Mesoporous silica nanoparticles: A multifunctional nano therapeutic system

Abstract

Efficient and safe drug delivery has always been a challenge in medicine. The use of nanotechnology, such as the development of nano drug delivery systems (DDS), has received great attention with high enthusiasm owing to the potential that nanocarriers can theoretically act as “magic bullets” and selectively target affected organs and cells while sparing normal tissues. The family of nano DDS includes conventional nano drug delivery materials such as lipids and polymers that have been scaled to the nanometer size range. With the rapid development of synthesis and characterization techniques for engineered nanomaterials, new DDS platforms have emerged, including inorganic based nanocarriers, such as mesoporous silica nanoparticles (MSNP). MSNP are able to act as a multifunctional delivery platform that is capable of delivering therapeutic elements to a variety of disease models (especially cancer) at cellular and in vivo levels. Furthermore, MSNP have shown to be exceptional delivery platforms capable of protectively packaging hydrophobic and hydrophilic drug molecules as well as other therapeutic elements for controlled on-demand delivery. In addition, MSNP have demonstrated the capability to image the delivery site for theranostic purposes. These functionalities have led to the development of MSNP as novel multifunctional nanocarriers, and therefore provide them with unique advantages compared to other nanocarriers.

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Insight, innovation, integration

Nanocarriers offer a new approach to drug delivery, providing a range of features including cargo protection and increased dose delivery to targeted sites. However, with the complexity of human disease that may lead to the in vivo inefficiency of drug treatments, the development of biologically functional nanocarriers at the intact animal level, and ultimately in patients, is crucial. There have been many attempts to functionalize and design nanocarriers so as to increase their capabilities. Unfortunately, the criteria of a successful nanocarrier are vast, and few nanocarriers have shown exceptional clinical potential. Inspired by the unique properties of mesoporous silica nanoparticles (MSNP), our review expands on the versatility and flexibility of MSNP and how its platform can be used for therapeutic purposes.

1. Introduction

Due to the rapid growth in nanotechnology, the study of drug delivery and disease treatment has been revolutionized. By molding nanomaterials into vesicles, numerous nanocarriers have been developed to securely deliver drugs and various other therapeutic agents specifically into targeted sites (Fig. 1). Many of the conventional nano drug delivery systems (DDS) (e.g. liposomes, micelles, and polymer-based) have reached the later stages of development, and a few have even received FDA approval (Table 1). Over the last two decades, the development of synthesis and characterization techniques has blossomed for engineered nanomaterials, including the ability to manipulate molecules and supramolecular structures for beneficial functions. This has led to the emergence of new DDS, such as inorganic nano delivery systems, for therapeutic and/or diagnosis purposes.^1–4 Compared to the conventional DDS, most inorganic-based DDS (e.g. mesoporous silica nanoparticles, MSNP) are still in their pre-clinical stages of development, with a few exceptions. The inorganic DDS which have reached the furthest stages of clinical trials are gold nanoparticles (GNP) used in drug delivery and hyperthermia-based treatments. These are discussed further in later sections. Through the use of both conventional and new DDS, one expects to combat the major issues in drug delivery: (1) unfavorable pharmacokinetics and biodistribution which lead to unwarranted side-effects (e.g. chemotherapy), (2) premature drug degradation in the blood, and (3) inefficient uptake at targeted sites that leads to low drug efficacy. Another important development is that nanocarriers have become highly integrated and multifunctional so as to include a range of applications such as on-demand release, specific tissue/cell type targeting, in vivo imaging and diagnosis, and photothermal treatment.^1–4

Fig. 1 The scheme shows the leading nanocarriers for drug delivery and their general stages of development. The top row shows the representative conventional nanocarriers such as liposomes, micelles, dendrimers, and polymers. The bottom row shows novel inorganic nanocarriers such as carbon nanotubes, quantum dots, iron oxide, gold, and mesoporous silica nanoparticles.

Table 1 Representative drug delivery systems on the market or in clinical trial

Nanocarrier platform	Delivered therapeutic agent	Stage in development	Drug name	Indications
Liposome	Doxorubicin	Approved	Doxil	Kaposi's sarcoma, recurrent breast cancer, and ovarian cancer
	Vincristine	Approved	OncoTCS	Non-Hodgkin's lymphoma
	Daunoxome	Approved	Daunorubicin	Kaposi's sarcoma
Polymeric micelle	Paclitaxel	Phase II clinical trial	Genexol-PM	Non-small-cell lung cancer (NSCLC)
Albumin (protein–drug conjugate)	Paclitaxel	Approved	Abraxane	Metastic breast cancer
Gold	Recombinant human tumor necrosis factor (rhTNF)	Phase II clinical trial	AurImmune™	Pancreatic cancer, melanoma, soft tissue sarcoma, ovarian, and breast cancer

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The ultimate goal of nano DDS is to generate therapeutically and clinically useful formulations for treating diseases in patients. While actively studied in academia, the search for optimal nanocarriers is still underway and the subject demands innovation and ingenuity in order to translate them into human trials. For a nanocarrier to be successful, it should ideally meet the following criteria: (1) be composed of biocompatible/biodegradable/bioexcretable material, (2) high drug and cargo loading capacity, (3) a site-specific delivery mechanism to spare normal cells and tissue, (4) zero premature drug release, and (5) a controlled release mechanism to provide an effective dose to the target site.⁵ To date, there is a limited amount of nanocarriers which can achieve such an accomplishment.

Although there are only a few FDA-approved nanoparticle based nanomedicines in the clinic (Table 1), these novel designs are already impacting medicine and have the potential to change conventional treatment and/or diagnosis. While most inorganic DDS remain in the preclinical stage, the successful demonstrations of MSNP's efficacy as well as the high biocompatibility and safety profile at the cellular and intact animal level are extremely encouraging from the perspective of moving this platform into clinical trials. Moreover, MSNP's platform can support additional design features that will make it possible to obtain controlled drug release and perform theranostics. This review paper will touch upon the advantages of various leading nanocarriers and discuss the challenges impeding their progress. The main focus will be placed upon the emerging role of MSNP as a multifunctional nanocarrier and how its platform can overcome the major issues that nanocarriers may encounter.

2. Nanocarriers and the ongoing challenges

2.1. Conventional nanocarriers

In the arms race to create the ideal nanocarrier, a great deal of effort has been spent on organic, or “soft”, carriers due to their innate biocompatibility and biodegradability. The majority of these delivery systems are made from conventional biomaterial, such as lipids and polymers, and synthesized to produce carriers in the nanometer size range. One of the most prominent of these are liposomes, lipid-based carriers comprised of a lipid bilayer that forms into an amphiphilic spherical complex (Fig. 1).^1,2,6 Liposomal nanocarriers have proven to be capable of encapsulating drugs and delivering them into targeted sites. For example, they have been very promising in terms of chemotherapeutic drug delivery to fight cancer since lipid-based nanocarriers are composed of phospholipids which can readily diffuse through cancer cell membranes and intracellularly deliver a concentrated dose of drugs.⁶ Moreover, since liposomes are comprised of phospholipids, they are inherently biocompatible and can be easily degraded, making them a good system from the safety perspective. Thus, liposomal drug complexes such as Doxil and Daunoxome have been approved for the administration of chemotherapeutic drugs doxorubicin and daunorubicin, respectively.² Another prominent “soft” carrier that has shared the spotlight with the lipid-based nanocarriers is the polymer-based nanocarrier.^2,4,7 Similar to the aforementioned liposome nanocarriers, polymer-based nanocarriers are composed of organic material that is generally biocompatible and biodegradable. Accordingly, poly(lactic-co-glycolic acid) (PLGA), one of the leading polymer-based nanocarriers, has been approved by the FDA to act as a therapeutic device.^3,7 Although conventional nanocarriers have reached clinical trials and FDA approval, many of these platforms still face pressing challenges. For one, the instability of liposomes under physiological conditions results in noticeable leakage of cargo in vivo due to the stringent conditions within the blood that weaken the molecular interactions between the phospholipid components.⁷ Other major drawbacks include lack of control over drug release rate and lack of means to overcome biological barriers (e.g. skin, blood–brain barrier, etc.).⁸ Recently, dendrimer platforms, composed of numerously branched macromolecules such as polyamidoamine, have been more thoroughly investigated as soft nanocarriers for their ease of additional modification to reap functional benefits (e.g. targeting molecules and imaging agents).¹ Although encouraging, dendrimer platforms are more costly than other nanoparticles and require more steps for synthesis which poses a challenge for large-scale production. Despite the challenges faced by conventional particles, they are still the most commonly explored nanocarriers and will be viewed as having high clinical potential due to their innocuous nature.

2.2. Inorganic nanocarriers

With the increasing search for more robust nanocarriers with novel functionalities, researchers have begun looking into inorganic material. Inorganic nanocarriers entail a number of characteristics that make them suitable for drug delivery, including robust and durable core material that allows them to more securely encapsulate their cargo compared to conventional nanocarriers; a wide range of functionalities such as addition of targeting ligands; and largely available material which cuts down production cost.^2–4 Moreover, nanocarriers made of inorganic materials posses unique, intrinsic properties such as imaging modalities and photothermal capabilities that are not normally seen in lipid and polymer-based nanoparticles.^4,9–11 Gold nanoparticles (GNP) are currently the most successful inorganic nanocarriers with one platform, CYT-6091 (also called AurImmune™), in the second stage of clinical trials. CYT-6091 consists of colloidal GNP conjugated to tumor necrosis factor (TNF), a cytokine which has been shown to have remarkable anti-tumorigenic effects.^12,13 Human clinical trials revealed that administration of TNF alone led to exceedingly toxic side-effects, primarily as a result of the limited biodistribution to the actual tumor site. CYT-6091 GNP were able to accumulate in the affected area by taking advantage of the leaky neovasculature seen in solid tumors, resulting in the sequestration of TNF in the tumors and no toxic side-effects as seen in independent TNF treatment.^12,13 Besides being used solely for drug delivery, GNP have also demonstrated photothermal capabilities which can be used for hypothermia-based treatments of tumors.^13–15 After administration of GNP, near infrared light (NIR) can be used to harmlessly penetrate the tissue and activate the GNP which will transform the light energy into thermal energy and kill nearby surrounding tumor tissue via a photothermal-ablation process. This GNP platform has also reached clinical trials for treating head and neck cancer.¹⁵ Another example is carbon nanotubes (CNT), which have been exploited as drug nanocarriers, especially in oncology, due to their tunable surface as well as for their use in photothermal ablation of nearby cancer cells.^9,16 CNT, which are made of graphene sheets rolled into seamless cylinders, have large surface areas which can been functionalized to include a variety of therapeutic molecules such as anti-cancer drugs (e.g. paclitaxel), small interfering RNA (siRNA), and targeting proteins. Quantum dots (QD) and iron oxide nanoparticles are two other classes of inorganic nanocarriers which have received attention for their constituent imaging abilities.^11,17 The advantage of using QD and iron oxide over conventional nanocarriers is their intrinsic ability to act as imaging agents for diagnostic roles and monitoring of drug delivery. QD have fascinating optical properties which enable them to act as fluorescent probes for imaging and detection.¹¹ Likewise, iron oxide nanoparticles have become increasingly potent contrasting agents for magnetic resonance (MR) due to their paramagnetic properties and have been used for cancer detection.¹⁷

Although inorganic nanocarriers show such rich diversity in function, their biodegradability and biocompatibility properties may be a key question that needs further investigation before the usage in clinic. For instance, QD which are usually composed of cytotoxic heavy metals (e.g. Cd), have been limited in their translation towards clinical application due to their heavy metal dissolution which has been linked to toxicity.¹¹ Recent studies on primates showed no major biochemical or histological abnormalities after 90 days of QD administration.¹⁸ However, chemical analysis revealed that most of the heavy metals remained in the liver, spleen and kidneys after 90 days, suggesting that the breakdown and clearance of QD is relatively slow which could lead to bioaccumulation-induced systemic toxicity. Similarly, raw CNT have also been impeded in their clinical advancements due to studies revealing that the unmodified CNT can induce inflammation, fibrosis, and biochemical/toxicological changes in the lungs.¹⁹ This toxicity could be largely reduced by appropriate surface modification and/or dispersal improvement. It is notable to mention that there are a few inorganic nanocarriers (i.e. MSNP and GNP) which show little to no toxicity. GNP, especially, have more clinical success than other inorganic nanocarriers due to the inertness of gold. Many successful GNP platforms have demonstrated minimal toxicity both in vitro and in vivo.²⁰ Although MSNP have not yet reached the clinical stages of GNP, their exceptionally low toxicities and delivery capabilities show great promise for future translational studies. The following sections will be dedicated to discussing MSNP as a highly promising inorganic nanocarrier system and how it satisfies the desired criteria mentioned earlier to overcome the major challenges faced by DDS.

3. Emergence of mesoporous silica nanoparticles: a multifunctional system

3.1. Why mesoporous silica nanoparticles?

The nascence of mesoporous silica nanoparticles (MSNP) can be traced back when the mobile crystalline material-41 (MCM-41) was successfully synthesized using a cohydrolysis method.^21,22 In the last ten years, MSNP-related bio-application has become one of the most attractive areas in nanobiotechnology and nanomedicine for various disease diagnosis and/or therapy, including bone/tendon tissue engineering, infectious diseases, diabetes, inflammation, and cancer.²³ MSNP have been successfully developed as a multifunctional platform to deliver therapeutic/diagnostic agents (e.g., drug, siRNA, imaging probe, etc.) in studies involving a variety of cell types and animal models.^23–29 When compared to the properties of conventional and other inorganic nanocarriers, MSNP have emerged as intermediary nanocarriers in the sense that they possess similar biocompatibility as conventional nanocarriers as well as the durability and versatility of inorganic nanocarriers. The intrinsically low toxicity of MSNP differentiates it from many other inorganic nanomaterials including other forms of silica-based nanomaterials such as fumed silica and nano quartz that are associated with poor biocompatibility and toxicity due to their highly reactive surface. In fact, we have recently demonstrated that the intrinsic safety of Stober and mesoporous silica, which are produced under low temperature synthesis conditions, is due to the absence of high energy, strained 2 or 3-member siloxane rings, which are frequently present in high temperature silica types (e.g., quartz and fumed silica) and lead to toxicity due to surface reconstruction that opens these rings to display vicinal silanols. In vitro studies have demonstrated MSNP to have zero or very low cytotoxic effects in various healthy and cancer cell lines.²⁴ Abiotic studies also support MSNP having good biodegradability by showing the gradual decomposition of MSNP in simulated body fluids at 37 °C.³⁰ In experiments involving mice, MSNP were shown to be biocompatible, biodegradable, and bioexcretable.^24,30–32 The mice data also demonstrated the safety of using MSNP as a nanocarrier, resulting in over 90% of injected MSNP bolus in mice being found in urine and feces.³³ In another animal experiment, mice treated with 120 mg kg⁻¹ of free MSNP on a weekly basis for three weeks via intravenous injection showed no difference in their histology and blood analysis when compared to normal mice, signifying the biological safety of using MSNP.³⁰

In regards to therapeutic capabilities, MSNP have been shown to be exceptional nanocarriers for a wide variety of drugs and biomolecules both in vitro and in vivo.^{22–24,30,33,34} To name a few, MSNP have been shown to successfully deliver small interfering RNA for gene knockdown, plasmid DNA for transfection, and many anticancer agents such as the hydrophobic drug camptothecin (CPT), paclitaxel and the popular hydrophilic chemotherapeutic agent, doxorubicin.^24,30,33,34 Although both the biocompatibility and delivery abilities of MSNP are notable, what makes MSNP distinguished from other carriers is its flexible platform that endows the system with a range of functionalities and modifications. Lastly, MSNP show great promise in terms of practicality. The advancements in sol–gel chemistry and surfactant-templated synthesis have made the production of MSNP relatively easy, with few simple purification procedures.^26,29,35 With these new synthetic processes and the abundance of silica precursors, MSNP can be produced at low costs and in large quantities.

3.2. Size and shape tunability

As mentioned previously, MSNP have been greatly advanced due to the rapid development of sol–gel chemistry and surfactant-templated synthesis.^24,26,29,36 Briefly, the sol–gel synthetic process begins with organosilane precursors such as tetramethylorthosilicate (TMOS) and tetraethoxysilane (TEOS) which undergo hydrolysis and condensation reactions, leading to the formation of a new phase (sol) (a and b).

(a) Hydrolysis: OH

⁻

+ Si(OR)

₄

→ Si(OR)

₃

OH + RO

⁻

(b) Condensation: SiO⁻ + Si(OH)₄ → Si–O–Si + OH⁻

The particles within the sol then condense into the gel phase. The major process used for synthesizing MSNP relies on surfactant templates. Initially, a silicate source is mixed with a surfactant such as cetyltrimethylammonium bromide (CTAB) in a heated aqueous solution under basic conditions. Once the nanoparticle is formed, the template is removed resulting in a silica particle consisting of a regular arrangement of hexagonal array of pores.²⁴

These advances in sol–gel and surfactant-templated synthesis of porous materials have led to the creation of MSNP in a variety of sizes and shapes optimized for drug delivery. This flexibility in nanocarrier structure is a commodity not frequently seen in other particle types. Early cellular studies demonstrated that the abundance of cellular uptake is governed by particle size in different cell types, and it was demonstrated that MSNP of ∼50 nm in diameter may be optimal in terms of cellular uptake.^23,37 Since most nanocarriers are particulates, they are recognized and effectively removed by the phagocytic cells in the reticuloendothelial system (RES).³⁸ At the intact animal level, particle accumulation in the RES organs (e.g., spleen and liver) has shown to increase monotonically with greater particle diameter in studies using particles ranging from 700 nm to 3 μm.^23,38 In a tumor-bearing animal model, experimental evidences also showed that the nanoparticle size (primary particle size and hydrodynamic size) could lead to considerable variation in the magnitude of the enhanced permeability and retention (EPR) effect, which is a phenomenon that increases a particle's tendency to accumulate in tumor sites due to the abnormally large fenestrations of tumor vasculature and inefficient lymphatic drainage in tumor tissue.^1–3,38,39 Different primary sizes of MSNP can be achieved by deliberately tuning the conditions during the synthesis, such as silica precursor concentration, temperature, and stirring speed. Another approach for synthesizing smaller particles (e.g. ∼50 nm primary size) involves using a co-templating agent method for particle synthesis. This involves addition of an optimal amount of Pluronic F127 to CTAB (the standard surfactant used for most MSNP synthesis). The Pluronic F127 changes the structure of the CTAB micelles, which affects their micelle packing behavior and therefore leads to a smaller particle size. Moreover, Pluronic F127 also improves the dispersion of the hydrophobic silica precursor, TEOS, and coats and stabilizes newly formed small MSNP, helping to protect particles from agglomeration and oligomerization.³⁰ In addition, the pores within MSNP can be adjusted through various templating methods to ensure a high volume for drug entrapment.^24,40 Recently, hollow MSNP have also been synthesized to increase pore volume for greater drug loading capacities.⁴¹

In addition to primary size and pore size, findings have shown that shape also plays a decisive role in a carrier's success.^38,40,42 In terms of particle circulation in blood and bioavailability, nanoparticles moving along with the blood flow may collide with the blood vessel walls if the particle's internal momentum force is greater than the surrounding fluid force when a directional change in the vessel occurs. The collisions increase the likelihood of interactions with macrophages, and thereby decrease the bioavailability of the cargo-laden nanocarriers.^37,43 Long aspect ratio DDS, such as rod-shaped particles, possess higher stability of fluid forces owing to their aerodynamic and hydrodynamics properties resulting from their unique shape.^37,43 In this regard, rod-shaped MSNP were successfully synthesized and tested.^40,42 For example, MSNP of sphere and rod shape with aspect ratios ranging from 1–4 were made to demonstrate the effect of particle shape on cellular uptake.⁴² MSNP with aspect ratios of 2.1–2.5 showed more rapid cellular uptake via small GTPase-dependent macropinocytosis with larger quantities compared to shorter and longer rod-shaped MSNP.⁴² Consequently, MSNP synthesized in this optimal aspect ratio were able to deliver a more toxic dose of camptothecin and paclitaxel to cancer cells than MSNP synthesized with aspect ratios of 1 (sphere), ∼1.5 (shorter rod), and ∼4 (longer rod).⁴² Through the use of MSNP library containing sphere-shaped and different rod-shaped particles (aspect ratio 1–10), another experiment showed that the aspect ratio of MSNP not only governs the rate and abundance of particle uptake, but also cell proliferation, apoptosis, cytoskeleton formation, adhesion and migration in A375 human melanoma cells.⁴⁰ Recently, the same group further demonstrated that the in vivo biodistribution and clearance were also dependent on the aspect ratio of MSNP. At 2 hours post-intravenous injection, short rods (aspect ratio = 1.5) were mainly biodistributed in the liver, while long rods (aspect ratio = 5) were predominately biodistributed in the spleen.⁴⁴

These findings have placed nanocarrier design in a whole new perspective. By controlling the physical parameters of the particles, nanocarriers can be tuned to optimize delivery for specific biological problems. In this sense, organic nanocarriers are relatively limited; they are mainly composed of a spherical core structure, and cannot easily be physically tailored to fit specific delivery needs. In comparison, due to the rapid developments of sol–gel and template-mediated particle synthesis techniques, MSNP of varying size and aspect ratio can be synthesized to address issues of loading capacity, particle blood circulatory time, and cellular uptake.

3.3. Surface functionalization

Another key design feature which MSNP possess is the ability to modify their exterior as well as interior surfaces. Nano-surface modification has proven to be beneficial for improving nanocarrier drug delivery and provides a range of functionalities.³ One of the key characteristics attributing MSNP with extensive functionalization capabilities is their high surface area-to-volume ratio as well as their porous structure.^24,35 Secondly, advancements in co-condensation and post-synthetic methods have also made it possible to introduce a myriad of organic molecules to the silanol groups on the exterior surface of MSNP through covalent or electrostatic interactions. In one example, polyethyleneimine (PEI) polymers were attached to the surfaces of MSNP to improve nucleic acid delivery.³⁴ The silanol surface was first grafted with negatively charged phosphonate groups which could then electrostatically bind to cationic PEI polymers. This PEI moiety established a net positive charge on the particle, and thus permitted the MSNP to securely adhere to negatively charged small interfering RNA (siRNA) and achieve gene knockdown in cancer cell lines.³⁴ Another example shows that the addition of polyethylene glycol (PEG) polymers to the silica surface allows the nanocarrier to reside in the blood and escape from RES uptake for a more extensive period of time, and therefore improve its delivering capabilities.⁴⁵ The surface of MSNP has also been shown to support complex functionalities such as PEI–PEG copolymers.³⁰ To achieve this, MSNP were first coated with cationic PEI polymers, in which a portion of these PEI polymers were then covalently attached to PEG. Accordingly, the MSNP gained both the steric hindrance effect from the PEG as well as the electrostatic repulsion effect from the PEI polymers, resulting in enhanced particle dispersion in physiological solutions and decrease macrophage uptake.³⁰ Furthermore, the versatile MSNP surface can also support active targeting vectors to increase the specificity of drug delivery and reduce damage in normal tissues. Active tumor targeting was achieved by conjugating antibodies to MSNP. For example, MSNP surfaces were functionalized with anti-HER2/neu mAb (monoclonal antibody), a growth factor receptor overexpressed in breast cancer (Fig. 2a).⁴⁶ The inclusion of anti-HER2/neu mAb enhanced the selectivity of the MSNP to the targeted cancer cells and improved cellular uptake via receptor-mediated endocytosis. Since the polymer modifications on the exterior leave the interior pore accessible, the MSNP maintain their cargo carrying capabilities. Recent research has revealed that the interior can also be functionalized to accommodate for specific cargo molecules. The interior pores have been modified with a variety of organic molecular structures and have been encapsulated with drugs, nucleic acids, and proteins for therapeutic purposes.^24,47–49 These interior alterations have shown to have a large effect on drug loading and delivery capabilities. For example, the water-soluble drug, doxorubicin (Dox), was efficiently packaged into MSNP via an electrostatic mechanism. To test the effects of various interior functionalities, Dox was loaded in MSNP decorated with OH, COOH, phosphonate, or NH₂ groups. The loading capacities of OH–, COOH–, and phosphonate–MSNP were 1.2%, 4.2%, and 8.4% (w/w), respectively. This contrasts with amine-decorated particles that showed low <0.1% (w/w) drug binding capacity.⁵⁰ While the NH₂ modification exhibited a lower loading capacity, this modification proved to be useful in increasing the release rate of positively charged drug molecules that electrostatically bind to the silica surface. This principle was successfully used in a MSNP-based nanovalve system where rapid drug release is desired for a concentrated dose delivery.⁵¹

Fig. 2 Multifunctional platforms of MSNP and unique functionalities. (a) MSNP conjugated to anti-HER2 mAb demonstrates specific targeting to breast cancers and improved cellular uptake via receptor-mediated endocytosis.⁴⁶ (b) MSNP loaded with doxorubicin and capped with pH-sensitive nanovalves show no release under neutral conditions, but efficient drug release once entering the acidic endosomal compartments of cancer cells.⁵¹ (c) Drug resistance can be overcome by MSNP carrier that simultaneously delivers Pgp siRNA and doxorubicin (Dox) in MDR cancer cells. The synergistic killing effects were achieved by efficient drug delivery as well as successful knockdown of Pgp, a drug efflux pump, and therefore an increased cellular concentration in MDR cells.⁵⁰ (d) MSNP-coated gold nanorods loaded with cancer drug demonstrate a synergistic killing effect on A549 lung cancer cells through nanoparticle-mediated drug and hyperthermia-based treatment.⁵⁶ (e) Through the design of multifunctional MSNP that contain cancer drug and superparamagnetic iron oxide nanocrystals encapsulated inside MSNP that also carried fluorescent tag, targeting group and hydrophilic modification capable of preventing aggregation, drug delivery, tumor targeting, magnetic resonance and fluorescence imaging, magnetic manipulation, and cell targeting can be simultaneously achieved in the same particle.⁵⁵ In the animal model, MSNP incorporated with iron oxide are capable of magnetic resonance imaging (MRI) in vivo.⁵⁹

3.4. Controlled drug release

One other major advantage of using MSNP as a drug delivery system is a controlled cargo release mechanism.²⁹ Although nanocarriers such as PLGA have been stated to have a controlled release of drugs through steady surface erosion, stimuli-mediated and on-demand release systems are required if one wants to achieve concentrated dosage release to targeted sites and prevent premature cargo leakage.¹ The pores of MSNP have shown to be securely capped by a variety of supramolecular complexes, also known as “nanovalves”, that can be triggered for release by an autonomously occurring intracellular event (e.g. pH change or presence of enzyme) or by an external stimulus (e.g. light, heat, or magnetic field).^24,51,52 Notably, incorporating nanovalves composed of an immobilized stalk and a cyclic molecule as a capping agent onto MSNP have demonstrated remarkable lysosomal pH-responsive controlled drug delivery capabilities (Fig. 2b). In this study, pH-sensitive nanovalves composed of aromatic amines as the stalk and β-cyclodextrin as the cap covered the pores of MSNP and prevented doxorubicin drug content from being prematurely leaked.⁵¹ Under normal physiological conditions (pH of 7.4), the stalk securely binds to the β-cyclodextrin; however, once the particle entered the cancer cells and were incorporated into lysosomes, the acidic endosomal compartments (pH < 6) weakened the stalk and cap interaction, allowing doxorubicin to release from the pores and induce potent cytotoxicity within the cancer cells. Besides these autonomous nanovalves, external stimuli-responsive nanovalves have been synthesized in the MSNP system to achieve more precise controlled release, even after the particles are added into the biological systems. One such case involves MSNP containing doxorubicin as well as iron oxide nanocrystals, capped with heat-sensitive nanovalves.⁵² Usage of an oscillating magnetic field, which serves as the non-invasive external stimuli, is able to heat the iron oxide nanocrystals and thus open the valve and control the subsequent drug release.⁵² Not only was this nanovalve-MSNP platform able to release a concentrated dosage high enough to induce apoptosis to cancer cells, but it also securely encapsulated the drug until stimulated for release at the target site, and therefore theoretically reducing inadvertent damage to normal tissue.⁵²

Although these examples demonstrate the drug entrapment and controlled drug-release capabilities of MSNP incorporated with nanovalves, there are numerous other supramolecular complexes which can be used to enhance MSNP as drug carriers.²⁹ These structures include nanoimpellers which can actively expel the cargo after excitation by light at 400 nm–450 nm, and bistable rotaxanes that release the entrapped cargo when the tetrathiofulvalene (TTF) component is reduced with mild ascorbic acid.²⁴ Inability to encapsulate drug cargos while circulating the blood can lead to systemic toxicity and also inefficient dose delivery.

3.5. Multifunctional MSNP

With such vast functionalities that can be incorporated onto the MSNP surface and interior, researchers have been prompted to fabricate a custom designed multifunctional system using MSNP (Fig. 2). Although most nanocarriers are used primarily for single drug delivery, MSNP have the potential to deliver various drugs and biomolecules at once.^{28,34,50,53–57} It has been recently demonstrated that MSNP can be used as a carrier for siRNA in order to silence cancerous genes responsible for the multiple drug resistance (MDR); a phenomena that renders modern chemotherapy treatment ineffective to resilient cancers.^34,50,53 Due to the instability of siRNA and rapid degradation by ribonucleases within the blood, MSNP surface was first grafted with a negative charge which could electrostatically bind to cationic polymers of appreciable molecular weight (e.g. PEI or dendrimers), giving the particle a net positive charge, and thus permitting the MSNP to securely bind to negatively charged nucleic acids without inducing pronounced cytotoxicity. By attaching the siRNA molecules on the exterior surface, the interior pores were left accessible for drug entrapment. Following experiments demonstrated that the MSNP system can be used to successfully co-deliver siRNA targeting MDR genes (e.g. P-glycoprotein and Bcl-2) together with cancer drugs, resulting in the silencing of Pgp (drug exporter gene) or Bcl-2 (anti-apoptosis gene) and accordingly restoring doxorubicin sensitivity and inducing MDR cancer cell death (Fig. 2c).^50,53

MSNP design has also begun including diagnostics and therapeutic agents simultaneously. To achieve this, imaging probes were placed within MSNP in coherence with drugs or other therapeutic agents.^31,55,56 Imaging modalities have the potential to play an important theranostic role in medicine since they permit the monitoring of drug delivery efficiency as well as the visualization of affected areas. One such multifunctional system incorporated RGD peptides, fluorophores, and gadolinium compound into a MSNP platform.⁵⁴ When put to the test, this multifunctional MSNP was capable of targeting specific cells through the RGD peptides, which bind to integrins abundantly expressed in many cancers, in addition to optical and magnetic resonance imaging (MRI) resulting from the unique properties of the fluorophores and gadolinium chelates, respectively.⁵⁴ In other MSNP multifunctional studies, MSNP-coated gold nanorods were used as a light-mediated multifunctional theranostic carrier for cancer treatment.⁵⁶ The incorporation of gold nanorods is a promising platform for cancer theranostics due to tunable localized surface plasmon resonance (LSPR) which allows the particles to act as probes for cancer cell imaging.⁵⁸ In addition, the LSPR property brings the possibility of photothermal treatment. After loading these particles with doxorubicin and administering them to A549 human lung cells, exposing the affected area to NIR light causes the gold nanorods to release a relatively large amount of thermal energy to nearby cancer cells. The delivery of MSNP-coated gold nanorods loaded with doxorubicin demonstrated an enhanced cancer cell killing effect through a synergistic effect of chemotherapy and hyperthermia-based treatment (Fig. 2d).⁵⁶ Moreover, one MSNP multifunctional platform has also been shown to encapsulate cancer drugs, superparamagnetic iron oxide nanocrystals, fluorescent tags, as well as targeting groups on the surface. With such functionalities, this multifunctional platform achieved capabilities of drug delivery, magnetic resonance, fluorescence imaging, and cell targeting simultaneously (Fig. 2e).⁵⁵

4. Conclusion

The development of nanocarriers has profoundly affected drug delivery. However, more effort and innovation is needed in order for them to reach clinical implementation. This usually involves an iterative design approach by which the engineered nanomaterials are custom-designed for the task, followed by abiotic/in vitro/in vivo testings and optimization processes. Only after thorough planning, assessment, and necessary redesigns of the carrier have been performed, can a pharmaceutically adapted candidate be created, as demonstrated by the examples in Fig. 3. Major challenges faced by leading nanocarriers today include premature cargo leakage, unwanted RES organ capture, biodistribution, and biocompatibility. In addition, complex diseases such as cancer require engineered nano systems which can specifically target affected tissues and deliver a toxic dose of drugs or other therapeutics. Recently, MSNP have emerged as a promising inorganic nanocarrier platform due to its biocompatibility as well as its versatility. MSNP have been demonstrated to support a range of polymer and biomolecule conjugations, as well as many design features which endow it with a list of beneficial functionalities such as active and passive tissue targeting, imaging for theranostic purposes, and hyperthermia-based treatments. Furthermore, in vitro and in vivo studies have demonstrated the safety of MSNP, revealing that the particles are biocompatible and can be biodegraded or excreted. The safety data as well as the establishment of the link between physicochemical properties of MSNP system to biological profiles (toxicity and pharmaceutical activity) allow us to design safe and functional nano carriers that are now moving the nano safety to consideration of proactive strategies at the design and development stage of new products. Although more investigation is required before implementation of MSNP as drug carriers, the MSNP platform shows high potential for therapeutic uses, especially for oncology and should be strongly considered for ultimate application in clinic.

Fig. 3 Implementation of iterative design to improve in vivo tumor biodistribution for MSNP anticancer drug delivery system. Three generations of MSNP types (NP1 to NP3) were synthesized with different parameters. Biodistribution of NIR dye-labeled MSNP in the tumor xenograft model were studied in nude mice after intravenous administration. To visualize cancer growth, cancer cells that stably express luciferase gene were used to grow xenograft. Animal images were obtained following the particle injection. NP1 was captured by the liver and spleen. NP2 showed less prominent liver uptake, with a sustained systemic distribution (longer circulation time), indicating the ability of PEG coating to decrease particle opsonization and removal by the RES. A similar reduction in RES uptake and increased circulation time were obtained for NP3, which showed prominent particle uptake in the tumor tissue at 24 h, suggestive of a strong EPR effect when expressed as a percentage of the total mass of the particles administered, <1%, 2%, and 12% of NP1, NP2, and NP3 load. Tumor-bearing mice received intravenous administration of doxorubicin-loaded particles. Dual color fluorescence to show the tumor localization of doxorubicin in relation to the tumor blood vessels detected by a CD31 biomarker. The smaller particles (50 nm) with PEI–PEG copolymers that resulted in monodispersed particle suspension under physiological conditions were able to accumulate in tumors via the improved EPR effect with higher efficacy. This resulted in greater delivery of the cancer drug Dox and therefore significantly higher tumor inhibition abilities in vivo. Doxorubicin delivery by the optimized MSNP carrier (NP3) reduces systemic toxicity and side effects compared to the free drug.³⁰

Acknowledgements

We thank the funding support by the U.S. Public Health Service Grant RO1 CA133697. We thank Dr Zhaoxia Ji at the UC Center for Environmental Implications of Nanotechnology (UC CEIN) for providing us insightful suggestions for this manuscript.

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