Review pubs.acs.org/ac
Theranostics in the Growing Field of Personalized Medicine: An Analytical Chemistry Perspective Niall Crawley and Michael Thompson* Department of Chemistry and Institute for Biomaterials and Biomedical Engineering, University of Toronto, 80 St. George Street, Toronto, Ontario M5 S 3H6, Canada
Alexander Romaschin
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Keenan Research Centre and Clinical Biochemistry, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada “cure”, often involving a drug or treatment that would exhibit near complete effectiveness against that condition.1 Despite the prevalence of this traditional approach, there has been significant recognition that some diseases are not conducive to this strategy. Many of the most troubling diseases of our time are heterogeneous in their expression.1 So much so, that our best efforts at treating them result in therapies that are effective for only a certain subpopulation of the afflicted. Furthermore, the complexity of the progression of these diseases limits our ability to produce treatment options that are viable in more than one stage of disease development.2 For example, when a patient is diagnosed with cancer, the cancer is phenotyped, the heterogeneity of the tumor is assessed, and the cancer is identified to be in a certain stage of development.3−5 The treatment regimen selected for the patient is specific to the way the cancer has manifested itself and the stage it is in, such that two patients with the same cancer may be undergoing very different treatment programs.6 The more medical science understands diseases like cancer and HIV, the more it becomes apparent that the “one size fits all” approach to treatment, though it may have been effective for some diseases in the past, will not be effective against these, the most devastating diseases of our time.7 Out of this came the call for new, more specific and individualized methods of treatment.2 Is there an effective and economical way to offer personalized treatment? As clinical diagnostic tools provided the detailed specifics of a patient’s condition, it was thought that combining a therapeutic aspect into diagnosis might produce personalized treatment protocols and may improve prognoses versus standard treatment.8 This growing area became known as “theranostics”, a portmanteau of therapeutics and diagnostics coined by Funkhouser in 2002.9 By his definition, Funkhouser intended the word “theranostic” to be used as an adjective describing “a material that combines the modalities of therapy and diagnostic imaging”. This is the root of one of the two main definitions of the term. Theranostics is defined as the combination of diagnostic and therapeutic agents/capabilities on a single platform such that both modalities are delivered,10 and the disease can be treated
CONTENTS
Theranostic Nanoparticles Molecular Imaging Modalities Used in Nanoparticle-Based Theranostics Magnetic Resonance Imaging Computed Tomography Single Photon Emission Computed Tomography Positron Emission Tomography Ultrasound Optical Techniques Theranostic Nanoparticle Platforms Vesicles Micelles Drug Conjugates and Complexes Dendrimers Microbubbles Carbon Nanotubes Example Core−Shell Nanoparticles Other Theranostic Processes Radiation Therapy Photothermal Ablation/Hyperthermia Photodynamic Therapy The New Theranostics: Where Does the Analytical Chemist Fit in? Inline Sensing Optical Sensing Electrochemical Sensing Acoustic Wave Detection Existing Devices Similar to the TCD The Future Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References
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ntil recent times, the standard protocol in medicine has been to diagnose a disease or disorder and then employ a form of therapy to treat it. Therefore, medical research has focused on characterizing a type of disease, followed by a © 2013 American Chemical Society
Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2014 Published: December 6, 2013 130
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agents, or a specific ratio of both.10 Ideally, the contrast agents would allow monitoring of the treatment process even in circulation on passage to the target site. Nanoparticles may be coated with drug molecules, or they can be loaded internally.10 The surface can be functionalized with targeting moieties, such as a substrate for a receptor that is known to be overexpressed in a certain type of cancer.17 Targeting ligands may be combined so that one ligand may allow the nanoparticle passage into the cell by endocytosis, and the other then targets an internal receptor before drug release.13 The surface may also be functionalized with immuno-cloaking substances such as polyethylene glycol (PEG).6 At present, there are only a few nanoparticle-based theranostic platforms involved in clinical trials.18,19 Despite being a very popular area of research and the most popular form of theranostics, no medical nanoparticle product is in widespread use.6 There are still many concerns that need to be addressed before theranostic nanoparticles can be brought to market including cost of materials and production costs, innate toxicity of the nanoparticle components and the safety of new formulations, long-term health effects, formulation stability, discovering new biomarkers to target in different diseases, and issues with intellectual property rights.20−22 This section will review the most successful theranostic nanoparticle platforms, including a description of the common imaging modalities used to monitor therapy that will precede the discussion of the types of nanoparticles. The reader is encouraged to contemplate the possibility (or lack thereof) of using these imaging techniques in an inline system arrangement. The nanoparticle formulations reviewed include vesicles, micelles, drug conjugates and complexes, dendrimers, microbubbles, carbon nanotubes, and core−shell nanoparticles (iron oxide, inorganic quantum dots, gold, and silica). Nanoparticlebased radiation therapy, photothermal ablation (PTA) or hyperthermia, and photodynamic therapy (PDT) will be discussed in brief. Molecular Imaging Modalities Used in NanoparticleBased Theranostics. Imaging techniques used for medical purposes typically require the use of a contrast agent that is injected into, or on occasion ingested by, the subject and is allowed to collect in target tissues. The contrast agents can be functionalized with targeting moieties themselves for active targeting, or they can be allowed to circulate and collect on the basis of the physical properties of the vascularization of the target tissue for passive targeting.23 In most theranostic applications, contrast agents are attached to functionalized nanoparticles that may have targeting moieties. There are currently no techniques that allow visualization of the contrast agents as they circulate.13 They are allowed to collect before the phenomenon (magnetic field with radiofrequency pulses, X-ray or γ radiation, etc.) used for imaging is activated. These are very useful techniques that can probe biological systems down to subcellular levels. The way these imaging modalities are employed today provides a snapshot of what is occurring within a patient. The diagnosis is made on the basis of a static image, and monitoring of treatment is made with a series of static images.24 With inline sensing systems, it could be possible to monitor treatment in real-time with a dynamic technique such as CCD video. These techniques will be discussed later in this paper. The techniques presented in this section are the main imaging modalities used in collaboration with nanoparticles. They are the principle imaging techniques of theranostics and
and monitored, simultaneously. This is the definition that will be adhered to in this Review. The other more broad definition is simply the use of an appropriate diagnostic measure to personalize a separate therapeutic intervention.11 This second definition was applied to most of the early developments deemed “theranostic” in the literature. Typically, in biology and the medical application of genomics, proteomics, and metabolomics, diagnostics involving polymerase chain reaction (PCR) were used to generate very specific, individual diagnoses of conditions like genetic disorders. Then, nucleic acid or gene therapies with virulent agents were used to treat the disorder.12 With the emergence of nanotechnology, it was evident that chemists, physicists, and engineers alike were directing their attention to applying theranostic properties to new nanomaterials. The two fields go together very well, so well in fact, that there was a boom in the literature beginning in 2010 with papers on nanoparticles for theranostic applications. The heavy majority of the papers written between 2002 and 2012 in the field of theranostics was written after 2010 and concerns itself with the use of nanoparticles. This Review will briefly cover the most prevalent nanoparticles and their imaging systems. The purpose of this article is more than a review of theranostics and nanoparticles. It is our intention to also pose questions and discuss the challenges of the future of this field. We attempt to show how, in our opinion, the analytical chemist can be featured prominently in the upcoming successes of this field. This is a “hot” area of research, and there is much that the analytical chemist can contribute moving forward. We will also introduce a new subfield based on the first definition of theranostics above. Instrumental or macro-theranostics describes the development of macro-scale instruments capable of both diagnostics and therapy. In particular, we introduce theranostic circuit devices (TCDs) that consist of extracorporeal circulation of the blood and the use of an inline imaging system with a therapeutic cartridge component to clean and remove harmful molecules or cells from the bloodstream.
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THERANOSTIC NANOPARTICLES Nanoparticles can be made of many different materials including metals and metal oxides, carbon, synthetic polymers, peptides and proteins, and lipids. In some cases, a bubble can act as a nanoparticle.10 The size scale of the particles is an obvious factor in common, but there are other characteristics that most nanoparticles share. To be a theranostic nanoparticle, the particle must be multifunctional in terms of carrying a therapeutic agent, as well as carrying a contrast agent for imaging. The particle itself may perform a therapeutic function and/or it could be made of a material that is already conducive to imaging.13 The size of the particles allows for renal clearance, and this can help improve circulation times in the blood pool.14 In fact, the plasma circulation time of a nanoparticle can be tuned by adjusting different physicochemical properties such as the surface functionalization of the particle.10 Also, many tumors have irregular, leaky, dilated blood vessels that allow a particle of nanosize to pass into and collect in the tumor due to poor drainage of the tissue by the lymph system.15 This is known as the enhanced permeability and retention effect (EPR).15,16 Nanoparticles generally have high surface area-to-volume ratios, and so, their surfaces can be loaded with different moieties to bestow a variety of chemical properties upon the particle.6 The surface can be loaded with a drug, contrast 131
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Figure 1. An example of each of the imaging modalities used with nanoparticle-based theranostics. Accompanied by an image of a mouse indicative of the capabilities of each instrument. [Reprinted with permission from Janib, S. M. et al. Adv. Drug Delivery Rev. 2010, 62, 1052−1063 (ref 10). Copyright 2010 Elsevier.] (a) MRI [Reprinted with permission from Ventura, A. et al. Nature 2007, 445, 661−665 (ref 206). Copyright 2007 Macmillan Publishers Ltd.]; (b) CT [Reprinted with permission from Baiker, M. et al. Med. Image Anal. 2010, 14, 723−737 (ref 207). Copyright 2010 Elsevier.]; (c) PET [Reprinted with permission from Radu, C. G. et al. Nat. Med. 2008, 14, 783−788 (ref 208). Copyright 2008 Macmillan Publishers Ltd.]; (d) SPECT [Reprinted with permission from Fu, D.-X. et al. Nat. Med. 2008, 14, 1118−1122 (ref 33). Copyright 2008 Macmillan Publishers Ltd.]; (e) Optical imaging [Reprinted with permission from Le, L. P. et al. Gene Ther. 2006, 13, 389−399 (ref 209). Copyright 2006 Macmillan Publishers Ltd.]; (f) Ultrasound [Reprinted with permission from Snyder, C. S. et al. BMC Cancer 2009, 9, 106 (ref 210). Copyright 2009 VisualSonics.].
the anatomy. It is favorable that MRI does not use radiation for imaging as CT, SPECT, and PET do.26,27 This being said, MRI is very expensive and it cannot be used on patients with metalbased surgical devices such as pacemakers, reconstructive elements, and implants. The biggest disadvantage of MRI for theranostics and nanoparticles is its low sensitivity. MRI can detect concentrations as low as 10−3−10−5 M, but at its best, this sensitivity is 4 orders of magnitude worse than the next closest technique sensitivity of the ones in this Review.26,27 The problem is that, to improve sensitivity, MRI requires very high concentrations of contrast agent. At present, these agents are too toxic at the concentration levels required due to accumulation in the body and slow clearance.10 Considering MRI as a potential inline sensing method, there are too many issues for it to be effective. The costs involved, the size scale of the instrument, and the low sensitivity (relying on contrast agents) are the biggest limiting factors. Computed Tomography. An X-ray attenuation coefficient represents a numerical representation of how well a beam of Xray radiation will pass through an object.28 On the chemical level, elements with high atomic numbers have many electrons and so they have high electron densities. The higher the electron density, the more difficult it will be for an X-ray to pass through as the X-ray loses energy upon collision with an electron that can absorb its energy. Biological components like bone, muscle, fat, water, and air will have differing elemental compositions, and thus, they represent different environments of different attenuation coefficients for a penetrating beam of Xray radiation.28 On the basis of the absorption of X-rays on their way through the tissues, an image is produced mapping tissue densities proportional to attenuation. The X-ray source is
are the most heavily researched for theranostic applications at present. These techniques are magnetic resonance imaging (MRI), computed tomography (CT), single photon emission computed tomography (SPECT), positron emission tomography (PET), ultrasound, and optical/fluorescence imaging (Figure 1). Magnetic Resonance Imaging. When an external magnetic field is applied over a biological system, the hydrogen nuclei of water will form a low energy spin-aligned ground state. By applying radiofrequency pulses, the hydrogen nuclei are perturbed and absorb energy to become spin-unaligned. As they relax to the aligned state, the nuclei emit the energy they absorbed. The local environment around the nuclei will alter their relaxation time. It is these differing relaxation processes that alter the energy emitted, which is detected and utilized to form an image.25 Contrast agents used in MRI are designed to shorten the time required for the water hydrogen nuclei to return to the aligned state. There are different relaxation parameters known as T1 and T2, and a contrast agent will usually affect only one of these parameters. For example, gadolinium- or manganesebased contrast agents that are paramagnetic will affect the T1 relaxation parameter, while iron or iron oxide-based agents that are superparamagnetic will affect the T2 relaxation parameter.25 One of the most well developed types of nanoparticles is the core−shell nanoparticle made of superparamagnetic iron oxide (SPIOs). SPIOs are often imaged with MRI because of the inherent relaxation properties of the nanoparticle itself.10 MRI has the best spatial resolution in comparison to the other imaging techniques mentioned. It can resolve images down to about 25−100 μm, and it can map detailed aspects of 132
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section of tissue.32 The reason two cameras are employed is that this configuration can collect images in 360 degrees twice as fast as one camera. By scanning the whole body, the crosssectional, two-dimensional data can be combined to form a three-dimensional image.31 SPECT is frequently compared to PET because the two techniques are similar in many ways. There are strong points to both techniques. Both gamma scintigraphy techniques are quantitative, which cannot be said of MRI and optical techniques, and they both have very little background interference and require virtually no signal amplification due to the use of γ radiation.31 Nothing else in the body except the radionuclide is emitting γ radiation, and the radiation is at such high energy, relative to other forms of radiation, that the signal is distinct and clear when it is detected. An advantage of SPECT is that it is less expensive than PET because it is more widely used and because the radionuclides used for SPECT are easier to prepare and have longer half-lives than those of PET.31 Also, SPECT can detect multiple radionuclides at once enabling different tissues to be imaged and studied simultaneously. SPECT has a good level of sensitivity relative to the optical techniques at about 10−10− 10−11 M, but this is an order of magnitude less sensitive than PET.26,27 However, both techniques are still sensitive enough to provide information down to the subcellular level. A disadvantage of SPECT is that it has low spatial resolution compared to the other techniques discussed. It can resolve 1−2 mm at best. This is also true of its sister-technique PET.26,27 Also, the gamma scintigraphy techniques are subject to concerns about the use of radiation as an imaging method and the costs of operation. The SPECT and PET techniques use γ radiation, which is much more energetic and damaging than X-ray radiation, and like all the techniques discussed to this point, the costs of operation are high, though SPECT and PET are slightly more affordable than MRI or CT.26,27 (PET is extremely expensive due to the need for a cyclotron to produce the isotope of interest, and then, the short half-life of the label requires immediate synthesis and injection.) In theranostic terms, the gamma scintigraphy techniques can be used with any nanoparticle that can carry a radionuclide. The radionuclides are the contrast agents attached to a nanoparticle carrying a drug load to a tissue of interest via a targeting moiety. As with most imaging techniques in theranostics at present, the gamma scintigraphy techniques are used to locate the nanoparticles within the body and to see whether or not the targeting method has succeeded.10 An advantage of using SPECT imaging moving toward an inline theranostic system is that SPECT instrumentation has been made fairly small. In a hospital setting, a SPECT machine can be quite large when used for full body scans. SPECT is also available in a unit small enough to stand in the corner of a room. It is not benchtop, but it is fairly small.33 This is an important feature considering that a theranostic circuit device should be about the size of a dialysis machine and must incorporate some form of inline sensing. This will be discussed further in the second half of this paper. Positron Emission Tomography. The main difference between PET and SPECT is that in PET the radionuclide emits a positron that travels a very short distance before annihilating an electron to produce two γ rays.34 It is these γ rays that leave the site of the positron−electron annihilation event in opposite directions, which are detected by two gamma cameras. The trajectories should theoretically be 180 degrees
rotated around the body and can produce a three-dimensional map of tissue density with great detail.10 CT scans have very good spatial resolution of about 50−200 μm; they are good at differentiating between tissues (even soft tissue), and even though this technique requires X-ray radiation, the amount used and the type of radiation is less harmful than that that of SPECT or PET.26,27 The sensitivity of CT has not been studied extensively enough to have a number value associated with it at present. The main issues with CT are its high cost, the fact that it uses radiation, and the nonspecificity of some tissue types which can only be enhanced by high concentrations of contrast agents.26,27 For the most part, CT contrast agents have low molecular weights and can be cleared rapidly.29 To enhance tissue specificity, core−shell nanoparticles and liposomes have been researched because these can hold high concentrations of electron dense elements like iodine in the form of iodinated polymers or molecules, they can remain in the blood for longer periods of time, and they are eliminated without releasing the high concentration of contrast agents within.29 Because they can remain in the blood for longer periods than contrast agents alone, nanoparticles have been considered for use in vascular CT including nanoparticles loaded with iodine, bismuth or gold, and gold-based core−shell nanoparticles alone.30 CT is a good imaging technique for theranostics at present, but in an inline system, CT would be hindered by its high cost and the size of the instrument, along with concerns over radiation. X-ray by itself may be a good imaging tool for inline sensing as the instrumentation required can be made relatively small. Radiation would be a concern, but handled appropriately, it would be as safe as at the hospital or in the lab. X-ray would provide snapshots of the blood or tissue being examined and could possibly be time-resolved. X-ray will have the appropriate sensitivity to differentiate between cells in the blood for example. However, the cost is high and this would limit development. Single Photon Emission Computed Tomography. SPECT is one of two commonly used forms of gamma scintigraphy; the other being PET. SPECT is more straightforward in concept as a camera that detects γ radiation directly is responsible for producing the image of the tissue of interest.31 The technique relies on the administration of a radionuclide, such as Ga-67, Tc-99m, or I-131 among many others, to the patient intravenously. The radionuclide could be left in soluble ionic form if the biodistribution properties of the radionuclide permit its collection in an area of interest. In many cases, the radionuclide is included in a larger radioligand that possesses chemical properties that will aid in directing the radionuclide to the appropriate tissue. An example is the attachment of F-18 to deoxyglucose to form fluorodeoxyglucose.32 Once the radionuclide has circulated and has collected in the tissues of interest, a gamma detector is rotated around the body. In medicine, it is common to see two gamma cameras, each covering 180 degrees around the body, rotate back and forth passing over the entire body.32 This would be employed for a test like a bone scan where the whole body is imaged. This instrument could also be used to scan selected areas. One camera can be used to scan a selected area as well. The cameras detect the location of the radioisotope within the section of the body being scanned on the basis of the area of the detector struck most by the emitted γ rays relative to the positioning of the detector over that section of the body. A rotating scan can pinpoint the location of the collected radioisotope in a cross133
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Figure 2. Structural representations of the general theranostic nanoparticle platforms reviewed. [Reprinted with permission from Janib, S. M. et al. Adv. Drug Delivery Rev. 2010, 62, 1052−1063 (ref 10). Copyright 2010 Elsevier.]
apart, but in practice, they are not exactly 180 degrees.34 Because the γ rays leave the event site at approximately the same time, the detectors are set with a specific time interval on the picosecond scale within which the γ rays should arrive. If one detector detects a gamma ray and the other does not detect one within this time frame, those γ rays are not from the event of interest and they are excluded from the computer analysis.35 Computer modeling of the approximate trajectories of the γ rays from the event allows backtracking of the trajectories to a common origin. This determines the position of the event and the position of the collection of radionuclide.35 The route of administration of radionuclide is the same for PET as for SPECT. In PET, the radionuclide has to undergo positron emission decay and so certain radionuclides like F-18, Cu-64, and In-111 are used.35 Any radionuclide that emits γ radiation one way or another can be detected by SPECT, but only those radionuclides that emit positrons first, before gamma emission, can be detected by PET. These radionuclides tend to have smaller half-lives and cost more to prepare.31 PET produces three-dimensional images directly, unlike SPECT that produces two-dimensional images first before combining them. PET has better sensitivity than SPECT by an order of magnitude and is one of the most sensitive techniques reviewed. It can detect radionuclide concentrations down to 10−11−10−12 M.26,27 Unfortunately, PET suffers from the same spatial resolution limitations as SPECT at 1−2 mm resolution, and it is more costly, uses γ radiation, and can only image one radionuclide at a time.36 In terms of the use of PET for inline sensing, it has also been scaled down to a size similar to the smallest version of SPECT. In the clinical setting, PET and CT have been paired for simultaneous use in order to corroborate data and fill in the gaps of one method with the advantages of the other.36 Again, issues of radiation and cost must be considered. However, the premise of combining different imaging techniques might be more effective in an inline experiment because of the complexity of inline scanning of a biological material like whole blood. Ruminating on the techniques discussed, it will take considerable effort and finance to make any of these techniques suitable for application in an inline sensing system. It is debatable as to whether or not these techniques are suitable for inline sensing at all. Ultrasound. Ultrasound imaging is accomplished by placing a transducer that produces high frequency sound waves of greater than 20 kHz on the skin over the area of interest. The sound waves pass into the body and eventually meet dense tissue that reflects the sound back to the transducer. The sound
from one area of the skin arrives at the transducer at a different time compared to another area. This depends on the spatial arrangement of the tissues relative to the surface of the skin and the transducer. The different sound relay times are used to produce an image of the underlying tissue. This image can be three-dimensional, and it can show movement in real time.26 For contrast with ultrasound, the contrast agent needs to change the acoustics of the tissue of interest. The nanoparticle used most often with ultrasound is the microbubble. The gas inside the microbubble can significantly alter acoustic properties.37 Ultrasound is considered low resolution with a spatial resolution of 50−500 μm.26,27 This is worse than MRI or CT but better than the gamma scintigraphy techniques and optical methods. The advantages of ultrasound are numerous. It is fast, simple, and effective. It does not use radiation, and it is completely noninvasive. The unit is small, portable, and costs very little compared to the other techniques reviewed.26,27 Ultrasound does have inline sensing capabilities, but its sensitivity and the effectiveness of its resolution would limit its use to detection on the large scale. Telling cells apart, for example, would be very difficult with ultrasound. A somewhat similar idea using acoustic waves produced by laser light known as photoacoustic flow cytometry is described in the optical techniques section. This new method could be used on the cellular scale. Optical Techniques. There are many optical imaging techniques available for medical and theranostic applications. In fact, this is the broadest category of imaging modality reviewed. In many ways, the subtechniques of this type of imaging are the most suitable for theranostic and, subsequently, inline sensing applications. Because this category is so vast, some general characteristics of optical imaging are presented herein with a more detailed analysis of individual techniques being more appropriate for the second half of this Review. Optical imaging includes absorption-, emission-, and scattering-based spectroscopic techniques. The common absorption techniques include UV, IR, and visible light spectroscopies where the amount of absorbance of light in these respective wavelength ranges is measured.10 The emission techniques rely on low-energy photon emission from the sample to produce an image. This can come in the form of bioluminescence or fluorescence from a probe or contrast agent. Quantum dots are often used for their tunable fluorescence properties and are one of the common nanoparticles used with optical imaging. The scattering techniques, including Raman spectroscopy, rely on the scattering of the 134
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liposome.42 Vesicles in the form of liposomes are well-studied, and they have been successful in the clinical setting. Polymersomes are newer in terms of their materials, but the vesicle concept is tested and proven. Vesicles are highly versatile in terms of both the types of moieties that can be encapsulated in and bound to the vesicle, as well as the type of bonding that can be used. Depending on the composition of the vesicle and the anticipated target environment, a vesicle can be tuned to encapsulate either hydrophobic or hydrophilic drugs or contrast agents.43 They can also be modified with covalent or noncovalent bonding, allowing for reversible interactions for drug release or irreversible covalent bonds for contrast agents.44 These special moieties can be bound to the surface of, held in the bilayer, or encapsulated within the vesicle. One example of a successful clinical liposome formulation is Doxil which is doxorubicin encapsulated in liposomes which are shielded by nonimmunogenic polyethylene glycol.45 This formulation improves the therapeutic index of doxorubicin by increasing tumor accumulation. It lengthens circulation times and improves safety versus the free version of the drug. There are other experimental formulations in clinical trials that have shown some useful qualities and benefits to the patient.46 Liposomes in general have a history of use with many different targeting moieties, drugs, and contrast agents. Also, when functionalized with many combinations of these three moieties, there is little loss of stability. They have been functionalized with iodine for CT, manganese and gadolinium for MRI, many radionuclides for PET or SPECT, and echogenic gas like sulfur hexafluoride for ultrasound.47−51 They have even been combined with quantum dots to allow optical imaging by fluorescence.47,48 Polymersomes are produced by the self-association of hydrophobic or hydrophilic blocks of diblock or triblock copolymers with different morphologies.52 For one example, diblock polymers may have alternating areas of hydrophilic and hydrophobic character. In an aqueous environment, the hydrophobic blocks will come together in a fashion similar to protein folding, and simultaneously, the hydrophilic blocks will associate, together forming a double layer. The hydrophobic blocks line the inside of the layer (or membrane), facing each other, and the hydrophilic blocks line the outside surface and the innermost surface of the vesicle. Thus, the inner core is an aqueous environment; the membrane layer is hydrophobic, and the outside environment is aqueous.53−55 The polymers utilized can be any diblock or triblock copolymer, for example, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polyethylene glycol, etc.52,56−58 It is also easy to control the synthesis parameters of a polymersome, allowing better control over thickness and the stability of the membranes compared to a similar liposome. This is a desirable property when considering how to enable drug release in vivo.10 Prashant et al. present an interesting example of a possible use for a polymersome.59 They describe using a polymersome made of polylactic acid and D-α-tocopherol polyethylene glycol 1000 succinate to enclose iron-oxide nanoparticles for MRI imaging. The main advantages were increased contrast agent density at the site of interest and reduced toxicity because the polymersome prevented the free iron-oxide from accumulating elsewhere in the body.59 The downside to vesicles is that they are difficult to produce because of complexity, and the materials used to make them are
incident light to varying degrees and in different directions based on the properties of the material that is irradiated.10 In comparison to the other techniques reviewed, optical methods are generally inexpensive, safe, and highly sensitive. These methods require less specialized equipment; there is no high energy radiation produced, and these methods can provide functional information from concentrations of 10−9−10−12 M for fluorescence and as low as 10−15−10−17 M for bioluminescence.26,27 These are some of the most sensitive techniques reviewed. The general disadvantages of optical methods include their low spatial resolution of 2−5 mm, issues of tissue penetration, and interference from surrounding tissues. Optical methods can only penetrate about 2 cm into tissue using light from the visible to near-infrared region of the electromagnetic radiation spectrum.38 In the UV and visible ranges, there are significant issues of background noise, autofluorescence, photobleaching, and tissue scattering. The use of infrared spectroscopy can be difficult in biological systems due to the strong IR absorbance of water. Near-infrared light of wavelengths of 700−900 nm does reduce many of the problems with the spectroscopy at the other wavelengths and can provide a greater depth of penetration.39 Theranostic Nanoparticle Platforms. This section will be a topical overview of the major theranostic nanoparticles that are currently being utilized in research and clinical applications (Figure 2). Nanoparticles in general have been studied for over fifty years. It is knowledge of these particles as drug delivery vehicles, combined with the more recent advances in nanotechnology and imaging, that has spurred the very recent boom in theranostic applications.40 At this point in time, there are a few well-established base nanoparticles. Some of these have been known for quite some time and are biomimetic by design like micelles and vesicles. Others are quite recent and have been studied extensively at a much more rapid pace like quantum dots and carbon nanotubes. Once a base nanoparticle is selected, the majority of the work is centered around designing ligands and multidentate polymers that will often carry a targeting moiety and the contrast agents for imaging.13 Sometimes, these ligands can carry the drug payload when the particle is solid like the core−shell variety, and in other cases, the drug can be loaded inside the particle. Almost all of the new research in this area is about the development of derivatives of these surface polymers and ligands. The physicochemical properties of these ligands are tuned to allow binding of a new drug or the binding of more contrast agent or to adhere more permanently to the nanoparticle.13 The recent literature is full of papers on the next adjustment or a better polymer or ligand. The main issue is that all of these ligands and polymers have to be bound to the surface of a nanoparticle in order to deliver them to the site where they are designed to work. The base nanoparticles have not improved significantly in the past few years, and there are still problems with clearance and toxicity that have not been adequately addressed. Indeed, the negative issues connected to toxixity have resulted in the foundation of a relatively new area of study: nanotoxicology. Vesicles. Vesicles for use as a theranostic nanoparticle platform can come in two forms: liposomes or polymersomes. Liposomes are similar to the vesicles found in nature where the outer surface is a phospholipid bilayer.41 Polymersomes are made up of synthetic polymer materials so that the final product has similar amphiphilic character to that of a 135
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antitumor drug, to chimeric polypeptide molecules using thiol chemistry. This allowed nanoparticle self-assembly from the smaller polypeptide molecules, and the formulation increased plasma circulation time and cellular internalization of the drug.67 There are many different synthetic polymers that have been studied for use as a drug conjugate. The most common and well-studied of these is N-(2-hydroxypropyl)methacrylamide or HPMA. HPMA is stable, nontoxic, and nonimmunogenic and can be functionalized with targeting moieties, contrast agents, and drugs by either covalent conjugation or copolymerization.68 Depending on the formulation, these polymer conjugates can aid in active or passive targeting, in vitro or in vivo imaging, decreasing toxicity of the drug, and improving circulation times.23 By the conjugation method, there have been HPMA polymer conjugates produced with doxorubicin and gemcitabine drugs and I-131 for imaging via the gamma scintigraphy techniques or CT. However, there are some problems with conjugation, as it is difficult to control the extent of conjugation and there can be bonding efficiency issues.69 By using copolymerization to functionalize a synthetic polymer, it is easier to have many more functional moieties together in the same polymer. For example, Wang et al. introduced an HPMA-based copolymer that featured an arginine−glycine−aspartic acid (RGD) recognition sequence for active targeting, gadolinium for MRI imaging, and In-111 for gamma scintigraphy. This polymer can utilize two imaging modalities to complement one another after the polymer has collected at the site of interest by active means.70 There is one major difference between drug conjugates and drug complexes. Drug conjugates utilize covalent bonding as a means to functionalize a polymer, whereas drug complexes use reversible interactions allowing drug to be released once the nanoparticle has reached the target site. These complexes can be synthetically made like almost all the conjugates, or they can be naturally occurring molecules.10 In fact, proteins and peptides are frequently used for complexation, and one of the only theranostic nanoparticle platforms available on the market, called Abraxane, is based on a natural protein.71 It relies on reversibly binding paclitaxel to albumin nanoparticles of about 130−150 nm in size. At equal doses, the nanoparticle complex proved more effective than the drug alone, while reducing toxicity, reducing unintentional activity in nontarget tissues, and improving circulation time of the drug because of the much higher molecular mass of the complex as opposed to the drug alone.71 Drug conjugates and complexes are limited by the complexity of their production and the safety of the materials used to synthesize them. The synthetic polymers can be toxic at the amount required for therapy.10 Dendrimers. Dendrimers are nanoparticles based on synthetic polymers. The identifying feature of a dendrimer is its shape. Initially, dendrimers are formed by standard polymerization reactions and begin as a planar molecule. As the polymerization reaction carries on through multiple generations, the planar molecules branch out and eventually become spherical and large. The polymer backbone and many side chains form a web-like structure within the sphere and on its surface. The polymers branch in many directions like the canopy of a tree. This has been described not only as a branched but also as a hyper-branched structure that yields many potential bonding sites for targeting moieties, contrast agents, drug molecules, and chelators. These can be bound
toxic. The latter is truer of polymersomes than liposomes, but liposomes can be toxic as well.10 Micelles. Micelles are well studied as drug and imaging agent carriers, and they have seen success in clinical applications. They have many advantages including easy formation, size uniformity, stability, synthesis from a variety of amphiphilic materials, increased solubility of hydrophobic molecules by encapsulation, and the ability to incorporate moieties for targeting, imaging, and drug delivery into the same nanoparticle.60 Polymeric micelles are made of block copolymers like vesicles, except micelles are formed by one layer where the hydrophobic blocks associate inside the micelle to form a hydrophobic core while the hydrophilic blocks associate to form the outer surface known as the corona. Polyethylene glycol or N-(2-hydroxypropyl)methacrylamide (HPMA) are often used as the hydrophilic block of the copolymer because, once in position, they form a nonimmunogenic corona imparting biocompatibility. Because of their single layer structure, in order to functionalize the micelle with hydrophobic molecules like certain drugs, these moieties have to be bonded to the polymer before micellar formation by covalent attachment directly to the polymer or covalent bonding to an anchor species or entrapped upon formation.61 There have been a number of drugs bound to the inner core of micellar nanoparticles. One example is Genexol-PM which has been part of a number of clinical trials for the treatment of different types of cancer. This formulation consists of paclitaxel encapsulated in a micelle made of monomethoxy poly(ethylene glycol)-block-poly(D,L-lactide).62 In terms of contrast agents, research has focused on developing polymers that can chelate potential imaging agents. Polymers like DTPA-stearylamine (SA) or DTPA-phosphatidylethanolamine (PE) are designed to have the hydrophobic tail group encapsulated in the hydrophobic core of the micelle, while the chelator becomes part of the corona.63,64 Polychelating amphiphilic polymers or PAPs are designed to anchor to the surface of the micelle and chelate multiple imaging agents. For the most part, the chelated species are radionuclides like In111 and Tc-99m for gamma scintigraphy or gadolinium for MRI.65 There have also been micelles made of polymers containing iodo-groups so that the membrane itself is laden with CT active agents. An example that summarizes this area quite well is the work of Trubetskoy et al. who used both DTPA-SA and DTPA-PE to chelate In-111, before being incorporated into the micellar layer of polymer polyethylene glycol−phosphatidylethanolamine. The In-111 allowed the imaging of the lymphatic system of a rabbit by gamma scintigraphy.66 One disadvantage of micelles is that their stability becomes questionable when they begin to disperse through the body. Their stability depends on the critical micelle concentration for the polymer utilized, and when the concentration of polymer in the environment drops below that concentration, synthetic micelles become susceptible to exchanging components with biological membranes.10 Drug Conjugates and Complexes. The main two types of drug conjugates are synthetic polypeptides and other synthetic polymers. Conjugates are made by chemical modifications to the polymer or to the drug. The drug is covalently bound to the carrier molecule, and as a result, the entire nanoparticle performs the therapeutic function.10 One example of a synthetic polypeptide conjugate from Dreher et al. is a polypeptide formed by covalently binding doxorubicin, an 136
dx.doi.org/10.1021/ac4038812 | Anal. Chem. 2014, 86, 130−160
Analytical Chemistry
Review
the bubbles passively aggregate and then coalesce at the tumor site. Using ultrasound, the bubbles act as the contrast agent, but the ultrasound also causes the bubbles to burst, which releases the doxorubicin.81 In a similar way, Lukianova-Hleb et al. showed that microbubbles can also physically destroy tumor cells. Using gold nanoparticles that are targeted and concentrate inside tumor tissue, it is possible to produce plasmonic nanobubbles with a laser pulse. The rapid expansion of the bubbles lyses the cell membranes of some of the tumor cells and can then be imaged by ultrasound. After imaging, another laser pulse bursts the bubble and causes further disruption in the tumor tissue.82 Carbon Nanotubes. Carbon nanotubes (CNTs) are small sheets of carbon in graphite-like arrangements that are rolled into hollow tubes. The tubes can consist of only a single layer forming a single wall, or other layers of carbon can be added to form multiwalled tubes that are more robust. The properties of CNTs make them very interesting in terms of their potential use in theranostic medicine. The properties of the CNT can be tuned by altering their size and the number of walls. For example, they can be synthesized to exhibit a high absorption coefficient and a broad excitation profile that covers a wide range of wavelengths.83 CNTs have not been studied to any great degree as a contrast nanoparticle with optical imaging techniques; however, the tube itself has shown potential as a contrast agent in photoacoustic imaging and especially nearinfrared and Raman spectroscopy.84 Strong absorption in the NIR range has enabled CNTs to be used for photothermal ablation. Gambhir et al. described detection of CNTs in vivo by NIR and Raman signals from subcutaneous tumors in mice.85 CNTs have also been functionalized with contrast agents like gadolinium for MRI and In-111 chelates for PET.86 One property that has been a major area of interest in research around this nanoparticle is the fact that CNTs are readily taken up by cells through both endocytosis87 and passive diffusion.88 The exact reasons and the mechanisms for this phenomenon are still unknown, but it is known that changing the functionalization on the surface of the CNT will change the route by which it is taken into the cell. The potential for drug delivery applications is large, and as such, most of the research into the medical use of CNTs has revolved around loading and delivering drugs and targeting. The issues with CNTs are that their carbon surface is inert; so, covalent conjugation chemistry will not function with a newly synthesized CNT before treatment, and they are not watersoluble.89 Noncovalent anchoring by π−π interactions and aromatic stacking is a way around the former issue.90 Liu et al. described a case in which doxorubicin was loaded onto a singlewalled CNT by aromatic stacking leading to a very high loading efficiency equivalent to about 4 g of doxorubicin per gram of CNT.90 Furthermore, aromatic stacking is dependent on the pH of the environment, and so, anticipated pH changes as the CNT travels through the cell could be used to release the large payload. For water solubility, some large amphiphilic molecules like sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) are used to coat the surface of the CNT. These molecules interact with the surface by noncovalent interactions, and it has been shown that the SDBS has a lower energy of interaction because of the contribution to aromatic stacking by its benzene ring. PEGylated phospholipids have been used frequently for the purpose of water-solubility but
inside the sphere because it is vastly porous. As a result, dendrimers are known to have high loading efficiencies.72 Monitoring the polymerization reaction and stopping the process after a select number of generations can tightly regulate the shape and size of the dendrimer. This also controls the molecular weight and chemical composition, which are important parameters for concerns like circulation time, clearance, functionality, biocompatibility, and toxicity. With any nanoparticle synthesis, polydispersity is a major concern. The tight control that is possible with dendrimer formation and growth creates significantly less deviation in size.73 In most of the recent literature, polyamido-amine (PAMAM) is the polymer of choice for dendrimer formation. Many researchers have attached a wide variety of targeting moieties, contrast agents, and drugs. To give an example of the versatility of the dendrimer platform, contrast agents using many forms of imaging have been used with dendrimers. Optical imaging using 5-fluorouracil,74 MRI imaging using the gadolinium chelate GdDPTA,75 gamma scintigraphy using Br-76,76 and CT imaging using iodine in 3-[(N,N-dimethylaminoacetyl) amino]-L-ethyl2,4,6-triiodobenzenepropanoic acid have all been successfully demonstrated.77 Targeting agents like folate, peptide sequences, and antibodies and drugs like doxorubicin have also been used successfully in a dendrimer platform. It is evident that dendrimers are highly modifiable and are very flexible in their application. They have been studied for years already and have been part of many studies and clinical trials, yet no dendrimerbased product is out on the market. The major problem with dendrimers is that the polymers used for their construction tend to be too toxic for use in humans. The concept is very successful and useful, but its materials are limiting its use.10 Microbubbles. As the name suggests, microbubbles and nanobubbles are gas-filled spherical nanoparticles made of a shell of varying chemical composition. These bubbles are usually
