Inorganic Frameworks as Smart Nanomedicines

Latvia

Series Preface: Pharmaceutical Nanotechnology

Alina M. Holban, University of Bucharest, Bucharest, Romania

Due to its immense applicative potential, nanotechnology is considered the leading technology of the 21st century. The science and engineering of nanometer size materials is currently employed for the development of numerous scientific, industrial, ecological and technological fields. Biology, medicine, chemistry, pharmacy, agriculture, food industry and material science are the main fields which have benefited of the great technological progress developed in nanoscience.

In the pharmaceutical field, nanotechnology has revolutionized traditional drug design concept and the art of drug delivery. The idea of a highly specific nanoscale drug for the targeted therapy of diseases is now considered a feasible treatment for severe health conditions.

Some scientists believe that pharmaceutical domain has been reborn by the important impact of nanotechnology. The field of pharmaceutical nanotechnology has the potential to offer innovative solutions for all diagnosis, therapy and prophylaxis domains. Application of nanotechnology tools in pharmaceutical research and design is likely to result in moving the industry from ‘blockbuster drug’ model to ‘personalized medicine’. Nowadays the main focus of clinicians is to treat patients individually, not general diseases which are usually difficult to diagnose or incorrectly diagnosed. There are compelling applications in pharmaceutical industry where suitable nanotechnology tools can be successfully utilized. By designing and modifying drugs at nanoscale, pharmaceutical nanotechnology could be useful not only for the development of completely new therapeutic solutions, but also to add value to existing products. This possibility opens successful perspectives for pharmaceutical companies for existing markets, but also for new markets.

Since the interest for pharmaceutical nanotechnology is impressive we face today a true dilemma of data unavailability, due to the multitude of existing information which can be highly inaccurate and contradictory. This is because of the lack of an efficient model for sorting out the plethora of nanotechnology tools that exists and strategically correlate those with potential opportunities into different segments of pharmaceutical research and design.

This series is trying to cover the most relevant aspects regarding the great progress of nanotechnology on the pharmaceutical field and to highlight the currently emerging trend of pharmaceutical nanotechnology towards personalized medicine concept.

The ten volumes of this series are wisely structured to offer relevant information regarding basic concepts and also to reveal newest approaches and perspectives in pharmaceutical nanotechnology.

Nanoscale fabrication, optimization, scale-up and biological aspects of pharmaceutical nanotechnology, introduces the readers into the amazing field of nanoscale design and empathize on the biological requirements of nanostructured pharmaceutical formulations for advanced drugs.

Design and development of new nanocarriers, the most recent progress made on the field of nano-delivery is discussed. Modern nanostructured drug carriers employ innovative solutions for the detection and treatment of various diseases in a personalized and efficient manner.

Design of nanostructures for theranostics applications, highlights the impressive impact of nanotechnology in the development of combined diagnosis and therapy – theranostics – concept.

Design of nanostructures for Versatile Therapeutic Applications, offers a dynamic solution for immune modulation, treatment of diseases by natural-based products and infection control, while employing nanostructured solutions to achieve top results.

Nanostructures for the engineering of Cells, Tissues and Organs: From design to applications, a highly investigated and debated field – tissue engineering, is dissected. Here is shown how nanotechnology advanced research and applications in the manipulation and engineering of cells and tissues in vitro.

Organic materials as smart nanocarriers for drug delivery, deals with the specific world of organic nanomaterials, revealing their wide applications, types and advantages in drug delivery.

Inorganic frameworks as smart nanomedicines, the main focus is to discuss the variety and properties of inorganic nanostructures for therapy and drug-delivery in the context of an improved personalized medicine.

Lipid Nanocarriers for drug targeting, deals with recently developed lipid nanostructures and the advances made in drug targeting.

Drug targeting and stimuli sensitive drug delivery systems, dissects smart stimuli responsive nanosystems employed to specifically detect various biochemical conditions and control the release of drugs.

Fullerens, Graphenes and Nanotubes, reveals major findings made on widely applied drug-design nanosistems, namely Fullerens, Graphenes and Nanotubes.

All ten volumes are nicely illustrated and chapters are organized into a logical manner to be accessible to a wide audience. The series is a valuable resource of new and comprehensive scientific proof on the intriguing and emerging field of pharmaceutical nanotechnology which could be of a great use for scientists, engineers, pharmaceutical representatives, clinicians and any non-specialist interested user.

Preface

Inorganic Frameworks as Smart Nanomedicines

This book highlights the recent progress in the field of inorganic materials as smart nanomedicines. Different types of inorganic nanomaterials (such as silica, gold, zinc oxide, iron oxide, magnetosoms, quantum dots, and others) are reviewed from a biomedical point of view. Special attention was also paid to toxicity assessment of nanopharmaceuticals and regulatory framework applied in their case for developed and developing countries.

The book entitled Inorganic Frameworks as Smart Nanomedicines contains 15 chapters, prepared by outstanding researchers from Ireland, Brazil, Turkey, Portugal, Uruguay, Latvia, India, Pakistan, Chile, Spain, United States, Finland, and France.

Chapter 1, entitled Silica-based nanoparticles as drug delivery systems: Chances and challenges, prepared by Didem Sen Karaman et al., provides an overview of the chances and challenges for silica-based nanoparticles in intravenous drug delivery. Once injected, silica-based nanoparticles encounter blood cells and complement proteins. Complement protein binding, hemolysis, and coagulation are challenges for the drug delivery systems, because this reduces their efficacy and also challenges their safety.

Chapter 2, Gold nanoparticles for cancer diagnostics, spectroscopic imaging, drug delivery, and plasmonic photothermal therapy, prepared by Mena Aioub et al., explores the design and use of AuNPs for their implementation in single cell detection methodologies, which allows for the real-time evaluation of drug delivery, drug efficacy, cell cycle perturbation, and cell death mechanisms. Additionally, the contributors discuss the therapeutic applications of AuNPs in the context of cancer treatment through tumor targeting and photothermal therapies.

Chapter 3, Porous silicon for biomedical applications, prepared by Gonzalo Recio-Sánchez et al., reviews the key properties of porous silicon and the most significant applications of porous silicon in the fields of biosensing, bioimaging, drug delivery, and tissue engineering. The different processes commonly used to tailor the behavior of porous silicon for specific applications are also described in detail.

Chapter 4, Green fabrication of metallic nanoparticles, prepared by Habiba Ramzan et al., presents recent progress related to the process of fabrication and source of metallic nanoparticles that differentiates the properties of nanoparticles. Many different methodologies were examined under chemical and physical methods, but the revolutionary step was taken and biological methods were used, which are proved ecofriendly and more efficient. Applications of metallic nanoparticles are widespread in electronics, medicine technology (MRI, chemotherapy, etc.) and pharmaceutical (drug production and drug carriers) applications.

Chapter 5, Interaction of green nanoparticles with cells and organs, prepared by Moniba Rahim et al., provides an overview of green synthesized nanomaterials having biomolecules on their surfaces interacting differently with human cells than nongreen nanomaterials. Depending upon their surface molecules, they interact differently with different organs and cells of human beings. Their internalization in the cells depends on the cell types and cell receptors present on the cell surface. Due to biomolecules attached to their surfaces, these materials become highly biocompatible, soluble, non-toxic, site specific and highly stable in the human body.

Chapter 6, Biomedical applications of zinc oxide nanoparticles, prepared by Ayan Kumar Barui et al., describes the recent advances of zinc oxide nanoparticles (ZnO-NPs) for various biomedical applications, including delivery of biomolecules (drug, gene, etc.), cancer therapy, angiogenic therapy, antibacterial application, tissue engineering, bioimaging, biosensing, etc. Additionally, in light of promising medicinal applications, pharmacokinetics, bio-degradability and clearance studies of ZnO-NPs are also concisely discussed. Finally, this chapter gives an overview of the present global market of ZnO-NPs, followed by their plausible challenges and future directions in biomedical applications.

Chapter 7, Iron oxide/oleic acid magnetic nanoparticles possessing biologically active choline derivatives: Synthesis, morphology, antitumour and antimicrobial properties, prepared by Alla Zablotskaya et al., discusses a recently developed synthetic procedure, based on binding of the first biologically active substance with a magnetic core and other active substance (ligand), and subsequent immobilization on the modified surface of nanoparticles creating plasma membrane-like structures. Magnetic fluids revealed superparamagnetic properties and affected tumor and microbial cells.

Chapter 8, Silver-containing nanoparticles in the research of new antimicrobial agents against ESKAPE pathogens, prepared by Graciela Borthagaray et al., presents the development of new silver-containing nanoparticles with antimicrobial activity, considering their synthesis and characterization methods. Moreover, an enumeration of different techniques for the evaluation of the silver nanoparticle antibacterial activity are presented and commented upon.

Chapter 9, Core–shell nanoparticles as a drug delivery platform for tumor targeting, prepared by Namdev L. Dhas et al., gives an up-to-date overview about core–shell nanoparticles and their various applications as a drug delivery systems for targeting tumors. Furthermore, core–shell nanoparticles are classified depending on the material used for the preparation of core and shell, along with their method of preparation and characterization. Details about target-specific surface modification of core–shell nanoparticles are also described. Lastly, factors affecting pharmacokinetic and bio-distribution profiles of core–shell nanoparticles, along with biomedical applications and their future prospective are describe in detail.

Chapter 10, Bismuth metallic nanoparticles, prepared by Catherine Gomez et al., presents different strategies to synthesize spherical metallic bismuth nanoparticles. Two pathways are referenced: the top-down and bottom-up approaches. All procedures are critically compared to show how they could be improved and directed towards greener strategies. The different capping agents (polymers, hydrophilic, or hydrophobic monomers) used to control size, morphology, and stability of these nanoparticles are discussed, as well as the choice of solvent. The relevance of heating techniques (classical heating or irradiation under microwaves) and purification are compared to green processes. Finally, bismuth metallic nanoparticles are discussed in different medical applications, such as theranostic applications as X-ray contrast agent or X-ray radiosensitizers.

Chapter 11, Magnetoliposomes for dual cancer therapy, prepared by Ana Rita O. Rodrigues et al., describes the synthesis, characterization, and applications of magnetoliposomes in cancer therapy, both by antitumor drug delivery and hyperthermia, besides the ability to attain magnetic guidance to the therapeutic sites of interest.

Chapter 12, Quantum dots: A novel fluorescent probe for bioimaging and drug delivery applications, prepared by N. Ilaiyaraja et al., presents recent progress in the field of quantum dots from the point of view of their excellent photoelectronic properties, including broad excitation spectra, tunable fluorescence, and multi-color fluorescence that are lacking in the traditional organic fluorescents. Recent developments have led to synthesis of low toxic QDs for in vivo applications. However, the further research is needed to produce more biocompatibility and toxic free nanoparticles using efficient and los-cost methods.

Chapter 13, Toxicity assessment of nanopharmaceuticals, prepared by Pinar Erkekoglu et al., provides knowledge on the safety and toxicity of nanopharmaceuticals and summarizes the in vitro and in vivo testing strategies for the evaluation of the oral, inhalation, and dermal toxicity of nanomaterials.

Chapter 14, Regulatory framework of nanopharmaceuticals in developing countries: An analysis of current rules in Brazil, prepared by Bárbara Juliana Pinheiro Borges et al., provides an analysis of the current rules of nanopharmaceuticals in Brazil, with the objective of putting forward multilateral cooperation, including intellectual property rights and regulatory issues. Although Brazil have already authorized registering and marketing of nanopharmaceuticals, currently it has no specific regulation. This leads to difficulties for defining which tests pharmaceutical companies should perform and government agency should request in order to guarantee their safety.

Chapter 15, A review of the regulatory framework for nanomedicines in the European Union, prepared by Sebastian F Vencken et al., describes the history of nanomedicine regulation by the institutions of European Union and its member states. The inclusion of nanotechnology into the development and manufacturing of drugs and medical devices has brought about major gains in efficacy and accuracy. However, with these gains the novel field of nanomedicine has also introduced many unknowns, the majority of which concern their safety to human health and the environment.

Chapter 1

Silica-based nanoparticles as drug delivery systems

Chances and challenges

Didem Şen Karaman and Helene Kettiger,    Åbo Akademi University, Turku, Finland

Abstract

Silica-based nanoparticles are used as excipients in pharmaceutical technology. Recently, mesoporous silica nanoparticles have emerged as drug delivery systems. Their porous structure enables the high drug-loading of drugs with poor water solubility. The silica matrix protects entrapped drugs against enzymatic degradation. Furthermore, the premature release of drugs is hindered by pore-gating strategies. Adding a targeting ligand to the silica-based nanoparticles directs them to diseased cells and can diminish side-effects in healthy cells.

Silica-based nanoparticles are preferably injected intravenously, since this administration route lacks the disadvantages of oral, dermal, and pulmonary delivery. Once injected, silica-based nanoparticles encounter blood cells and complement proteins. Complement protein binding, hemolysis, and coagulation are challenges for drug delivery systems, because this reduces their efficacy and also challenges their safety. Consequently, hemocompatibility testing is a must for nano drug carriers.

This chapter provides an overview of the chances and challenges for silica-based nanoparticles in intravenous drug delivery.

Keywords

Silica; nanoparticles; drug delivery; mesoporous silica; hemocompatibility; hemolysis; coagulation; complement

Chapter Outline

1.1 Introduction: Mesoporous Silica Nanoparticles as Drug Delivery Systems 1

1.2 Chances of Mesoporous Silica Nanoparticle-Based Drug Delivery Systems 6

1.2.1 Promising Drug Carriers Among the Nano Drug Delivery Systems 6

1.2.2 Therapeutic Efficacy: Variety of Drug Release Mechanisms From Mesoporous Silica Nanoparticles and Intracellular Delivery of Mesoporous Silica Nanoparticles 10

1.3 Challenges of Silica-Based Nano Drug Delivery Systems 13

1.3.1 Hemocompatibility: An Introduction 13

1.3.2 Hemolysis 16

1.3.3 Coagulation 20

1.3.4 Complement Activation 24

1.4 Conclusion 25

Acknowledgment 26

Abbreviations 26

References 27

Further Reading 40

1.1 Introduction: Mesoporous Silica Nanoparticles as Drug Delivery Systems

Drug delivery is a multidisciplinary discipline that aims to deliver a pharmaceutical drug to a specific site of action. This interdisciplinary field spans from manufacturing a galenic form to delivering a therapeutic amount of a drug to its intended site of action (Parveen et al., 2012). The most common dosage forms for oral drug delivery are tablets, where an active pharmaceutical ingredient is combined with excipients. Although widely used, these drug delivery systems suffer from serious limitations, such as high dose requirement, low bioavailability, and off-target effects (side effects). Therapeutic plasma levels are usually maintained by repeated administration of the drug. Drugs with a narrow therapeutic window are especially critical in terms of toxicity and efficacy (over- or under-dosing), due to unavoidable drug level alterations in the body. Having the right drug at the right place in the right concentration remains a challenge in pharmaceutical technology (Coelho et al., 2010).

Drug molecules for oral drug delivery are classified by the Biopharmaceutics Classification System (BCS). The BCS divides drugs into four groups, and defines the terms solubility and intestinal permeability (Amidon et al., 1995). Class I includes substances with high permeability and high solubility. Class II consists of substances with high permeability and low solubility. Class III houses the substances with low permeability and high solubility, and Class IV consists of low permeable and low soluble substances. Here, highly soluble is defined as the highest dose strength to be soluble in <250 mL water at a pH between 1 and 7.5. Highly permeable is defined as the gastrointestinal absorption of >90% of the administered dose, based on a mass balance or in reference to the intravenous injection. An approximate estimate of 40% of drug compounds are identified as poorly water soluble (Merisko-Liversidge and Liversidge, 2008). By miniaturizing the drug crystals (e.g., nanomilling) to the nano size, the solubility of such drugs could be dramatically enhanced (Li et al., 2016). Another approach to enhance solubility is to transform the drug into an amorphous state and consequently make it more water soluble than in the crystalline state (Salonen et al., 2005).

Nano drug delivery systems (nano-DDS) aim to overcome the aforementioned limitations of poor water solubility and low permeability, by delivering the drug in a controlled manner. A nanoparticle is defined as a particle of any origin with a size between 1 nm and a few hundred nanometers (Krug and Wick, 2011). A nano-DDS can be targeted (e.g., by attaching specific antibodies or peptides), and consequently be taken up by specific cells. Targeting the nano-DDS can help dramatically to reduce side effects and the amount of drug can also be reduced, which leads to an overall decreased exposure of a cytotoxic drug (Bhagwat and Vaidhya, 2013). Recently, so-called intelligent nano-DDSs have been developed, that release their cargo on demand, e.g., in the presence of a certain enzyme or upon a pH-drop (Giménez et al., 2015; Tian et al., 2013). The recent progress in the engineering of inorganic or organic/inorganic nanoparticles make them especially more attractive for their applications in drug delivery, compared to traditional organic material–based nanoparticles, such as liposomes (Y. Chen et al., 2013b). Currently, 43 nano drug formulations have been approved (Weissig et al., 2014), and some examples are given in Table 1.1. Most of them are liposome-based formulations, such as Doxil (Barenholz, 2012). However, the intrinsic instability and low drug-loading capacity of organic nano drug formulations restrict their further clinical translations and leave them in the preclinical stage (Pepic et al., 2014).

Table 1.1

Adopted from Wicki, A., Witzigmann, D., Balasubramanian, V., and Huwyler, J. (2015). Nanomedicine in cancer therapy: challenges, opportunities, and clinical applications. J. Control. Release Off. J. Control. Release Soc. 200, 138–157. doi:10.1016/j.jconrel.2014.12.030

Silicon is the most abundant element (27.2%) in the earth’s crust after oxygen (45.5%) (Jurkić et al., 2013). By virtue of its chemical and physical characteristics, silicon and its oxide forms take place in variety of industries. Among these fields, biomedical applications attract great attention and are growing rapidly. For instance, silicon-based materials have been employed as dietary supplements, implants, dental fillers, and contact lenses (Jaganathan and Godin, 2012). The fundamental characteristics of silicon dioxide (silica)-based nanoparticles, such as size, optical properties, high surface area, low density, adsorption capacity, capacity for encapsulation, biocompatibility, and low toxicity, make them an especially attractive inert solid in diverse biomedical applications (Bitar et al., 2012).

In the beginning of the 1990s the first preparation of mesoporous silicate material was conducted by Yanagisawa et al. (1990). In addition, Mobil Oil scientists reported mesoporous molecular sieves designated as M41S in 1992 (Beck et al., 1992). These mesophase silicates were prepared by the reaction of monomeric silicate and CnTMA surfactants (Beck et al., 1992, 1994; Van Speybroeck et al., 2009). MCM-41 is the initial member and the most widely studied material of the M41S family. It is based on a hexagonally packed, rod-shaped micelle structure (Karge and Weitkamp, 1998). Another member of the family is MCM-48, which has a cubic structure and has been proposed as a three-dimensional and unconnected network of rod micelles. Lamellar structured materials (MCM-50) were also prepared by using the same liquid crystal templating system as the other materials. After the investigation of mesophase silica materials, much effort has been put towards the improvement of their features and also on promoting their use in different fields. All these investigations have helped to improve mesoporous silica materials in the particulate form. The nano-form of mesoporous silica (MSN) has gained great interest for use in pharmaceuticals, where they show a great potential as nano-DDS.

The biocompatibility of mesoporous silica material, the large specific surface area and pore volume, controllable particle size, and flexible surface functionalization possibilities meet the requirements to construct an efficient drug delivery system, which are listed below (Tiwari and Tiwari, 2013):

1. The carrier material should be biocompatible

2. High loading/encapsulation capacity of chosen drug molecules

3. No premature release of drug

4. Cell type or tissue specificity and targeted delivery ability

5. Achievement of effective local concentration of drug by controlled release of drug molecules

One of the first attempts to use mesoporous silica particles (MCM-41 type) as a reservoir for drugs was done by Vallet-Regi et al. (2001. In the study, ibuprofen was employed as a guest molecule and loaded into the pores. Subsequently, the drug release in simulated body fluid was investigated. Since then, the exploration of mesoporous silica particles as drug carriers continued with increasing interest. More sophisticated systems, such as on-demand release or so-called theranostics, which combine an optical/magnetic tracer and a therapeutic agent, are available (Baeza et al., 2015; N.-T. Chen et al., 2013a; Chen et al., 2014; Kempen et al., 2015). These promising composites are currently one of the hottest topics both for in vitro and in vivo research in the drug delivery field. Despite the existence of well-established in vitro research of MSNs, the in vivo assays of MSN regarding bio-safety, imaging, and therapeutic efficiency to encourage the translation into preclinical stage was only started in 2008 by Wu et al. (2008). The main reason for such a delay is the complexity of the in vivo physiological environment, which makes it difficult to imitate and produces less reproducible results. Furthermore, the design of MSNs should be well addressed to be used in the physiological environment because the bio-behavior of MSNs is strongly related to their preparation that varies the size, shape, and surface chemistry of the particles (Desai et al., 2014; Şen Karaman, 2016).

To facilitate the use of MSN as nano-DDS, different design strategies are employed, since tuning the morphology (size and shape), pore size and internal properties of pores, and surface functionalities of MSNs have great impact on improving their capability as drug carriers. For instance, when MSNs are administered by intravenous injection the biodistribution and blood circulation time depend on the MSN physicochemical properties (Huang et al., 2011). The surface chemistry of the MSN also limits in vivo efficacy, due to unspecific protein adsorption, which leads to recognition and elimination by the reticuloendothelial system (Kettiger et al., 2013). Biocompatibility of the carrier itself is of vital interest. Biocompatibility is defined by IUPAC as the ability of a material to perform with an appropriate host response in a specific application and the ability to be in contact with a living system without producing an adverse effect (Terminology for Biorelated Polymers and Applications (IUPAC Recommendations 2012), 2012). Silica is classified as generally regarded as safe by the United States Food and Drug Administration. Several nano-safety studies include silica as a nontoxic reference material or no adverse events in vivo were found (Ryu et al., 2014). However, several concerns about nanosilica have been raised recently (Lin et al., 2006; Napierska et al., 2010). Studies tried to relate the physicochemical properties of nanosilica with the biocompatibility. For most of the cases, the toxicity was apparent at very high concentrations, or it was related to a highly positive surface charge, which covered the initial nanoparticles, and could not be consequently used to claim whether silica is biocompatible or not. For silica-based nanoparticles used as intravenous drug delivery systems, the biocompatibility also involves compatibility with the blood system. This special part of biocompatibility is termed hemocompatibility. So far, MSNs and also nonporous silica nanoparticles (SNPs) that are used as imaging agents (Ow et al., 2004) have shown hemocompatibility issues in vitro (Kettiger et al., 2015; Slowing et al., 2009). Although silica-based nanoparticles are frequently called the new nano drug carriers or highly biocompatible materials, there is a surprising lack of data about hemocompatibility. Hemocompatibility is often measured by the hemolysis assay, but other cells such as thrombocytes, leukocytes, and plasma proteins should also be considered before SNPs and MSNs are deemed hemocompatible.

Today, many research groups are working on closing these knowledge gaps to advance MSN from bench to bedside, by studying their pharmacokinetics and biocompatibility, including biodistribution, degradation, excretion, blood-circulation lifetime, cellular uptake and cytotoxicity, blood compatibility, etc. (He and Shi, 2011). The successful demonstration of MSN efficacy, biocompatibility, as well as safety profile in vitro and in vivo investigations encourages many researchers, however, standardization in testing of their therapeutic efficacy, biocompatibility, and biodistribution of silica-based nanoparticles is still missing (Mamaeva et al., 2013). Standardization is strongly required to allow inter-laboratory comparison of silica-based drug delivery systems.

Despite the lack of standardization, silica-based drug carriers possess promising features and, consequently, recent advances in MSN-based theranostic designs can readily contribute to human health and personalized medicine platforms. Stronger cross-disciplinary collaborations are still required to develop MSN-based smart nanomedicine and ultimately benefit from their abilities as nano-DDS. The interplay of chances and challenges is of vital importance to advance silica-based nanoparticles further towards in vivo and first-in-man trials.

1.2 Chances of Mesoporous Silica Nanoparticle-Based Drug Delivery Systems

1.2.1 Promising Drug Carriers Among the Nano Drug Delivery Systems

Reported adverse drug-related (ADR) hospitalization was estimated up to 12.8 % among the unspecified patient profile through Europe (Bouvy et al., 2015). There are various reasons for the occurrence of ADR hospitalization, such as practice error, impaired homeostasis in elderly, or lack of compliance due to polypharmacy (Kwon et al., 2013). Aside from this, the cause of adverse drug reactions might be due to lack of specificity of a drug molecule that causes undesired interactions of a drug with healthy tissues or cells. To overcome the problem, delivering and maintaining effective drug concentration at its target is required. For example, in the case of vancomycin treatment, high dose is required to have an acquired effectiveness, which can easily lead to severe nephrotoxicity (van Hal et al., 2013). Additionally, hydrophobic drugs cannot be administered in a straightforward manner, due to their poor solubility in aqueous media, such as treatment Class II (low solubility and high permeability) and Class IV (low solubility & low permeability) drugs (e.g., anticancer drugs). Accordingly, achieving effective concentration of therapeutic agents at the target site is not always feasible (Kwon et al., 2013). For this purpose, formulation scientists try to improve strategies to overcome this hurdle by employing novel drug delivery systems.

Mesoporous silica nanoparticles (MSN) are one of the promising candidates of nano-DDSs, especially for Class II and IV drugs, since they can improve the dissolution rates of the poorly water soluble drugs (Van Speybroeck et al., 2009). The beneficial properties of MSNs include: (1) large surface area, provides a high potential for drug adsorption; (2) high pore volume, allows large amount of drug confinement in the carrier matrix; (3) well-ordered pore distribution in their structure favors the homogeneity and reproducibility of the drug adsorption and release stages; and (4) high density of silanol groups on the surface ease the chemical modification of the pore walls and the surface of the carrier. This allows a better control over drug loading and release (Manzano and Vallet-Regí, 2010). The aforementioned features of MSNs consequently improve the solubility, permeability, bioavailability, and pharmacokinetics of the drug, which are important for the performance of nano-DDS, as depicted in Fig. 1.1.

Figure 1.1 Physicochemical properties of mesoporous silica nanoparticles (MSN) and their effect on various processes in drug loading and biocompatibility.

For successful drug loading into MSN, it is of vital interest that the cargo component i.e. drug molecules are retained in the carriers and release only upon delivery to the site of action in the body. Over the last decade, many approaches have been developed to load drugs into MSNs. In the case of poorly water soluble drugs (Class II and Class IV), which are most of the recently discovered drugs (Lipinski, 2000), loading into MSNs can be carried out by organic solvent immersion, incipient wetness impregnation, or the hot melt method (Mellaerts et al., 2008). The extent of drug loading in the MSN depends on various parameters. Firstly, the surface area of the MSN (the larger the surface area, the more space for hosting drug molecules is available); secondly, the affinity of the drug to the MSN (native or surface-modified silica favors or disfavors the adsorption/binding of the drug, depending on the ability to form molecular interactions); thirdly, the pore volume of the MSN to prevent recrystallization of the drug in the pores; and, lastly, the concentration of drug in solution. The chosen drug loading method affects the extent of loaded drug, the homogeneity of drug distribution as proposed in Fig. 1.1, and the state of the loaded drug (crystalline/amorphous) in the mesoporous structure (McCarthy et al., 2016).

The organic solvent immersion method for drug loading has been extensively reported (McCarthy et al., 2016). In this approach, the substrate (i.e., MSN) is immersed in a drug solution and stirred mechanically for a certain period. Subsequently, the drug-loaded MSNs are collected and the solvent is removed by evaporation. The particles are then dried under reduced pressure (Xu et al., 2013). Solvent immersion allows for fine tuning of the amount of incorporated drug. Additionally, the drug can be loaded into MSN homogenously. In this approach, it is crucial to remove the solvent below acceptable limits, as mentioned in the guidelines to avoid possible toxicity (Impurities: Guideline for Residual Solvents: ICH). Additionally, post-processing such as filtration or centrifugation is needed. The second approach for drug loading is the incipient wetness approach. Here, a drug solution at the concentration close to the saturation of the drug, in the volume close to the pore volume of the substrate, is added to the MSN and mixed intensively. Afterwards, the MSN-drug mixture is left for drying at ambient conditions and subsequently placed under reduced pressure (Mellaerts et al., 2008, 2010; Van Speybroeck et al., 2009). This method is usually preferred when the quantity of drug is limited (Tochilin, 2016). The challenges of this method are that high drug solubility in the solvent and repeated impregnations are needed. The third method is the hot melt method and is a solvent-free approach in which the physical mixture of the drug and substrate is heated above the melting point of the drug and then mixed properly. The mixture is subsequently dried under reduced pressure (Kinoshita et al., 2002; Mellaerts et al., 2008). This method can also be carried out by controlled microwave irradiation, especially for the thermally sensitive materials (Gignone et al., 2014; Hillerström et al., 2014; Waters et al., 2011). The advantage of this method is that no solvent is needed. However, drug degradation, drug crystal formation, and the use of special set-ups are drawbacks of hot melt extrusion. Fig. 1.2 summarizes the three main approaches of drug loading into MSNs. Recently, supercritical fluid technology was also used to load water insoluble drugs into MSNs (Ahern et al., 2012; Patil et al., 2011). The method was introduced as a reproducible, controllable, and scalable alternative for drug loading to MSNs.

Figure 1.2 Different drug loading approaches on MSN with advantages and disadvantages of each method.

The physicochemical characteristics of MSNs are determinant factors for the drug loading capacity of MSNs (Rosenholm and Lindén, 2008) and are summarized in Fig. 1.3. The diameter of the mesopores on the MSN is one of the most important parameters, because they act as a size-selective parameter for the drug molecule. The pore dimension of the MSN is usually in the range of 2–4 nm; however, in recent reports, MSNs with pore sizes larger than 15 nm were prepared by researchers for the delivery of larger biomolecules (Kim et al., 2011). When the drug molecule is smaller than the pore cavity, the drug can be incorporated in the mesopores. However, the drug loading in the pore cavity of MSNs cannot take place if the drug molecule is larger than the mesopore diameter and would preferentially adsorb on the external surface of the MSN due to steric reasons. Briefly, the mesopores on MSNs act as molecule sieves and are determinant for the size of the drug molecules, which can be loaded into the carrier material. The accessible pores for the drug molecules are obtained when the ratio of pore diameter:drug molecule size is >1. For high drug loading, this ratio should be higher than 3 (Xu et al., 2013). In addition, the pore volume of MSN also has great impact on the degree of drug loading. In the literature, the MSNs with the highest total pore volume were reported to reach a very high drug load of over 1:1 in weight with the other major determining factor of pore size (Heikkilä et al., 2007). Another parameter is the porous structure of MSN. Recently, several studies have reported improved drug loading capacity of MSNs with the hollow structure design. Hollow MSNs possess a voidal core, surrounded by a shell with mesopores in one single unit. The hollow core acts as drug reservoir in the structure, whereas the mesoporous shell provides pathways for the release of encapsulated substances. A remarkable drug loading degree difference was obtained between a hollow MSN and its non-hollow mesoporous resembling (Geng et al., 2016). In addition, pore orientation on MSNs could facilitate the homogeneous incorporation of payloads into the matrix (Li et al., 2011).

Figure 1.3 The features of MSN that affect loading or release of drug onto or from the MSN matrix.

The appropriate chemical modification of the interior and/or exterior surface of the MSN is essential for both water soluble and poorly water soluble drugs, in order to enhance the loading degree of drug molecules, as it could affect the state of loaded drug molecules (Lodha et al., 2012; Rosenholm and Lindén, 2008; Zhang et al., 2010). Different methods are presented in the literature for the surface functionalization of MSNs including in situ synthesis modification, which are known as cocondensation and postsynthesis surface modification approaches (Rosenholm et al., 2011). In the cocondensation method, the functional groups are already introduced during the synthesis step, whereas postsynthesis functionalization approaches are carried out after the particles are synthesized. For instance, amine-modified materials showed alendronate (Class III drug, high solubility and low permeability) loading almost three times larger than unmodified counterpart, with 24 h solvent immersion method (Balas et al., 2006). Conversely, a similar approach with telmisartan (Class II drug, low solubility and high permeability), loading led to crystalline structure formation of the drug (Zhang et al., 2010). In this study, the amine-functionalized mesoporous silica and pristine mesoporous silica were loaded with the similar amount of drug and were analyzed with an X-ray diffractometer. According to the obtained patterns, a crystalline drug structure was observed, which is associated with improved drug loading capacity. However, this may affect the drug release in terms of solubility and dissolution, which can subsequently influence the bioavailability of the drug.

In summary, MSNs are promising drug carriers, as their loading capacity can be tailored according to the physicochemical properties of the drug of interest. In the literature, different types of MSNs have been reviewed by McCarthy et al. for improving drug dissolution, which was correlated with the extent of the achieved drug loading and the state of drug on MSN after the loading process (McCarthy et al., 2016). A summary can be found in Table 1.2. The pore diameter and pore volume, and the interaction between the drug and mesoporous silica substrate are attributed as the determinant parameters for the loading capacity of the MSN and also appropriate confinement of the drug molecules in an MSN matrix.

Table 1.2

MCM, Mobil Composition of Matter; SBA, Santa-Barbara Amorphous; FSM, folded sheets mesoporous materials; TUD, Technische Universiteit Delft.

Adapted from McCarthy, C.A., Ahern, R.J., Dontireddy, R., Ryan, K.B., Crean, A.M., 2016. Mesoporous silica formulation strategies for drug dissolution enhancement: a review. Expert Opin. Drug Deliv. 13, 93–108. doi:10.1517/17425247.2016.1100165.

1.2.2 Therapeutic Efficacy: Variety of Drug Release Mechanisms From Mesoporous Silica Nanoparticles and Intracellular Delivery of Mesoporous Silica Nanoparticles

The high drug loading capacity of MSNs without premature drug release, and protection of the guest molecule during the drug delivery process make them desirable for many therapeutic actions. In addition, MSN cellular internalization renders them as an attractive candidate for specific tissue and cell-targeted drug delivery. Therefore, targeted action is frequently aimed with the MSN nano-DDS. Through this, higher therapeutic effects can be obtained and lesser side effects are present, due to the reduced interactions with healthy tissues. In drug delivery systems, the major advantage of an MSN as carrier is the variety of cargo release mechanisms can be provided by altering the design of MSNs. In the literature, advances in the design of MSN as DDS for immediate, modified, and targeted drug delivery have been reviewed (Yang et al., 2012).

Immediate drug delivery systems are designed to give a fast onset of drug action, improving the solubility, and dissolution rate of drug. This can subsequently improve the permeability and bioavailom the ability of the drug (Perrie and Rades, 2012). In MSN-based immediate drug delivery systems, the dissolution process of the drug is considered as the rate-limiting step for the absorption of the drug. Drugs can be confined in amorphous form, which helps to improve the solubility of the drug. The drug dissolution process is influenced by the drug loading method, pore morphology, and pore dimension of MSNs (Wang et al., 2015). For instance, the drug dissolution rate from the MSNs with an interconnected pore structure was reported as being faster, compared to those with unconnected pore structure (Wang et al., 2014). In the literature, mesoporous silica substrates that have one-dimensional pore structure with cage-like pores are reported as the most promising pore geometry for providing a slow release of drugs from mesoporous host, under in vitro conditions (Andersson et al., 2004; Sun et al., 2015).

In the literature, the design of smart drug delivery systems has been promoted with surface-modified MSNs. The evolution of smart drug carriers encouraged the upgrading of release systems from sustained release systems to the controlled release systems with reversible, multifunctional, complicated, logical and selective switches. In the field, the first MSN with reversible responsive controlled release system was prepared in 2003 by Tanaka et al. After their investigations, MSN-based controlled cargo release systems have become attractive, especially in the fields of material science and medicine (Mal et al., 2003).

As mentioned earlier, frequent administration of a drug can be avoided with modified-release drug delivery systems. Among the modified-release drug delivery systems, extended drug release systems are commonly employed. Here, the drug release is extended over prolonged time periods. Due to their pores, MSNs are promising candidates for such formulations. The key characteristic of MSNs is their exterior and interior surface, which can be extensively modified. This has been proven to be beneficial in different drug release systems (Mai and Meng, 2013). For instance, the drug-loaded pores can be sealed with various gatekeepers, and such payloads will not be released until triggered by stimuli, which offers an opportunity for stimuli-responsive drug delivery systems for controlled release (Yang et al., 2014) (depicted in Fig. 1.3). The gatekeepers can be pH-responsive polyelectrolytes, supramolecular nanovalves, acid-decomposable inorganic materials, thermosensitive polymers, and enzyme responsive polypeptides (Kuthati et al., 2013; Liu et al., 2015). The development of a responsive MSN-based controlled release system is very important to improve drug efficacy and reduce drug side effects.

Drug release from the nonfunctionalized mesoporous silica is mainly diffusion controlled and happens with first-order kinetics, in which a constant portion of the drug concentration is available. First-order kinetics are usually observed in the absence of interaction between drug and mesoporous silica matrix. Parameters that influence the release rate are: the loaded drug amount, the effective pore size, the solubility of the drug molecule, the existing interaction between the drug and pore walls, and the state of the loaded drug (crystalline/amorphous). Variations in the interaction of drug molecules through the mesoporous silica matrix lead to slower first-order release kinetics for nonfunctionalized mesoporous silica. Another reason for such observation was explained due to the dissolution of silica in physiological environment, which may lead to the hydroxyapatite crystalline formation at the pore gates and cause to partially blocking of the pores (Rosenholm et al., 2011). Nonfunctionalized MSNs with different structural properties (conventional, hollow-structured, and hollow-structured with double shell layer around the voidal compartment) can be employed in order to alter the sustained release profile from the carrier. Significant differences in the sustained release profiles were obtained with this strategy (Chen et al., 2011).

Surface-functionalized MSNs have been highlighted in both sustained release and controlled drug delivery systems. Among them, MSN-based stimuli-responsive nano-DDSs that integrate targeting moieties are presently under extensive investigation (Moreira et al., 2016; Zhu et al., 2014). MSN are promising candidates for the delivery of a large payload of drug molecules without degradation or premature release of the drug to targeted cells since they can be readily internalized by cells without any cytotoxicity issue in vitro or any apparent negative health effects in vivo (Zhao et al., 2010). This property of MSNs is truly advantageous, in case the drug to be delivered is toxic or precise dosage control is requisite. Furthermore, it has been shown that MSNs dissolve in vitro and their degradation products (silicic acid) are inherently nontoxic (Chen et al., 2012).

MSN used for therapeutic applications can be readily taken up by living cells, and their cellular internalization is their most exploited property. The efficient internalization of MSNs in a broad range of cell lines has been presented by many researchers (Costanzo et al., 2016; Kotcherlakota et al., 2016; Vivero-Escoto et al., 2010). The uptake mechanism is influenced by different factors, such as morphology (size, shape), the surface functionalization of MSN, and their interactions with the cell membrane. The internalization of MSN into cells mostly occurs via endocytosis. In the endocytosis process, MSNs are entrapped in vesicles (endosomes) and in order to reach their intracellular target, endosomal escape is a prerequisite. For this purpose, different endosomal escape strategies are employed and also to release the payload of the MSN in the cytoplasm (Tu et al., 2012; Wang et al., 2013; Wu et al., 2013). Therefore, intracellular trafficking of MSNs after cellular internalization is highly decisive for exerting a therapeutic effet.

Although the intracellular delivery of several drugs by MSNs have been successful both in in vitro and in vivo, there are still many unsettled concerns for future clinical applications, such as the acute and chronic safety profile, long-term circulation properties, colloidal stability in complex biological fluids, and targeting efficacy. Detailed investigations of MSN cellular activities and their long-term biocompatibility in higher mammalian species is mandatory to move these promising nano-DDSs towards clinical trial in humans.

1.3 Challenges of Silica-Based Nano Drug Delivery Systems

1.3.1 Hemocompatibility: An Introduction

Silica-based nanoparticles are preferably injected intravenously, since this is the administration route where the nano-DDS does not need to cross a barrier to reach systemic circulation, compared to skin, oral, or pulmonary delivery (Bertrand and Leroux, 2012). Once injected, nano-DDSs should circulate in the bloodstream to reach their target. Accordingly, premature clearance from the systemic circulation is a challenge. When the nano-DDS is in the bloodstream, it immediately gets covered with plasma proteins and interacts with the blood cells. This interaction can be desired, but in most cases, the nano-DDS-blood interaction can disturb red blood cells, coagulation, and the complement system, as depicted in Fig. 1.4. Distortion of the blood system can lead to various fatal consequences, including anemia, thrombogenesis, or inflammation (Straat et al., 2012; Weisenberg and Mooradian, 2002). To assure the safe and long-term application of nano-DDSs, so-called hemocompatibility is a challenge to be considered.

Figure 1.4 Three main issues in hemocompatibility. If hemolysis, coagulation, and complement activation are disturbed in the presence of silica-based nanoparticles, this may lead to fatal consequences for the patient.

Hemocompatibility is defined as the interaction of foreign material with the blood system (Williams, 1987). According to ISO 10993-4, hemocompatibility consists of testing for thrombosis, coagulation, platelets, hematology, and complement system. Most of the procedures in vitro can be evaluated by microscopy, cell surface markers, or cell number counting. To date, the majority of the research in hemocompatibility has been conducted with medical implants, as they may be constantly exposed to the blood (e.g., stents). The research of producing hemocompatible surfaces of these devices started almost 70 years ago and has been discussed ever since (Braune et al., 2013; Ratner, 1993). Devices, initially deemed as hemocompatible, turn out to be problematic in vivo by causing complement activation, or thrombocyte aggregation; coronary stents still have a risk of causing thrombosis in human (van Oeveren, 2013). Furthermore, the ISO 10993-4 catalogue demands minimum test requirements, and these guidelines may not be suitable for nanoparticles. Seyfert et al. (2002) strengthened the use of standardized blood collection and anticoagulation, the time of exposure to the material, temperature of exposure, and flow dynamics to mimic the intended use of the medical device. For example, interpreting the results of the hemolysis assay is hampered by a lack of universally accepted criteria for determination of the test-result validity (Dobrovolskaia et al., 2008). Critical for inter-laboratory comparison is: the incubation time (normally varying between 1 and 4 h), the wavelength at which hemoglobin is quantified (preferably, cyanmethemoglobin at 540 nm is measured, as the most stable form of hemoglobin), and blood conditions (purified erythrocytes, whole blood, choice of anticoagulant, origin of blood sample). The sampling of platelets is also crucial for in vitro assays (Dhurat and Sukesh, 2014; Harrison et al., 2011). The platelets age rapidly outside the body and their reactivity decreases after one hour. This is why the time delay between drawing of the blood and analysis should be within 30 min to 2 h (Dhurat and Sukesh, 2014; Harrison et al., 2011). Since platelet rich plasma (PRP) preparation and the time elapsed after the blood is drawn significantly influences the outcome, it is furthermore difficult to compare results which are obtained from different laboratories. Additionally, the health status and diet of the donors must be checked, as healthy subjects have far less activated platelets than subjects with atherosclerosis. The use of the appropriate anticoagulant is also an important discussion (van Oeveren, 2013) Adenosine diphosphate (ADP), epinephrine, collagen, and others are used as inhibitors of platelet aggregation. Additionally, the use of an appropriate syringe is important, since high shear stress also induces platelet activation. The choice of the appropriate inhibitor should be driven by the choice of tested blood (e.g., PRP or whole blood) and the questions of the study (Braune et al., 2013). Ultimately, blood from human beings is preferred at most times. The Nanotechnology Characterization Laboratory offers hemocompatibility testing assays, which could serve as standardized protocols for nanoparticles (Dobrovolskaia et al., 2008; Neun and Dobrovolskaia, 2011).

Currently, the test systems relate the thrombogenicity or the hemolysis relative to a positive control, which is, in most of the cases, thrombin or water, respectively. There are no particle controls available, and hence it is not possible to say if one specific type of nanoparticle is more hemocompatible than another type. However, it would be of more use for development if relative comparisons are made to nanoparticles that are already on the market. This would facilitate comparison between the new nanoparticles and existing ones, setting them in relation to be more/less hemocompatible.

Generally, SNPs and MSNs are considered as safe and well tolerated in most of the in vitro and in vivo biocompatibility tests (Fruijtier-Poelloth, 2012; Kettiger et al., 2015) However, SNPs and MSNs have been shown to induce hemolysis and disturb coagulation in vitro, as detailed below. Hemolysis is often tested, as it is an easy assay and used to predict for their pathogenicity (Pavan et al., 2014). This partly explains the popularity of the assay. Since red blood cells lack endocytosis, it is also an interesting model to study cell membrane-nanoparticle interaction (Kettiger et al., 2016), since these interactions are believed to mainly contribute to the toxicity of SNPs (Kettiger et al., 2015; Slowing et al., 2009). The aforementioned reasons explain why complement and coagulation of SNPs and MSNs have been studied to a lesser extent. This is surprising, as consequences of a disturbed coagulation and complement activation can have fatal outcomes and hamper SNPs and MSNs to be translated into the clinics.

The interaction of foreign material with blood and blood vessels in vivo is always complex, and the authors want to stress that the assays discussed below give estimations on isolated blood components or cell cultures under artificial and, most importantly, static conditions. Hemocompatibility studies mainly focus on three types of adverse events, which include hemolysis, thrombogenicity/coagulation, and complement activation (Dobrovolskaia et al., 2008; Simak, 2009), as seen in Fig. 1.4. These three processes are mainly involved in acute inflammatory response.

1.3.2 Hemolysis

Hemolysis is the rupture of red blood cells followed by the release of intracellular material into the bloodstream (Slowing et al., 2009). It is a severe adverse event that biomaterial can induce in red blood cells (Dobrovolskaia et al., 2008; Pavan et al., 2013). Consequences of hemolysis are anemia, icterus, and hemoglobinuria. Red blood cell membranes can adhere to the endothelium in the circulation, leading to for prothrombotic and proinflammatory changes (Straat et al., 2012). As a consequence, patients with hemolysis suffer from acute and chronic vascular disease, inflammation, thrombosis, and renal impairment (Schaer et al., 2013).

The American Society for Testing and Materials (ASTM) proposed a standard test protocol to determine the hemolytic potential (ASTM E 2524-2008). Therein, the percentage of released hemoglobin from destroyed erythrocytes upon nanoparticle exposure is expressed as percentage to 100% hemolysis (mostly water or a hypo-osmolar solution). The released hemoglobin can be measured spectrophotometrically. Dobrovolskaia et al. have scrutinized the hemolysis assay proposed by the ASTM and found that it is crucial to include interference controls to exclude false-positive and false-negative results (Dobrovolskaia et al., 2008). The study is available as a protocol to determine the hemolytic property of nanoparticles on the webpage of the Nanotechnology Characterization Laboratory (NCL method ITA-1). Although hemoglobin release is the most common assay to determine hemolysis, other methods such as flow cytometry (count of cells, specific surface markers) (Thedsawad et al., 2011) or scanning electron microscopy (shape of red blood cells) are available (Joglekar et al., 2013).

The underlying mechanism for hemolysis of amorphous SNP is not fully understood and several hypotheses are published (Kettiger et al., 2016; Slowing et al., 2009; Zhang et al., 2012). The most accepted one is that the amount of surface silanol groups is responsible for the hemolysis in both SNPs and MSNs. It is supposed that an electrostatic interaction between the surface silanols (negatively charged at neutral pH) and the quaternary ammonium (positively charged) on the cell surface is the driving factor for hemolysis. Using artificial membrane systems, it was shown that the interaction is based more on van der Waals interaction, and that the electrostatic interaction is negligible (Kettiger et al., 2016; Vakurov et al., 2012). If the amount of silanol groups on the surface is decreased (e.g., via thermal treatment, which results in siloxanes on the surface), the hemolytic potential is reduced (Pandurangi et al., 1990; Zhang et al., 2012). Depending on the synthesis route, the surface of SNPs and MSNs can also vary. Normally, room-temperature and basic-driven formation of an SNP results in a high amount of silanols on the surface (also called Stöber-process for SNP or nonthermal template removal of MSN), whereas calcination or pyrolysis leads to the formation of siloxanes on the surface (Zhuravlev, 2000). Reactive oxygen species (ROS) are also said to cause hemolysis, but the outcomes are somehow contradictory: whether SNP create ROS is strongly dependent on the synthesis and pre-treatment of the particles (Zhang et al., 2012). Even if the underlying mechanism is still under debate, it was shown that postsynthesis reduces hemolysis (Kettiger et al., 2015; Yu et al., 2011; Zhang et al., 2012). Depending on the synthesis and pre-treatment of silica-based nanoparticles, the biological outcome can, accordingly, change dramatically.

Lin et al. have investigated the hemolytic effect of SNPs (Stöber process) ranging from 25 to 225 nm (Lin and Haynes, 2010). They found that with decreasing particle size, the hemolytic effect increased. However, the larger SNP also caused hemolysis, but only at higher concentrations (>200 μg/mL). They assumed that the higher hemolytic activity of the small SNP stems from the higher numbers of exposed silanols. However, the particles were administered in a weight/volume dose. It would have been interesting to see the hemolytic potential when the doses would be normalized by surface area (expressed as accessible surface/dose).

In contrary, larger SNPs (commercially available for very small ones, lysine-assisted SNP synthesis and Stöber process) induced hemolysis to a higher extent than their smaller counterparts (Thomassen et al., 2011). They studied the hemolytic effect of individual SNPs and low- and high-density aggregates with comparable sizes to the individual particles. The low-density aggregates showed a decreased hemolytic activity with increasing of their hydrodynamic size. However, the high-density aggregates induced hemolysis to a similar extent as the larger individual SNPs. They also found that larger SNPs induced hemolysis to a higher percentage as their smaller counterparts, when normalized by the contact surface area. These findings were confirmed by Rabolli et al. (2010). This study pinpoints, again, the importance of following the colloidal stability of an SNP suspension over time and in different media.

A previous study by us investigated the hemolytic effect of various SNP (Stöber process and no heat treatment for MSN) by varying their size (80 vs. 230 nm in dry state diameter), their surface charge (introduction of primary amines), and their porosity (nonporous vs. mesoporous) (Kettiger et al., 2015). All SNPs used were synthesized by the sol-gel method. The results revealed that only small SNPs with the native silica surface (silanol rich) and large MSN induced hemolysis. Due to their pores, MSNs are believed to have a reduced hemolytic potential, because less silanols are exposed and available to interact with the red blood cell surface (Slowing et al., 2009).

Zhao et al. investigated the hemolytic effect of MSNs with different sizes and surface groups (Zhao et al., 2011). They found that larger MSNs (600 nm) induced hemolysis to a stronger extent than their smaller porous counterparts. They concluded that the amount of silanols on the surface and the rigidity of the red blood cell membrane is responsible for the hemolytic effect. They further modified the surface with amine groups (i.e., positive charge), carboxylic groups (i.e., negative surface charge) and polyethylene glycol (PEG, i.e., neutral surface charge). The main findings were that surface-modified SNPs interact less with red blood cells, regardless of the modification (also for negatively charged ones), and that the PEGylation reduced hemolysis to the highest extent.

Yildirim et al. (2013a,b) measured hemolysis upon exposure to 80 nm MSN with different surface groups, including ionic, polar, and hydrophobic functional groups, keeping the molar ratio of the functional group at 15 mol%. Their findings are in agreement with Lin et al. and show that unmodified MSN induced hemolysis to the highest extent at any given concentration, followed by the hydrophobic surface-modified ones, whereas all polar surface groups reduced hemolysis to almost zero percent. Furthermore, they investigated the surface density of primary amines on the surface of MSNs and found that, at already 2.5 mol% primary amines, hemolysis was completely inhibited, even at higher concentrations of 1 mg/mL. They further concluded that the interaction cannot be solely based on the surface charge of the MSN, as they found complete suppression of hemolysis after introducing a negatively charged surface group.

Lin et al. have investigated the size- and pore integrity–dependent hemolysis of MSNs. As for the nonporous SNPs, a dose-dependent hemolysis was demonstrated for the MSN. A 25 nm MSN, however, showed a different behavior and is explained by the different pore size than their larger counterparts. The authors concluded that larger pores result in less silanols per outer contact surface; this is why the 25 nm MSN did not induce hemolysis to a higher extent. However, it could also have been an agglomeration effect, as described by Thomassen et al. After incubation of 6 days in buffer, the 25 nm particles had a much higher hemolysis, and this was attributed to pore collapse. The authors assumed that the cell-contactable silanols on the outer surface increases, which leads to higher hemolysis. When the particles were modified with poly(ethylene glycol) (PEG), no hemolysis was observed. The PEG layer shielded the surface silanols from the red blood cells.

Yu et al. have investigated the effect on the aspect ratio of MSN and SNP (Stöber synthesis and low temperature MSN) with both native and amino-modified surfaces (Yu et al., 2011). The aspect ratio of nanoparticles is defined as length-to-width ratio. A concentration-dependent hemolysis was observed, which was more pronounced when the particles had spherical or low aspect-ratio shape. The effect for rods with an aspect ratio of 8 was not present up to 250 μg/mL, but elevated to 50% at 500 μg/mL, whereas spherical particles showed hemolytic effects at concentrations higher than 100 μg/mL. With the amino-modified particles, the shape was no longer the predominant factor for hemolysis, and it seemed that all shapes, even rods with high aspect ratios, induced hemolysis at already lower concentrations (50 μg/mL). This is conflicting with other findings in the literature, where aminated SNPs lead to a reduction in hemolysis (Kettiger et al., 2015; Yildirim et al., 2013a,b). However, the authors did not specify the number of primary amines introduced with post-grafting, and it is highly likely that there is kind of a surface density of primary amines, where the hemolysis again starts to increase.

One of the most common linkers used to reduce interaction with biological environment is polyethylene glycol. He et al. investigated the effect of the PEG-chain (4, 6, 10, and 20 kD) length and the PEG density (0.05–3.75 wt%) of 150 nm MSN on hemolysis (He et al., 2010). PEG-10kD and 0.75% showed the lowest hemolysis, compared to the other formulations and the nonmodified MSN.

Ferenc et al. (2015) investigated the role of the mesostructured, surface functionality, and linker length on Santa-Barbara Amorphous (SBA) 15-type mesoporous SNP and found similar results: an increase in the linker-length reduced hemolysis dramatically, suggesting that the spacer between native silica surface and functional group has a shielding effect. Furthermore, native (−OH), amino-modified (−NH2), mercaptopropyl (−SH), ethylcarboxylic (−COOH), and undecanoic-modified MSNs were tested for hemolytic potential. They found that mesopores in general reduce hemolysis. Depending on the surface group, the hemolysis was higher (−NH2), and very low for the mercaptopropyl MSN.

The hemolytic properties also immediately change after preincubating the particles in a protein solution. Since the proteins are shielding the silanol groups, similar to adsorbed or bound polymers, hemolysis is diminished to almost zero, as shown by Paula et al. (Paula et al., 2012). Martinez et al. found that binding representative proteins from the human blood plasma, such as serum albumin and others, can suppress the hemolytic effect of MSNs (in a dose-dependent manner) because of the formation of the protein corona.

Combining the aforementioned studies, it is evident that nonporous, native SNPs induce hemolysis to a higher degree compared to their surface-modified and porous counterparts. However, depending on the surface group and surface group density (charge density), it is also possible to induce hemolysis, but this has been rarely shown. PEGylation seems to diminish hemolysis to near zero. It would have been interesting to see how drug-loaded MSN (hence MSN with filled pores) would influence the hemolysis, as drug-loading of hydrophobic drugs may render the surface more hydrophobic.

1.3.3 Coagulation

Coagulation is a process to stop bleeding in the case of a vascular injury. The process of sealing a wound can mainly be divided into three processes. Firstly, the vasculature contracts, which results in a lower blood flow. Secondly, platelets are activated and aggregate at the site of injury to loosely seal the wound. Thirdly, this loose clot is stabilized by fibrin. Additionally, other platelets and red blood cells are included in the fibrin