Biodegradable and Renal Clearable Inorganic Nanoparticles

Personalized treatment plans for cancer therapy have been at the forefront of oncology research for many years. With the advent of many novel nanoplatforms, this goal is closer to realization today than ever before. Inorganic nanoparticles hold immense potential in the field of nano‐oncology, but have considerable toxicity concerns that have limited their translation to date. In this review, an overview of emerging biologically safe inorganic nanoplatforms is provided, along with considerations of the challenges that need to be overcome for cancer theranostics with inorganic nanoparticles to become a reality. The clinical and preclinical studies of both biodegradable and renal clearable inorganic nanoparticles are discussed, along with their implications.


Toxicity Concerns of Inorganic Nanoplatforms
Inorganic nanoplatforms face larger challenges in the process to clinical translation when compared to organic systems. Most of these nanoparticles are made from materials that are wellcharacterized in their bulk state. However, the nanoscale of these structures creates additional properties that must be considered when analyzing their toxicity. [ 12,22 ] The same properties that give nanoplatforms their promise in medicine may also be the most crucial to consider in toxicological evaluations.
For example, traditional cytotoxicity assays may not be proper techniques for nanoparticles, as the particles themselves Personalized treatment plans for cancer therapy have been at the forefront of oncology research for many years. With the advent of many novel nanoplatforms, this goal is closer to realization today than ever before. Inorganic nanoparticles hold immense potential in the fi eld of nano-oncology, but have considerable toxicity concerns that have limited their translation to date. In this review, an overview of emerging biologically safe inorganic nanoplatforms is provided, along with considerations of the challenges that need to be overcome for cancer theranostics with inorganic nanoparticles to become a reality. The clinical and preclinical studies of both biodegradable and renal clearable inorganic nanoparticles are discussed, along with their implications.

Introduction
With one in four deaths in the United States being attributed to cancer, the diagnosis and treatment of cancer is one of the main focuses of the biomedical world. Great progress has been made in the last few decades, with overall death rates declining by 20% since 1991. [ 1 ] However, with over 500 000 deaths due to cancer in 2014 alone, there is still an enormous need for the development of better cancer treatments today. Personalized medicine has recently come to the forefront of cancer therapy discussions, representing a paradigm shift from generalized treatments to ones that are developed specifi cally for an individual's cancer thumbprint. By targeting the molecular characteristics of each unique cancer, a treatment regimen can ideally be developed to allow for a maximized therapeutic index. However, challenges exist in the development of these biomarkertargeted agents. [ 2 ] The fi eld of nanotechnology holds great promise in cancer imaging and therapy, as novel nanoplatforms for biomedical have been found to chemically reduce the dyes (e.g. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)) used in classical assays, resulting in inaccurate toxicity determinations. [ 23 ] The majority of existing toxicological data results from in vitro studies, but will need to be validated with in vivo experimentation for a reasonably long time period. [ 24 ] Most importantly, a standardized method of toxicity determination for nanoplatforms is necessary to facilitate further clinical translation. Indeed, the scientifi c community as a whole has recognized the need for nanoplatform safety standards, initiating the implementation of databases for techniques of nanotechnology characterization and measurement. [ 25 ] One recent study evaluated the toxicity of phospholipid micelle-encapsulated CdSe/CdS/ZnS quantum dots (QDs) in rhesus macaques. [ 8 ] Results showed that after ninety days blood and biochemical markers were within normal ranges, indicating that the immediate effects of as-studied QDs are not the primary concern-the longer-lasting impacts are crucial. The majority of the QDs were found to be retained in the RES even after the ninety-day trial. These long-term effects of nanoparticles within the body are perhaps the most fundamental areas to be studied. Since the FDA has declared that all injected imaging agents must be cleared in a reasonable amount of time, engineering of nanomaterials to allow for biodegradation or renal clearance is crucial to avoid longterm retention. [ 10 ] However, this goal is counteracted by the fact that nanoparticles are often inherently stable, even maintaining their fl uorescent abilities after two years in vivo in a QD-related study. [ 26 ] Many techniques and novel nanoplatforms have been explored in the last few years for biologically safe theranostic nanomedicine, resulting in two categories of promising inorganic nanoplatforms: biodegradable larger sized nanoparticles and renal clearable ultra-small sized nanoparticles ( Scheme 1 ).

Iron Oxide Nanoparticles
Iron oxide nanoparticles (IONPs) hold great promise in widespread medical applications as magnetic resonance imaging (MRI) contrast agents and in drug delivery, hyperthermia, and immunoassays. [ 27 ] When compared to QDs which contain heavy metals, iron-based nanoparticles are known to be less toxic given that iron ions (the biodegradation byproduct of IONPs) are essential as a trace element and the iron transport pathways and regulation of iron homeostasis are quite well understood. [ 28 ] Preclinical studies indicated that iron levels in rats returned to normal values within three weeks or more after intravenous administration of oleic acid-pluronic-coated iron oxide nanoparticles. [ 29 ] Research further showed that cirrhosis and hepatocellular carcinoma were not evident until the liver iron concentration reached over ten times the natural level, which was far beyond any required dose. [ 30 ] Since the human body naturally contains a substantial level of iron, the small concentration of free iron that results from the degradation of iron oxide nanoparticles is not thought to have biologically detrimental impacts. Ferumoxides (Endorem in Europe, Feridex in the USA, particle size: 120-180 nm) and ferucarbotran (Resovist, particle size: ≈60 nm) are two negative magnetic resonance contrast agents based on iron oxide nanoparticles that were previously approved for clinical use. [ 31,32 ] Most current clinical trials related to IONPs are focusing on the anemia drug ferumoxytol (Feraheme, AMAG Pharmaceuticals), which is an iron replacement product containing non-stoichiometric magnetite coated with polyglucose sorbitol carboxymethylether (particle size range 17-31 nm). [ 33 ] In March 2015, the FDA ordered stricter warnings and contraindications for Feraheme, stating that potentially fatal allergic reactions can occur with this drug in patients who have had an allergic reaction to any intravenous iron replacement product. [ 34 ] Besides larger sized iron oxide nanoparticles, ultra-small superparamagnetic iron oxide nanoparticles have also been developed as an alternative magnetic resonance contrast agent for patients at risk for nephrogenic systemic fi brosis. [ 35 ] By combing high-resolution MRI with 2-3 nm sized lymphotropic IONPs, successful detection of small and otherwise undetectable lymph-node metastases in patients with prostate cancer has also been achieved. [ 36 ] Although iron oxide nanoparticles with different sizes have been approved for use in the past ( Table 1 ), they are no longer available (due to the toxicity concerns) with the exception of the oral iron oxide contrast agent, Lumirem/Gastromark (approved by the FDA in 1996).

Porous Silicon and Silica Nanoparticles
Like iron, silicon is a trace element within the human body. Porous silicon nanoparticles have been formulated to be biodegraded into renal clearable components while effectively evading the RES ( Figure 1 ). [ 9 ] The intact particles themselves are on the scale of 130-180 nm, which would traditionally be eliminated through the RES. However, by degrading  Ultra-small superparamagnetic iron oxide nanoparticles (Clariscan) MRI contrast agent Discontinued due to safety concerns [59] Aminosilane-coated iron oxide nanoparticles (NanoTherm) Thermal therapy Phase 1 [60] Luminescent porous silicon nanoparticles Imaging and drug delivery Preclinical studies [9] Mesoporous silica nanoparticles Imaging and drug delivery Preclinical studies [7,[41][42][43] Ultra-small quantum dots Imaging Preclinical studies [53,54,61] Ultra-small gold nanoparticles Imaging Preclinical studies [6,48,50] Ultra-small fl uorescent silica nanoparticles Image-guided surgery Investigational New Drug approved (FDA) [4,5] into water-soluble silicic acid (i.e. Si(OH) 4 ) (Figure 1 a,b), the injected luminescent porous silicon nanoparticles (LPSiNPs) were found to clear noticeably within a week (Figure 1 c). In vivo near-infrared (NIR) imaging further confi rmed the clearance of LPSiNPs into the bladder at 1 h post injection (Figure 1 d). As developed LPSiNPs have been further demonstrated to effectively carry drug payloads, provide NIR luminescence, and self-destruct into the subparts that demonstrate very minor toxicity. Although solid evidence has been provided to support the degradation of LPSiNPs while circulating in the blood stream, a reliable imaging technique that can be used for visualizing the degradation process is currently unavailable. Engineering of LPSiNPs with tunable and controllable in vivo biodegradation rate is vital to prevent the self-destruction of LPSiNPs before delivering the drug payloads to the target of interest. Silica (or silicon dioxide) is "generally recognized as safe" by the FDA (ID Code: 14808-60-7), [ 37 ] which is highly desirable for future clinical translation. [ 4,5 ] Biocompatible mesoporous silica nanoparticles (MSNs) have been accepted as one of the most attractive inorganic drug delivery systems due to their high specifi c surface area and pore volume. [ 38,39 ] Very recently, a dendritic mesoporous silica structure with tunable pore size (ranging from 2-13 nm) was developed, and showed a faster biodegradation rate (byproduct: silicic acid) in simulated body fl uid for MSN with a larger pore size (≈10 nm, Figure 2 ). [ 40 ] In another study, mesoporous organosilica nanoparticles (MONs) with a large pore size (8-13 nm) were developed by using a micelle/precursor co-templating assembly strategy. [ 41 ] Hollow MONs with up to fi ve different organic hybridizations were further created for high intensity focused ultrasoundresponsive drug-release by the same research group. [ 42 ] Asdeveloped HMONs maintained signifi cant biocompatibility and demonstrated tunable biodegradation rates. Although radiolabeled-and antibody-conjugated MSN (or hollow MSN) with a smaller pore size range (2-3 nm) have been developed for in vivo tumor vasculature targeted imaging and drug delivery, [ 7,43 ] engineering of biodegradable MSN (with a larger pore size and controllable degradation rate) for in vivo tumor targeted imaging and therapy has not been achieved yet.   Biodegradation into nontoxic subparts is an ideal property for injected inorganic nanoparticle based drug delivery systems, as long-term toxicity of the carriers would be of less concern. However, the carriers also need to be stable enough in vivo to allow for suffi cient tumor targeting to be achieved. Remote stimuli (such as magnetic fi eld, light, heat or ultrasound) responsive drug delivery is another approach for reducing the potential toxicity, and has attracted increasing interest in the research community. [ 44 ] Although organic-based or hybrid nanoplatforms, such as dendron-virus complexes [ 45 ] and magnetoferritin, [ 46 ] have been developed for controlled assembly (or disassembly), delivery and release of compounds in vivo, the engineering of inorganic nanoplatforms that can be stimulated disassembled is still a huge challenge due to their rigid and crystallized nanostructure.

Quantum Dots
Ultra-small inorganic nanoparticles (less than 10 nm) together with suitable surface modifi cations can naturally be cleared through the renal system after administration. [ 47 ] Poly(ethylene glycol) (PEG) and ziwitterionic ligand coating are two of the widely used surface modifi cation techniques for creating renal clearable nanoparticles with a neutral surface charge. [ 10,48 ] Since the pioneer work of renal clearable QDs in 2007, [ 10 ] other inorganic nanoparticles such as ultra-small fl uorescent silica nanoparticles (C dots), [ 4,5,49 ] glutathione-coated gold nanoparticles, [ 6,50 ] and carbon nanotubes [ 51 ] have also been developed. So far, imaging or image-guided surgery are the major applications for these renal clearable inorganic nanoparticles.
QDs have found their way into preclinical biomedical applications as fl uorescent labels. [ 52 ] Their unique size-tunable light emission/absorption properties and high quantum yield make QDs one of the best inorganic nanoparticles for optical imaging, if they can be effectively concentrated within the region of interest. QDs with a fi nal hydrodynamic (HD) size of 5.5 nm were developed that allowed for effi cient renal clearance in preclinical studies ( Figure 3 a,b). [ 10 ] Many approaches have been taken to generate QDs that are both actively and passively targeted to specifi c biological targets. For example, maleimideactivated QDs can be conjugated to the thiols of reduced antibodies and ligands to generate targeted nanoparticles. [ 53 ] Due to the fast clearance rate (within hours), careful design considerations (particle size, surface charge, number of targeting ligands, etc.) need to be taken into account to generate renal clearable QDs that can still actively target to specifi c biological targets in vivo (Figure 3 c, d). [ 11 ] Recent advances in developing self-illuminating QDs by conjugating with Renilla reniformis [ 54 ] also make it possible to create renal clearable and self-illuminating QDs for future deep-tissue, multiplexed, in vivo imaging. Since QDs are traditionally made of heavy metals (such as cadmium), toxicity concerns have greatly limited their clinical translation, and so far no fi rst-in-human clinical trials have been approved by the FDA.

C dots
Ultra-small dye encapsulated fl uorescence silica nanoparticles, known as C dots (or "Cornell dots"), found their way into clinical  . Reproduced with permission. [ 10 ] Copyright 2007, Nature Publishing Group. c) A schematic illustration of ultra-small QDs functionalized with targeting ligands and NIR) fl uorophores. d) In vivo NIR fl uorescence imaging of GPI (a ligand which targets to prostate-specifi c membrane antigen [PSMA]) and 800-CW functionalized QDs (or QD-CW-GPI) in PSMA positive (LNCaP) and negative (PC-3) tumor models. Reproduced with permission. [ 11 ] Copyright 2010, Nature Publishing Group. trials in January 2011 (NCT01266096, NCT02106598). [ 4,5 ] These ultra-small dense silica nanostructures are modifi ed with near NIR dyes, cRGD, and radioisotopes, allowing for multimodality targeted imaging of cancer ( Figure 4 a). [ 4 ] With HD sizes of 6-8 nm, C dots have suffi ciently long in vivo blood circulation time (t 1/2 = 190 min) to allow for optical imaging to be feasible throughout an entire surgery procedure. [ 49 ] PEG surface modification changes the highly negative charged surface to be neutral, which prevents C dots from forming large aggregates when circulating in the blood, and allows them to be effi ciently cleared through the renal system. [ 49 ] The NIR emission of the encapsulated dyes allows for minimized signal attenuation and autofl uorescence from surrounding tissue, resulting in improved signal-to-noise ratio and a maximal contrast per administered dose. Iodine-124 (t 1/2 = 4.18 d) was also conjugated to the surface of the C dots for positron emission tomography (PET) verifi cation of the optical images (Figure 4 b). Initial results of clinical trials in patients with metastatic melanoma indicate no adverse effects due to the nanoparticles. [ 4 ] A more detailed follow-up clinical trial focused on the safety, pharmacokinetics, clearance properties, and radiation dosimetry of PET-optical hybrid 124 I-cRGDY-PEG-C dots (Figure 4 b). [ 5 ] Preliminary clinical trials showed that C dots could not only signifi cantly improve target-to-background ratio (when compared with free dyes), but also showed higher sensitivity and specifi city (when compared with 18 F-fl uorodeoxyglucose [ 18 F-FDG]), [ 5 ] indicating great potential for safe use of these particles in human cancer diagnostics in the near future.

Ultra-Small Gold Nanoparticles
Fluorescent ultra-small gold nanoparticles (AuNPs) with efficient renal clearance capability have also been developed recently. [ 6,48,55,56 ] Glutathione coated 2-3 nm sized AuNPs were chelator-free labeled with gold-198 ( 198 Au) to form Glutathione (GS)-[ 198 Au]AuNPs ( Figure 5 a,b). Over 50% of the particles were found to be excreted in the urine within 24 h, by tracking with single-photon emission computed tomography (SPECT) and optical imaging (Figure 5 c). [ 50 ] In a follow-up study, PEGylated AuNPs (PEG molecular weight: 1 kDa) were demonstrated to have three times higher passive targeting effi cacy when compared with GS-AuNPs ( Figure 5 d-f). [ 48 ] Although tumor necrosis factor bound colloidal gold nanoparticles have been approved for a phase I clinical trial (NCT00356980, NCT00436410), [ 57 ] and silica-Au nanoshell (also known as Auroshell) also gained approval in a clinical pilot thermal treatment for patients with refractory and/or recurrent tumors of the head and neck (NCT00848042, NCT01679470), the clinical translation of ultra-small renal clearable AuNPs is still in the early stages.

Challenges and Future Directions
Individualized treatment has become a goal of oncology research in recent years. Inorganic nanoparticles with biodegradable and renal clearable properties could have an optimistic outlook in future nano-oncology by offering safer and multifunctional nanosystems for both cancer diagnosis and therapy. Table 1 further provides a collection of representative inorganic nanoparticles that have been used in clinical and preclinical studies.
Toxicity concerns will continue to be one of the largest obstacles during the clinical translation of inorganic nanoparticles. A great deal of preclinical work has yet to be done in determining the best method for characterizing and reducing toxic effects. For inorganic nanoparticles that are mainly composed of metals that are foreign to the human body, renal clearance has been demonstrated to be the most practical means for evading undesirable toxic effects. Sophisticated control of particle size and surface coating is vital, since these characteristics determine the pharmacokinetics and biodegradation properties of these nanoparticles. Although a number of renally-clearable particles have been developed to date, [ 6,10,11,48,50 ] C dots are the only renal clearable inorganic nanoparticles that have gained Investigational New Drug approval by the FDA for fi rst-in-human clinical trials. [ 4,5 ] One of the major challenges facing renal clearable inorganic nanoparticles is the specifi c accumulation in tumor tissues in vivo. The fast clearance in vivo through the renal system indeed is benefi cial for reducing the toxicity concerns, but in vivo active targeting to tumor tissue needs to be further explored for  I-cRGDY-PEG-C dots revealed probe activity in bladder (*), heart (yellow arrow), and bowel (white arrowhead). Reproduced with permission. [ 5 ] Copyright 2014, American Association for the Advancement of Science.
their application to be viable. [ 11,55 ] We believe the use of these renal-clearable nanoparticles has the potential to revolutionize future cancer detection, image-guided surgery and treatment provided that a satisfi ed balance between in vivo targeting and renal clearance can be achieved. Biodegradable porous silicon (or silica) based nanoparticles may hold greater potential for future clinical translation when compared with other inorganic drug delivery systems considering the successful clinical translation of C dots, [ 4,5 ] the well-established surface modifi cation chemistry, [ 58 ] the demonstrated tumor targeted drug delivery capabilities, [ 7,43 ] and their potential in alleviating the long-term RES accumulation concerns.

Conclusions
In conclusion, the clinical and preclinical studies of biodegradable and renal clearable inorganic nanoparticles are explored in this short review article. Although toxicity concerns still remain as the major barriers to further translation of inorganic nanoparticles from bench to the bedside, progress has been made in demonstrating the reduced toxicity effects by engineering biodegradable and renal clearable nanoparticles while maintaining their desirable imaging or therapeutic functionalities. Personalized treatments are not so much a dream as a real possibility with the great advances that have been made in the fi eld of nano-oncology. We believe inorganic nanoparticles with biodegradable and renal clearable properties could hold great potential to revolutionize future cancer diagnosis and therapy.