. One obvious advantage of nano-scale particles over larger particles is their ability to cross biological barriers, accumulate selectively in tissues and, moreover, at least for some classes of NPs, the ease by which the particle surface can be tailored to harbor targeting ligands such as folic acid. However, the very same properties may also give rise to unexpected effects and toxicities caused by the translocation of particles to distant sites in the body. The challenge in terms of utilizing NPs for targeted drug delivery in cancer patients is thus, in principle, the following: how to target NPs exclusively to cancer cells, allowing the NPs to release their payload, and then become excreted or, alternatively, biodegraded in a programmable manner, while avoiding the uptake and accumulation of NPs in bystander cells, including cells of the immune system, our main surveillance system against foreign intrusion (microorganisms, as well as particles). We have highlighted recent developments of folate mediated uptake of NPs for several main classes of NPs, as well as noted other tumor and tumor vasculature targeting strategies. Resolving the issue of targeted drug delivery without side-effects in normal tissues promises to be an exciting journey, with potentially great benefits for cancer patients. This will require not only the careful design of NP carriers to optimize their pharmacological profile while mitigating any potential toxicities but also an increased understanding of the fundamental process of endocytosis/phagocytosis and its regulation in cancer cells versus normal cells.

Graphical abstractfigure legend

This review discusses the benefits and limitations of folate-targeted nanoparticles for anticancer therapy. Garcia-Bennett et al.

  A c c e p t e d M a n u s c r i p t endocytosis machinery are utilized as a portal of entry to deliver large payloads of chemotherapeutic drugs. Receptor-mediated endocytosis is a very effective, energyconsuming cellular import mechanism that utilizes the selectivity mechanisms provided by a series of unrelated receptors, combined with the ubiquitous clathrin-and/or caveolin endocytosis systems, to ensure the specificity of transport. Additionally, folate-targeted

  methotrexate or aminopterin) to achieve better results. Moreover, FOLR1 itself has become a promising target of novel biological therapeutics, such as the monoclonal antibody farletuzumab (MORAb-003), which has shown considerable promise in the treatment of latestage ovarian cancer patients who have developed resistance against conventional anti-cancer drugs such as cisplatin.[START_REF] Konner | Farletuzumab, a humanized monoclonal antibody against folate receptor alpha, in epithelial ovarian cancer: a phase I study[END_REF] It is unclear whether the process of receptor-mediated endocytosis may be more active in cancer versus normal cells, but the possibility exists that tumor cells exploit particularly effective endocytosis mechanisms as a result of generally hyperactive metabolic pathways.The putative role of caveolins (CAV1-3) and caveolae-related import mechanisms in cancer cells is currently intensively investigated. Furthermore, the identification of receptors and receptor-mediated uptake mechanisms other than FOLR1 to target tumor cells specifically is currently poorly developed. The identification of additional ligand/receptor pairs that show A c c e p t e d M a n u s c r i p t significant tumor-specific expression and utilize the same endocytosis route as FOLR1, may be highly rewarding for diagnostics, cancer imaging, and therapeutic applications of NPs.

A c c e p t e d M a n u s c r i p t 1 -

 1 ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) depending on the linker group. The use of PEG allows further conjugation to other therapeutically relevant

A c c e p t e d M a n u

  s c r i p t to assess the biocompatibility and cellular targeting ability of these materials.Lu et al. have

  studies and clinical development as a result of their low toxicity, relatively facile and cost effective functionalization with folate conjugates through a variety of routes, improved pharmacokinetic behavior of the pharmaceutical agents, feasible diagnostic tools offering A c c e p t e d M a n u s c r i p t non-invasive strategies, and, finally, in vitro and animal model in vivo evidence for an

A c c e p t e d M a n u

  s c r i p t suppression of the internalization of pristine NPs may favor selective uptake of drug-loaded, functionalized NPs.

  vasculature and are exposed to the tumor microenvironment. The size-change of the nanoparticles is triggered by proteases that are highly expressed in the tumor microenvironment, which degrade the cores of 100 nm gelatin nanoparticles, releasing smaller NPs from their surface. This interesting system, akin to a multi-stage rocket for space exploration, could provide a means to achieve deep tumor penetration of NPs and more effective treatment of cancer.

A c c e p t e d M a n u s c r i p t 20

 20

A c c e p t e d M a n u s c r i p t 21 lipids

 21 could potentially determine the toxicity of nanomaterials, including their thrombogenic and complement-activating potential.

u s c r i p t 36 Figure legends Figure 1 .

 36legends1 Figure legends

Figure 2 .

 2 Figure 2. Expression of folate receptor mRNA in normal and cancer tissues. Data derived

Figure 3 .

 3 Figure 3. Synthetic folate linkers with functional groups capable of direct tethering to

  Figure

  Figure

Table 1

 1 Some examples of folate-functionalized nanoparticles currently under investigation for targeted drug delivery or imaging.

		1-ethyl-3-[3-	Pancreatic	In vivo		92
		dimethylaminopropyl]carbodiimide	adenocarcinoma	Luminescence
		hydrochloride (EDC) with PEG		imaging
				(xenografted
				tumors in mice)
	Mesoporous Silica	NHS and EDC on amine	Breast cancer	In vivo	In vitro	29
	(100nm)	functionalized silica surface.		Fluorescence	Camptothecin
				imaging
				(xenografted
				tumors in mice)
	Liposomes	PEG-	KB cells		In vivo	93
	(70-90nm)				doxorubicin
	Dendrimers (≤5nm)	Activated ester of folic acid reacted	Immunodeficient		In vivo	94
		on acetylated terminal dendrimer	mice bearing		methotrexate
		groups	human KB tumors
	Single-Walled	Folic acid modified phospholipids	mouse ovarian	In vitro confocal		95
	Carbon Nanotubes		surface	imaging
	(SWCNTs)		epithelial cells

Acknowledgements

The authors are supported by the Swedish Research Council, the Academy of Finland (grant no. 140524), and the Seventh Framework Programme of the European Commission (EC-FP-7-NANOMMUNE; grant no. 214281).


H Deplaine

J L Gómez Ribelles

G Gallego Ferrer

Effect of the content of hydroxyapatite nano-particles on the properties and bioactivity of poly(L-lactide) -hybrid membranes

Keywords: A.Poly(L-lactide), A.Hydroxyapatite, A.Nano composites, B.Bioactivity, B.Surface treatment

Poly(L-lactide)/hydroxyapatite, PLLA, composite membranes for bone regeneration with different concentrations of nanoparticles have been prepared and their physicochemical properties and bioactivity have been determined. Hydroxyapatite nanoparticles act as nucleating agent of the poly(L-lactide) crystals, as detected by DSC, and as reinforcing filler, as proven by the monotonous increase of the elastic modulus of the microporous membranes with increasing nanofiller content. The bioactivity, which regards to the use of these materials in bone regeneration, was tested by immersing the samples in a simulated body fluid, SBF. A faster deposition of a biomimetic apatite layer was observed as increases the content of hydroxyapatite nanoparticles, thus membranes with a 15% (w/w %) of hydroxyapatite particles (relative to PLLA weight) present a homogeneous layer of hydroxyapatite on the surface of their pores after 7 days of immersion in SBF. An especial emphasis has been made on the influence of a plasma treatment on the bioactivity of the membranes. With this aim, the membranes were submitted to a plasma treatment previously to their immersion in a simulated body fluid.

It has been observed that the surface of a PLLA membrane after 21 days of immersion in SBF is still not completely covered by hydroxyapatite whereas the same sample treated with plasma show a smooth layer of biomimetic hydroxyapatite. The increase of bioactivity achieved with this treatment was less important in high hydroxyapatite content composites.

Introduction

Bone regeneration using tissue engineering techniques involve the use of bioresorbable scaffolds that should sustain the load applied "in vivo". Ceramic materials are brittle and stiff, and therefore macroporous materials made with them are fragile, while polymeric materials are unable to sustain the applied loads in many applications [START_REF] Gross | Biodegradable composite scaffolds with an interconnected spherical network for bone tissue engineering[END_REF].

Nanocomposites formed by a polymeric matrix reinforced with hydroxyapatite (HA), other ceramic materials such as tricalciumphosphate, silica or bioactive glass have been proposed in order to design porous materials with adequate mechanical properties for bone engineering [START_REF] Stevens | Biomaterials for bone tissue engineering[END_REF]. HA has the particularity to bind directly the bone without forming any surrounding connective tissue. HA has the same formulation of mineral bone (Ca 10 (PO 4 ) 6 (OH) 2 ). The rest of the resorbable ceramics are also based on calcium phosphate, but among them the quicker in degrading is HA [START_REF] Sastre | Biomateriales[END_REF]. Besides that, the presence of hydroxyapatite nanoparticles in the polymeric matrix can improve the bioactivity of the material, inducing osteogenesis when implanted "in vivo", due to osteoinductive and bone bonding ability [START_REF] Murphy | Effects of a bone-like mineral film on phenotype of adult human mesenchymal stem cells in vitro[END_REF]. This "in vivo" behaviour has been shown to be demonstrable by "in vitro" mineralization (Kokubo test). This experiment was proposed by Kokubo as an "in vitro" test to assess the bioactivity of a material by its capacity to generate a layer of HA on its surface when immersed in simulated body fluid (hereafter SBF) [START_REF] Kokubo | How useful is SBF in predicting in vivo bone bioactivity?[END_REF]. The mechanism proposed for this behaviour is that bioactive materials show negative surface potential when immersed in SBF, due to deprotonation at physiological pH of groups like -COOH, -OH, -TiOH (these groups have been shown to provoke hydroxyapatite precipitation in SBF) [START_REF] Oliveira | Nature-inspired calcium phosphate coatings: present status and novel advances in the science of mimicry[END_REF][START_REF] Kawashita | Apatiteforming ability of carboxyl group-containing polymer gels in a simulated body fluid[END_REF]. This negative charge attracts calcium ions, creating a Ca-rich layer with positive surface potential. This positive surface recently formed, can in its turn attracts negative ions in solution like phosphate, leading to the deposition of a Ca-poor layer. This process repeats itself generating the HA layer on the surface of materials [START_REF] Landi | Nucleation of biomimetic apatite in synthetic body fluids: dense and porous scaffold development[END_REF][START_REF] Kim | Process and kinetics of bonelike apatite formation on sintered hydroxyapatite in a simulated body fluid[END_REF][START_REF] Lu | Theoretical analysis of calcium phosphate precipitation in simulated body fluid[END_REF].

For this work poly(L-lactic acid), PLLA, was used, since it exhibits mechanical properties suitable for human bone applications. PLLA is a biocompatible and biodegradable polyester belonging to the group of poly α-hydroxy acids, and it can be easily processed into complex shapes. PLLA degrades by non-specific hydrolytic scission of its ester bonds thereby producing lactic acid which is a normal by-product of anaerobic metabolism in the human body [START_REF] Wong | Biomaterials. Biodegradable polymeric biomaterials: And updated overview[END_REF][START_REF] Wei | Polymer/Ceramic composite scaffolds for bone tissue engineering[END_REF][START_REF] Peibiao | In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(L-lactide)[END_REF]. In this work we present a series of PLLA/HA nano-composites membranes prepared by a freeze extraction process [START_REF] Ho | Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods[END_REF][START_REF] Deville | Freeze casting of hydroxyapatite scaffolds for bone tissue engineering[END_REF][START_REF] Nejati | Synthesis and characterization of nanohydroxyapatite rods/poly(L-lactide acid) composite scaffolds for bone tissue engineering[END_REF]. This method is based on phase separation, like liquid-liquid demixing, whereby the solvent and the polymer crystallises in the polymer-poor phase and the polymer-rich phase, respectively [START_REF] Budyanto | Fabrication and characterization of porous Poly (Llactide) (PLLA) scaffolds using liquid-liquid phase separation[END_REF]; thus leaving a porous morphology when the crystallised solvent is removed with the aid of a good solvent. These membranes were then modified by plasma treatment in order to enhance their bioactivity [START_REF] Qu | The effect of oxygen plasma pretreatment and incubation in modified simulated body fluids on the formation of bone-like apatite on poly(lactide-co-glycolide) (70/30)[END_REF]. As polymer containing carboxylic acids show high plasma susceptibility, the plasma treatment was applied as to help initiate the nucleation of a layer of HA. Oxygen plasma permits to lower the surface energy of PLLA, and consecuently increases the wettability as contact angle with water decreases after plasma exposure and the permeability of the membranes to SBF. Our AFM experiments allow concluding that the particles incorporated in the membranes do not show on the surface since they are covered by a thin layer of PLLA. This thin surface layer is directly attacked by the radicals and electrons of the plasma and thus HA particles show at the surface as observed in the SEM and AFM pictures. Apatite layer formation on the surface of the membranes was observed after different times immersed in SBF as a means for studying "in vitro" bioactivity, the apatite layer density increases with the time of inmersion. The plasma treatment, by increasing the wettability of the membranes, as well as the increase of HA content in the composites, allows nucleating HA only after 7 days of immersion in SBF, what is not possible for pure PLLA. Moreover thermal and mechanical properties of the membranes were studied.

Materials and Methods

Materials

PLLA from Purac biomaterials (The Netherlands) was used: it is a homopolymer of Llactide with an inherent viscosity of 1.8 dl/g. The molecular weight distributions were analyzed by gel permeation chromatography using a Varian liquid chromatograph equipped with refractive index, UV, light scattering and viscosity detectors.

Trichloromethane at 35 C was used as the eluent and the SEC columns were from Supelco (G6000-4000-2000 HXL). Thus, the optically pure PLLA has a number average molecular weight of 59,500 and a polydispersity index of 1.8. 1,4-dioxane was purchased from Scharlau Chemie, S.A.( Barcelona, Spain). Hydroxyapatite particles with particle size inferior to 200 nm were purchased from Sigma-Aldrich (Spain). The chemicals used in the preparation of SBF were from Sigma-Aldrich (Spain). All chemicals were used as received without any further purification.

Sample preparation

A series of poly-L-lactic acid/hydroxyapatite composite membranes with HA content 0, 5, 10, and 15% (w/w) were prepared by freeze extraction method. Freeze extraction is a modification of freeze drying process described by Ho et al [START_REF] Ho | Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods[END_REF]. The required quantity of HA powder was dispersed in dioxane by sonication, then a 15% weight PLLA (relative to solvent weight) was added to the mixtures, and stirred to complete dissolution. Four solutions with different content in HA were prepared, with 0, 5, 10, 15% particles by weight with regard to PLLA. The PLLA-HA solutions were poured into Teflon moulds and frozen in liquid nitrogen. Then, cold ethanol at -10ºC was poured on the frozen membranes in order to dissolve the crystallised dioxane. Dioxane extraction was conducted in a cold ethanol bath at -10ºC. Ethanol was replaced several times to ensure that most of the dioxane has been extracted from the samples, the PLLA is almost not plasticized and the pores do not collapse at room temperature. After extraction, membranes were dried in air atmosphere during 24h and then in vacuum to constant weight at room temperature. The membranes were cut into squares (1 cm x 1 cm) of 200 µm thickness. To obtain the desired thickness the samples were shaped included in resin O.C.T. TM compound from Tissue-Tek at -30ºC and cut by cryogenic microtome. After removing residues of O.C.T., samples were dried during three days.

The first day at room temperature, the second under vacuum and the third under vacuum at 40ºC. In order to analyze the effect of plasma treatment on the composites surface by AFM thin films were spin-casted from a 2% (2g/100ml) solution of PLLA in chloroform with a 10% (w/w) of HA (relative to PLLA weight) particles. Spin casting was performed on 12 mm glass coverslips at 2000 rpm for 30 s; previously to that process the mixture of the polymer solution and the particles was homogenised by sonication. Samples were dried in vacuo at room temperature before further characterisation.

Bioactivity test

SBF solution was prepared with the method of Müller et al. [START_REF] Müller | Preparation of SBF with different HCO -3 content and its influence on the composition of biomimetic apatites[END_REF] from eight salt solutions of, KCl (59.64 g/l), NaCl (116.88 g/l), NaHCO 3 (45.37 g/l), MgSO 4 .7H 2 O (49.30 g/l), CaCl 2 (14.702 g/l), TRIS (tris-hydroxymethyl aminomethane; 121.16 g/l) (used for pH adjustment, see below), NaN 3 (1 g/l), KH 2 PO 4 (27.22 g/l) into 650 ml deionised ultra pure water, in order to prevent salt precipitation and maintain the pH of the solution. Then the solution was poured in a 1000 ml flask and filled with deionised ultra pure water. For pH adjustment to pH 7.6-7.7 at 25ºC (which corresponds to pH 7.3-7.4 at 37ºC), HCl or TRIS was added.

[Table 1.] Samples were soaked in SBF for different times up to 21days. The SBF solution was not renewed during the first 7 days. Afterwards, the ion concentrations were adjusted to twice those of SBF (2xSBF) and the solution was renewed each 2-3 days, in order to provide more favourable conditions for apatite deposition. The samples treated with plasma were soaked in the SBF solution 1 hour after the treatment.

Oxygen plasma treatment

Surface modification was carried out with oxygen plasma (Plasma Electronic, model Piccolo, quartz cylinder, 2.45 GHz generator) at 300W in a 50 Pa vacuum, for 120 seconds for each side. Membranes were used for experiments immediately after exposure.

Differential scanning calorimetry (DSC)

Crystallinity was evaluated by thermal analysis with a Perkin Elmer DSC Pyris I. Two heating scans from 0ºC to 210ºC at 10ºC/min were performed, the first one in order to gain information on sample crystallinity after freeze extraction process, and the second one in order to observe the behaviour of the samples after erasing thermal history and cooling down from 210ºC to 0ºC at 10ºC/min.

Calcination experiments

The real content of HA in the composites was determined by weighting membrane residues after calcination. Calcination was carried out in a Conatec HC-300 ceramic oven. For each concentration (0-5-10-15 wt.% HA), three samples were heated up to 600ºC at 10ºC/min, and maintained at 600ºC for 10 minutes, in order to entirely thermally degrade the PLLA.

Dynamic mechanical spectroscopy (DMS)

The dynamic mechanical analysis of the membranes before immersing in SBF was determined by the Seiko Instruments Extrar6000 Thermal Analysis and Rheology System. The samples were heated first from 0ºC up to 120ºC at 2ºC/min, maintained at 120ºC for 60 minutes, and then cooled to 0ºC at 2ºC and heated again to 150ºC. Testing frequency was 1 Hz, the membranes presented rectangular dimensions 3 x 3 x 30 mm 3 .

Hydroxyapatite morphology analysis and characterization

For scanning electron microscope analysis, samples were mounted onto copper holders and gold sputtered. The morphology of the HA formed on the surface of the scaffold was characterized with a JEOL JSM 6300 scanning electron microscope in secondary mode under an acceleration tension of 15kV.

Atomic force microscopy

The effect of plasma treatment on the surface of the spin-casted films was observed by the amplitude images obtained with am Atomic Force Microscope, AFM, before and 24 hours after plasma treatment. NanoScope III from Digital Instruments operating in the tapping mode in air and NanoScope 4.43r8 software version was used. Si-cantilevers from Veeco (Manchester, UK) were used with force constant of 3 N/m and resonance frequency ranging from 66 and 94 kHz.

Contact angle measurements

A Dataphysics OCA instrument (DataPhysics Instruments GmbH, Filderstadt, Germany) and SCA20 software were used to measure the contact angle on the surface of PLLA films of drops of water. Different drops of ultrapure water were deposited directly on PLLA films, before and after a plasma treatment (1 and 24 hours after). The volume used and the time between the drop deposition and the capture of the image were constant for each measurements (V = 0.20ml, t = 30s).

Hydroxyapatite layer characterization

Element quantification was performed by Energy Dispersive Spectroscopy, EDS, with a JEOL JSM 6300 scanning electron microscope with an acceleration tension of 10kV.

For EDS analysis, samples were mounted onto copper holders and carbon sputtered. The samples were observed by SEM and EDS after 0, 7, 14 and 21 days in SBF with and without oxygen plasma treatment.

Results and discussion

Membranes characterization

The freeze extraction process produces a membrane with 10 µm micropores as shown in Figure 1. The micropores are homogenously dispersed on the surface of the membranes.

To assure the reproducibility of the technique, 3 different membranes were fabricated and observed by SEM. All membranes exhibited the same morphology. The pores are produced by the phase separation when both dioxane and PLLA crystallise from the solution. When producing a nano-composite membrane, the HA nano-particles are suspended in the solution. Crystallisation of PLLA entraps part of these particles but another part is expected to remain between different dioxane crystals or at the interfaces between dioxane and PLLA in the solid sample when immersed in liquid nitrogen.

[Fig. 1

. (left) and (right)]

The morphology of the membranes seems not to be affected by plasma treatment. After oxygen plasma treatment, as shown in Figure 2 some kind of abrasion defects can be seen on the surface of the membranes, which does not appear for the membranes without plasma treatment. Surface abrasion that was already found in plasma treated PLLA by Wang et al [START_REF] Qu | The effect of oxygen plasma pretreatment and incubation in modified simulated body fluids on the formation of bone-like apatite on poly(lactide-co-glycolide) (70/30)[END_REF] can allow HA particles to appear at the pores walls.

[Fig. 2

. (a) and (b)]

Although HA nano-particles cannot be seen by SEM, especially in the untreated membranes, probably because a layer of PLLA covers them, EDS analysis was able to show Ca and P peaks distinctive of the HA components as shown by the insets of Figures 2a and2b. The EDS analysis was repeated three times in different zones for three different samples, and all the signals present the same behaviour, proving that particle dispersion is homogenous. AFM amplitude images (Figure 3) confirm these results. On one hand, as can be seen in Figure 3, the HA particles are uniformly dispersed in the PLLA matrix. Although the original particles are of sizes below 200 nm (as specified by Sigma Aldrich) it seems that some of them tend to agglomerate in the preparation process forming aggregates with sizes up to 300 nm. On another hand, AFM pictures clearly show that there is a layer of PLLA covering the particles. The detail of an agglomerate of nanoparticles in Figure 3b is quite diffuse contrarily to what happens in the images took 24 hours after the plasma treatment, where the layer of PLLA covering the particles has been eliminated by plasma, and the particles are clearly exposed in the surface (Figure 3c). Even if there is a layer of PLLA covering the particles previously to the plasma treatment they can be detected by EDS because the beam electron trajectory of the EDS ray interacts with the material and can penetrate till 5 microns in the material [START_REF] Goldstein | Scanning Electron Microscopy and X-ray Microanalysis[END_REF].

[Fig. 3. (left) and (righ)]

Calcination experiments [Table 2]

The calcination experiments showed that the amount of HA incorporated into the membranes is close but lower than the amount of HA dispersed in the PLLA solution.

Membranes with 5, 10 wt.% HA (table 2) show 11-12% mass error, and 15 wt.% HA membrane mass error is 46%. These deviations can be an indication of an imperfect dispersion of particles, or the leaching of a part of the particles that remained in the solvent during phase separation.

Differential scanning calorimetry (DSC)

DSC experiments included a heating scan that gives information about the crystalline structure obtained after freeze extraction process. This scan was performed between 0 and 210ºC. Thus, at the end of this scan the sample is melted. A cooling scan followed to characterize the crystallization kinetics from the melting of the different nanocomposites. Finally a second heating scan was recorded to characterize the structure of the polymer crystallized from the melt.

[Fig. 4. left and right.]

The first heating scan shows the glass transition with a large overshoot characteristic of the physical ageing (or structural relaxation) process suffered by the polymer during annealing at 40ºC while drying in vacuum. The cold crystallisation peaks appear after glass transition. These exothermal peaks appear at lower temperature as the HA nanoparticles content increases (see Figure 4, left). Another crystallisation process appears at higher temperatures that is characteristic of highly nucleated samples that crystallize at very low temperatures [START_REF] Munehisa | Melting behaviour of poly(Llactic acid): X-ray and DSC analyses of the melting process[END_REF][START_REF] Sánchez | Influence of Low-Temperature Nucleation on the Crystallization Process of Poly(l-lactide)[END_REF], finally the melting process takes place at a temperature independent of sample composition. Cooling scan recorded after the first heating scan showed an exothermal peak (results not shown) that proves that PLLA partially crystallizes on cooling.

The second scan is representative of the samples crystallized after melting. Here the endothermal overshoot due to physical ageing disappears since the sample was not annealed below the glass transition (Figure 4, right). The crystallization and melting follow a trend similar to that of the first scan, although the crystallization peaks are clearly shifted towards higher temperatures. This behaviour can be explained by the formation of crystalline nuclei during physical ageing at temperatures below T g that has been proven in PLLA and in other semicrystalline polymers as well [START_REF] Lim | Processing technologies for poly(lactic acid)[END_REF]. An increased number of nuclei at the beginning of cold crystallization speeds up the process; as a result the observed crystallisation peak is sharper and shifted towards lower temperatures. The changes in shape and position for cold crystallisation peaks with increasing HA content demonstrate the nucleating effect of the HA nanoparticles [START_REF] Lebourg | Porous membranes of PLLA-PCL blend for tissue engineering applications[END_REF].

The baseline used for the determination of crystallinity in the sample is presented as an example in Figure 4 in the thermogram corresponding to the second scan of the 15% HA composite. The determination of crystallinity was done by an integration of the heat flow trace with respect to it. This linear baseline joints a point immediately above the glass transition and another one after melting. If the sample was completely amorphous at the beginning of the heating scan the area between the thermogram and the baseline would be zero. The calculus becomes quite uncertain since the temperature interval to which it is extended is very broad and can easily lead to small errors in the determination of the baseline used in the integration (for instance for small curvatures in the baseline of the calorimeter). This difficulty is especially clear in the first scan when the thermogram starts deviating towards the exothermal side immediately above the glass transition (Figure 4, left), which impedes any enthalpy calculation. In the second scan crystallinity values between 6 and 20% were found but the HA content dependence was non-systematic. The crystallinity, X (%), was determined based on the following equation:

100 1 . 93 (%) ⋅ ∆ - ∆ = c m H H X ,
where ∆H m and ∆H c are respectively the melting and crystallization enthalpies, obtained from the integration of the heat flow thermograms, and the constant 93.1 J/g is the melting enthalpy for a 100% crystalline PLLA [START_REF] Lim | Processing technologies for poly(lactic acid)[END_REF]. The onset of the glass transition seems to be independent to the filler content (Table 3). The difference between the values measured in the first and second scan should be due to the physical ageing process to which samples were subjected during drying before the first scan. A slight decrease of the enthalpic glass transition temperature with the HA content is observed (Table 3).

[Table 3]

Dynamic mechanical spectroscopy (DMS)

Dynamic-mechanical experiments are also highly dependent on the thermal treatment to which the sample is subjected. The samples were first subjected to heating from 0 and 120ºC in the first measuring scan. The temperature dependence of the storage modulus, E' shows the sharp decrease starting close to the calorimetric glass transition. The shape of the curve with a change of slope in the middle of the relaxation indicates that crystallization starts during the scan itself, as seen in the DMTA curves (Figure 5a).

This makes the values measured at temperatures above T g quite dependent on the thermal history in the scan itself. The modulus in the glass region measured in the first scan increases with the HA content of the composites (Table 1) showing the reinforcing role of the HA particles. To compare the behaviour of the different nano-composites with a more reproducible structure, samples were annealed at 120ºC for one hour to allow crystallization and cooled to 0ºC to start the second scan. Thus, the second scan corresponds to higher crystallinity samples. The main relaxation in the second scan is shifted to higher temperatures with respect to the first one. The value of E' in the glassy region increases with respect to the first scan due to the higher fraction of PLLA crystals. The reinforcing effect of the nanoparticles is clearly shown in the semicrystalline samples both below and above the main relaxation of the amorphous phase (Figure 5b) [START_REF] Uskokovic | Micromechanical properties of a hydroxyapatite/poly-L-lactide biocomposite using nanoindentation and modulus mapping[END_REF].

[Fig. 5. (a) and (b)]

Hydroxyapatite layer characterisation

SEM analysis shows the influence on the kinetics of HA deposition during increasing immersion times in SBF. As noticed in Figure 6, PLLA is able to nucleate HA in its surface, without any special treatment but at very slow rates compared to plasma-treated PLLA or PLLA reinforced with HA. At short times a few nuclei can be observed in the SEM pictures, they increase in number to form a rough layer of cauliflower-shaped crystals [START_REF] Hong | Preparation and in vitro characterization of scaffolds of poly(L-lactic acid) containing bioactive glass ceramic nanoparticles[END_REF]. The surface of the micropores appears uniformly coated by HA after an immersion time that decreases as the content of HA in the composite increases and with the plasma treatment. In order to show this behaviour with a reasonable number of Figures, Figure 6 shows the surface of a PLLA membrane after 21 days of immersion in SBF, a time at which coating is still not complete, whereas the sample treated with plasma show a smooth layer of biomimetic HA deposited on the surface. Although plasma treatment increases the bioactive potential of pure PLLA a faster process is needed to avoid degradation of the polymer before its osteo-integration. After 21 days in SBF or in the implantation site PLLA starts the degradation process and consequently its mechanical properties start to decrease too early making the material useless for bone tissue engineering. Consequently it is interesting to generate a layer of HA at shorter time of SBF inmersion (7 days in SBF) [START_REF] Kang | A comparative study of the in vitro degradation of poly(l-lactic acid)/β-tricalcium phosphate scaffold in static and dynamic simulated body fluid[END_REF], an effect that has been obtained in this work by the addition of the HA particles.

[Fig. 6. left and right]

Figure 7 shows the effect of the HA nano-filler content after 7 days immersion. After this time the PLLA samples show no signs of apatite layer even when treated with plasma, but the rate of deposition increases with HA content and samples containing more than 10 wt.% HA nano-particles show a smooth coating of the composite surfaces.

Plasma treatment further enhances the rate of deposition, and the thickness of the layer increases in such a way that biomimetic apatite fills the whole volume of the micropores (Figure 7). Results obtained after different immersion times in SBF (7, 14, 21 days) are similar. Figure 7 shows the typical cauliflower structure of the HA of the layer on the surface [START_REF] Vallés Lluch | Biomimetic apatite coating on P(EMA-co-HEA)/SiO 2 hybrid nanocomposites[END_REF].

Treated Untreated

[Fig. 7. (8 micrographs)]

[Table 4]

The water contact angle on PLLA before plasma treatment is 86.8º and it dramatically decreases to 22.6º one hour after plasma exposure. There is not too much difference between this value and that obtained 24 hours after plasma exposure, 25.1º, indicating that radicals recombination on the surface has been almost completed. The observed decrease indicates that the plasma treatment increases the wettability of the material, and so, the interaction between the ions in presence in SBF and the surface of the material. Consequently plasma treatment accelerates the process of precipitation of the ions on the surface of the material.

As can be seen on Table 4, Ca/P atomic ratio of the biomimetic HA layer has been calculated from EDS analysis. This ratio has been compared to the one of stoichiometric hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) Ca/P = 1.67, or physiological HA = 1.65. Globally the Ca/P atomic ratio calculated for all the samples is higher than the physiological HA one, ranging from 1.63 to 2.54 (table 4) [START_REF] Vallés Lluch | Biomimetic apatite coating on P(EMA-co-HEA)/SiO 2 hybrid nanocomposites[END_REF]. The membranes with HA incorporated at day 0, in which the Ca/P atomic ratio measured corresponds to the HA nanoparticles added as filler, have the highest ratio, and the ratio observed does not match that of stoichiometric hydroxyapatite claimed by the supplier. For the same immersion time membranes previously exposed to plasma present a ratio closer to the physiological one in comparison with membranes that had not been exposed to plasma. In general, the ratio that is closest to the physiological one is for the plasma treated membranes prepared with nano-filler after 7 days in SBF.

Conclusions

Freeze extraction allows obtaining PLLA/HA nano-composites with a good dispersion of HA particles and bioactive properties. The storage modulus increases with the filler content at temperatures above and below the glass transition of the amorphous phase.

Particles act as a nucleating agent for PLLA modifying the crystallisation kinetics whereas glass transition temperature of PLLA is independent of the filler content. The bioactivity is enhanced by the presence of the HA particles more than by plasma treatment; indeed the nucleation of a biomimetic HA layer on the surface of the nanocomposites when immersed in SBF is more effective than on PLLA membranes. This study allows combining two independent ways to increase the bioactivity of PLLA membranes: nanoparticles of HA content and plasma treatment. Our results show that membranes presenting the best interest for their use in bone regeneration are that with a 15% of HA nanoparticles and previously exposed to plasma. Indeed these membranes

Captions to Figures

Fig. 1 .

 1 Fig. 1. Scanning electron microphotographs of the surface of PLLA membranes before

Fig. 2 .Fig. 3 .

 23 Fig. 2. Scanning electron micrographs and Energy dispersive spectroscopy analysis (as

Fig. 4 .Fig. 5 .

 45 Fig. 4. First DSC heating scan of PLLA/HA nanocomposite with different filler content

Fig. 6 .

 6 Fig. 6. Scanning electron micrographs of pure PLLA membranes, without plasma

Fig. 7 .

 7 Fig. 7. Scanning electron micrographs of 0, 5, 10, 15 wt.% HA membranes without and

Acknowledgements

The authors acknowledge the support of the Spanish Ministery of Science and Innovation through the DPI2007-65601-C03-03 project (including the FEDER financial support). Dr. G. Gallego Ferrer acknowledges the support of the same Institution for the mobility grant JC2008-00135 in the "José Castillejo" program to do a research stay at the 3Bs Group from the Polymer Department of the University of Minho, Portugal. Furthermore the work of GGF and JLGR was partially supported by funds for research in the field of Regenerative Medicine through the collaboration agreement from the Conselleria de Sanidad (Generalitat Valenciana), and the Instituto de Salud Carlos III (Ministry of Science and Innovation).