Future Approaches of Nanomedicine in Clinical Science

Med Clin N Am 91 (2007) 963–1016

Future Approaches of Nanomedicine in Clinical Science Mary Brewer, PhD, Tierui Zhang, PhD, Wenjun Dong, PhD, Michael Rutherford, Z. Ryan Tian, PhD* Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA

Burgeoning applications of nanotechnology are altering practices in traditional medicine. Recently promoted by the National Institutes of Health [1], nanomedicinal research quickly is advancing technologies and revolutionizing strategies in clinical science by providing easy access to innovative nanodevices and nanosystems that are newly built based on the rational design and precise integration of functional nanomaterials. It is expected that many long-standing challenges in clinical science soon could be met through advancement and revolutionization. New nanomedicinal diagnostics could acquire critical information regarding the status of diseased (eg, cancerous) tissues and organs quickly and inexpensively with minimal sampling size, volume, and invasion. On that basis, new strategies in therapeutic and regenerative nanomedicines will enable clinicians to take effective actions in a timely fashion and patient-friendly manner.

Opportunities in diagnostic nanomedicine Early detection and diagnostics are in high demand in modern medicine. In vitro and in vivo detection and imaging are important tools of physicians in the quest for detection of early onset of diseases, such as cancer, and prevention of the like. New diagnostic methods combined with nanomaterials currently are under active study for detection of diseases at the cellular level.

* Corresponding author. E-mail address: [email protected] (Z.R. Tian). 0025-7125/07/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.mcna.2007.05.006

medical.theclinics.com

964

BREWER

et al

Nanoimaging Nanocarrier imaging agents and therapeutics can target areas with compromised barrier function or augmented permeability owing to pathophysiology issues. The blood-brain barrier (BBB) in certain central nervous system maladies, such as stroke, multiple sclerosis, Alzheimer’s disease, and cancer and infectious diseases (such as encephalitis and dementia from HIV infection), has shown increased permeability. Nanocarriers armed with imaging agents for diagnosis are found able to cross the BBB effectively. Any leaky vasculature, however, can be crossed by nanoparticles (NPs); therefore, patients who have concurring diseases must use caution so as not to transport these agents to undesired locations [2]. NPs as imaging agents in diagnosis, especially in diagnostic imaging and procedures, encompass one of the oldest and primary quests of the medical profession. Elucidative imaging from the organism level to the cellular level is of importance in diagnosis and in strategic and therapeutic planning. Monitoring the results attained from therapy and observations during patients’ healing process in many cases is performed through imaging processes of varying types. Nanotechnology is providing health care professionals with diagnostic imaging tools never before used. The nanoscale systems include liposomes; micelles; nanoemulsions; dendrimers; nanogels; albumin; and magnetic-, ceramic-, carbon-, and siliconbased particles. These contain, carry, and disperse contrasting agents and radiopharmaceuticals to targets for imaging. In vivo imaging can be performed by the use of imaging techniques that already exist, including single photon emission CT (SPECT), positron emission tomography (PET), MRI, fluorescence microscopy, CT, and ultrasound. Superparamagnetic NPs of metal and metal oxides of iron and cobalt already show promise as contrasting agents in MRI. Other NPs that show promise are colloidal gold and quantum dots (QDs), both of which exhibit advantages in clinical diagnostics. Colloidal gold can be used to enhance existing imaging techniquesdin vitro labeling of biologic systems, scanning probe technology, and fluorescence resonance energy transfer (FRET) measurements, to name a few. In diagnostic imaging, nanoscale size would allow the nanomaterials or nanoagents to be administrated through needles and virtually are nonocclusive.Using the single nanocarriers or groups of nanocarriers to transport systems, improved pharmacokinetics is demonstrated along with the efficacy to target specified sites of interest. Possible side effects resulting from toxicity associated with some imaging agents promisingly are minimized or eliminated as a consequence of the accumulation at designated target sites and less accumulation in healthy tissues nearby. In addition, some nanocarriers can help solve solubility concerns by linking a carrier to a molecule that has an affinity for specific binding sites. For instance, the solubility of compounds exhibiting hydrophobicity in aqueous environments can be either encapsulated within a carrier or functionalized with polymeric nanostructures that

NANOMEDICINE IN CLINICAL SCIENCE

965

even could make the biologic molecules exhibit certain hydrophilicity. Therefore, simply by functionalizing the agent, carrier, or both, the nanocarrier systems can remedy solubility problems and effectively target specified sites for enhancing the administration of the agents. In the most basic sense, liposomes simply are lipid shells with hydrophilic, aqueous interiors. Because of the hydrophobic exterior, liposomes may pass through the blood stream without the significant degradation by blood components, encapsulate hydrophilic contrast agents and radiopharmaceuticals, and be used extensively for in vitro imaging of cells and tissues. Recently, one group has reported using 99mTc-liposomes in SPECT imaging of human breast cancer tissues in which overexpressed vasoactive intestinal peptide (VIP) receptors abide. Their imaging used sterically stabilized liposomes to encapsulate the 99mTc-labeled hexamethylpropyleneamine oxime that was found to accumulate less in healthy breast tissues than in cancerous breast tissues with or without the presence of covalently attached VIP. Furthermore, when the liposomes were of an appropriate size, they would not extravasate the normal breast tissue. Therefore, significant accumulation of the contrast agent occurred in the abnormal tissue as compared with the normal. The accumulation of the liposomes and the effective targeting demonstrated in the study support future in vivo uses of such systems in breast cancer imaging and treatment [2]. This same group experimented with paramagnetic gadolinium (Gd)loaded liposomes in a series of MRI imaging experiments using mice. Their findings indicate the propensity for the liposome system to increase relaxation times compared with conventionally used, paramagnetic complexes, Gd-diethylene triamine pentaacetic acid (Gd-DTPA), and gadoterate meglumine. A threefold lower dose (0.3 mmol/kg) of the imaging agent Gd was required. Furthermore, their studies using the Gd complexes showed that the sterically stabilized liposomes maintained contrast quality for tumor imaging enhancement 20 hours after administration. Liposomal-mediated 99mTc-Gd contrasting currently is in preclinical stages of development with the techniques of SPECT, MRI, and PET [3]. Other studies show that the circulation half-life of liposomes consisting of dipalmitoylphosphatidylcholine; cholesterol; and uronic-acid derivative, palmityl-D-glucuronide (PGIcUA) was elongated in comparison with liposomes without PGIcUA. Greater circulation half-life implies less liver accumulation and greater tissue accumulation of the contrasting agent carried in the liposome with the PGIuCA [2]. This result also could be beneficial in the transport of other agents. Dendrimers usually are polymeric, highly branched complexes with a central core that has a large molecular weight of 1000 to 800,000 kd. They are synthesized from monomers via convergent or divergent polymerization reactions [4]. Commonly used monomers for radiopharmaceutical and drug delivery processes are polyamidoamine (PAMAM) [5,6], polyethyleneimine [6], polypropyleneimine [7], polyethylene glycol (PEG) [7], and

966

BREWER

et al

poly(L-glutamic acid) [7]. Each branch, also called a cascade, is added sequentially and labeled according to its synthesis relative to the core and other branches (that is, the core first is synthesized and labeled as generation zero, G0; the first branch generation 1, G1; the second G2; and so on). Because of the branching, dendrimers can be highly functionalized in each branch generation for possessing the potential of coupling with carbohydrates, peptides, and silicon. Limitations of dendritic transport systems are limited as a result of associated toxicity resulting from positive charges of surface and immunogenicity, especially in larger molecular-weight dendrimers [8]. Nanoscale dendrimer systems carrying Gd as a contrasting agent also are in preclinical stages of study. The development of dendrimers and dendrimer systems with a molecular weight less than 60 kd to carry Gd MRI contrasting agents to lessen retention times and the eliminate the problematic toxicity of their larger dendrimer and albumin counterparts is demonstrated. Excretion through the renal system and improved imaging of vascular structures resulting from decrease extravasation, which is faster than the commonly used Gd-DTPA, is documented [8,9]. QDs are nanoparticles with possible prominence in nanomedicinal diagnosis and treatment, mainly because of their multifarious applications and established synthetic protocol. Owing also to the stability, fluorescence capability, and multicolor emission and emission control, QDs are excellent candidates for in vivo imaging from the whole organism to the cellular level, especially in cancer imaging. Consisting of a few hundred atoms from group II-VI or III-V elements, these are semiconductor nanoscale crystals normally within the range of 1 to 10 nanometers in diameter. They emit between the ultraviolet (UV) and the near-infrared (NIR) spectral region and exhibit properties uncommon to organic dyes and fluorescent proteins. QDs have a size-dependent photoelectron band gap and, therefore, tunable photoluminescent qualities. Tuning is the process of selecting the emission wavelength [9,10]. Thus, QDs have potential for labeling biologic systems for optical or electrical detection in enzyme, antigen, and cellular imaging. One of the most promising applications of QDs is the detection and diagnosis of cancers in vivo. In vivo imaging using QDs after injection into an organism entails excitation with a long wavelength source and capture using a charge-coupled device camera. The use of QDs has fewer operational and equipment-related expenses compared with MRI and, as such, possibly could become the preferred imaging technique, particularly in the area of cancer research [2]. Imaging from the cellular level to the organs and tumors is believed potentially doable with QDs. Crucial to diagnosis and treatment of cancer is the effectual imaging of tumors. In a 2002 study of such a use, QDs were coated with a peptide, injected intravenously, and successfully targeted cancer accumulation in lung tissue in vivo. In a study involving tumor metastasis, B16F10 cells were labeled with QDs and injected into mice. The QDs were used to track

NANOMEDICINE IN CLINICAL SCIENCE

967

extravasation into the lung tissue and their potential use in vivo and in identifying metastatic behavior. In the same study, it was determined that the behavior of the QD-labeled cells was not different from that of the unlabeled cells. Besides these findings, the study showed that different QDs could be attached to cells that could be monitored over five separate populations [2]. An innovative type of QDs that has shown a propensity for in vivo detection of lymph node association is called the near-IR fluorescent type II QD. Near-IR QDs have an advantage of deeper tissue penetration capability and, therefore, can detect and image objects otherwise undetectable by QDs emitting only in the visible range. As an invaluable tool to surgeons in the resection of the sentinel lymph node, which is a common procedure in cancer diagnosis, this method provides images of the node with greater detail in real time. Plus, dissipation of fluorescence is an indication of complete extraction of all lymphatic tissue. Compared with other modalities, magnetic labeling of cells using superparamagnetic iron oxides (SPIOs) exhibits superb biocompatibility and great T2 relaxation time for enhanced differentiation between abnormal and normal cell groups. SPIOs can create magnetic moments of a magnitude sufficient to alter the magnetic moments of protons in neighboring cells that could lead to widespread dephasing in tissue [11]. Another MRI imaging agent, iron oxide NPs covered by dextran, is under study to determine if the toxicity could be reduced by some ligands that can bind to the surface of the NPs. Covalent coupling of insulin has proved able to improve the targeting toward desired tissues, thereby reducing the cellular internalization and toxicity. Recent studies suggest that insulin coupling on the iron oxide NPs can decrease in vitro cellular internalization. The insulin can bind to the cell membrane surface reseptors, thereby preventing the internalization and minimize the toxicity at the cellular level. Iron oxide nanocrystals also could be inserted into nanostructured matrices containing PEG-functionalized polyacrylamide and solid lipid NPs. Threefold relaxation time and prolonged periods of circulation half-life are resulted. Nanodetection The second common practice in clinical diagnosis, other than imaging, is detection or sensing. There is a crossover, an intermingling of nanomaterials used in diagnosis and detection. Many results from the research performed currently show the potential to create NP systems or nanoplatforms to perform both tasks, to detect and subsequently label the targeted cells or tissue for imaging. The nanodetection platforms include mainly nanodevices, such as sensors or multiplexed assays. NP-based detection systems may consist of biologic molecules, QDs, metal NPs, silicon- or carbon-based nanostructures, or perhaps an integration of several of these. Microarray platforms used commonly for detections have been improved for detecting lower limits and a wider range of biotargets. The literature is replete with studies of new

968

BREWER

et al

detection systems configured to target specific extra- and intracellular targets. NPs recently have been used to detect antibodies, nucleic acid, sites of tissue inflammation and tumor extravasation, and antioxidants based on physical or chemical criterion [12]. A good example is the use of self-assembling DNA-based dendrimer platform to detect nucleic acid sequences [13]. Separation-free detection systems in the past have shown the most effective results in FRET and fluorescence correlation spectroscopy based on gold-binding kinetics and related sensitivity. There exist, however, the ever-present complications of cross talk, poor signal-to-noise ratios, photobleaching of fluorescent dyes, and extensive and complicated photodetection systems necessary to discriminate the fluorescent tags [12,14]. To improve the bioassays currently in use, new QD-mediated biosensing methods are catalyzing much of the seemingly inexhaustible research. QD-based biodetection that is a good example of using biologic and inorganic components is the amperometric immunosensor of HBsAg. A gold (Au) electrode was placed into mercaptopropyltrimethoxysilane (MPS) sol gel followed by the coupling of gold NPs to the surface-modified electrode. The affinity between Au and sulfur (S) allows a self-assembly through chemisorption of the thiol groups onto the gold. To the surface of the Au NPs on the electrode, HBsAb was adsorbed to create an interface of bare Au electrode/MPS/Au NP/HBsAb. This interfacial specific bonding can be used to detect the presence of HBsAg via the characteristic redox reaction with HBAb. This immunosensor was tested against the traditionally used enzyme-linked immunosorbent assay (ELISA) method with similar detection limits and reproducibility [15]. Another NP detection scheme, consisting of QD-empowered biosensing assays for nucleic acid detection, has been created for detecting nucleic acid sequences. By manipulating the characteristic structural changes on binding to a target molecule, separation-free optical assays have been developed to form the fast and inexpensive multiplexed bioassays [16,17]. QDs’ flexibility and adaptability make them useful in multiple detection applications, especially in detection of cancer biomarkers. QDs can be linked covalently to immunoglobins for use in fluorescent assays to detect antigens and other biomarkers, such as Her2 on cancer cells. Being better than the commonly used fluorescent molecules that have typical poor stability toward the photobleaching, QDs have a better signal-to-noise ratio and, therefore, a better signal intensity or detection sensitivity [18]. In vivo applications of QDs in the detection show preliminary successes in detecting cancer in laboratory mice. When the monoclonal antibodies bind to the PEG surface for recognizing prostate tumor antigens, a PEG coating can increase the biocompatibility and protect the QDs from degradation [2]. Because of the versatility of the surface modification, QDs are regarded as highly useful biosensing assays. When binding to streptavidin, QDs of varying emission wavelengths are coupled to two biotinylated

NANOMEDICINE IN CLINICAL SCIENCE

969

single-stranded DNA. An example of a functionalized QD for biomedical detection is detecting respiratory syncytial virus (RSV), a serious respiratory tract infection. Because of the number of repeat infections and complications from the virus that may reoccur during a lifetime after first contracting RSV, prevention and early detection could diminish hospitalization, not to mention the suffering and pain of patients [19]. In the presence of the DNA target, QDs can be arranged in a codelike fashion, referred to as optical barcoding [9,18]. This is an effective method of performing multiple detections within populations of cells, screening drug compounds, and for use in multiplexed assays. Normally, when tagging individual targets in a cell population, the specific types for study can be transfected. When the cells are mixed with those of the general population, the barcoded cells can be located and differentiated according to the wavelengths of the QD’s emitted light [18]. Of the many applications of carbon nanotubes (CNTs) since their discovery in 1991 [20], a multiplicity of uses has been uncovered based on their properties of superb conductivity, electrical and thermal, along with their rigidity and resilience. These properties have given rise to CNT applications, such as biosensing and drug delivery [21]. Specifically, their use in biosensing escalated after a variety of biologic molecules was introduced successfully into their interior. Efforts currently are devoted to the creation of new interfaces, such as microelectromechanical systems (MEMS) and nanoelectromechanical systems (NEMS). The ultimate goal in this area is mass fabrication of MEMS or NEMS capable of detecting hundreds of targets simultaneously for timely diagnosis of diseases at minimal cost. CNTs, thus far, have been used to detect anions, proteins, enzymes, antibodies, and DNA and to transduce or amplify signals in sensing [22–26]. The success of CNTs as field effect transistors (FET) is catapulting CNT use further in biomedical sensing [27,28]. The CNTFETs, successfully used in antibody sensing [21,26], have been fabricated successfully, the CNTs serving as a conducting channel on a silicon wafer. When in contact with antibodies, a significant change in electrical conduction corresponding to the concentration of the antibodies can be sensed by the CNTFET nanodevice. It is suggested that the mode of conduction change is prompted by electron-donating characteristics of the amine group (–NH2) groups, which results in a reduction of the source/drain current of the FET [29]. In addition to the QD- and CNT-based chemical sensors, there is a wide range of nanodevices for highly specific detections in nanomedicine, many of which currently are under research, several in clinical trials. One of the latter sorts is a recently developed single-virus detector using nanowire (NW) FET. The NWs are modified with viral antibodies that can detect virus in a fluid. Likewise, the transistors can detect electrical conductance changes instantly once the virus binds on the wire arrays. The magnitudinal changes in the conductance and the binding time are the characteristics for each type of viruses. Therefore, the twofold detection scheme significantly

970

BREWER

et al

reduces the likelihood of the false positive in the target identification. Another nanodetection system is the novel use of an oxygen sensor. The development of a sensor, termed a reactive oxygen species (ROS) biosensor, is a new biodetection strategy for locating cells experiencing oxidative stress. These cells produce antioxidant responses detectable to a ROS biosensor. The biosensor is used to activate and impel DNA repair sequences within the cell [3,9,10].

Opportunities in therapeutic nanomedicine Research in nanomaterials-empowered drug delivery and therapeutics is attempting to advance toward a new technologic level not attained before, with innovative research results aiming to meet many long-standing challenges in the pharmaceutical, therapeutic, and drug-delivery industries. The quest to resolve the issues of bioavailability, solubility, chemical stability, and the amelioration of side effects from drug therapies is pursued fiercely and promisingly by current research developments. In light of soon-to-expire protein-based drugs (human growth hormone, insulin, erythropoietin, and so forth) and the competition from manufacturers of generic pharmaceuticals, the impetus is to invest in synthesis of novel NP drugs and drug platforms. Tackling Alzheimer’s disease, cancer, and common maladies and increasing demands for a better future to patients who have less pain and suffering are encouraging nanosceintists and governments worldwide to invest enthusiastically in so-called ‘‘nanodrug’’ research. Nanodrugs using liposomes Among the drug carriers, liposomes and lipid-based NPs are common. In gene therapy treatment, liposomes are potential carriers for DNA, RNA, and small interfering RNA (siRNA). Surface-modified liposomes carrying doxorubicin and antisense oligonucleotides systems successfully have targeted multidrug resistance-associated protein 1 (MRP1), messenger RNA, and Bcl2 RNA. On reaching the cell, the system can deliver the doxorubicin and the antisense oligonucleotides successfully, inhibit the synthesis of MRP1 and Bcl2, and provoke the apoptosis of the cancer cell by arousing the caspase-dependent pathway [2]. Liposomes for drug delivery have been used since the 1960s. There are several therapeutic agents currently under research for treatment of cancer: camptothecin, paclitaxel, and cisplatin; antibiotics: amikacin, ciprofloxacin, and vancomycin; biologics: DNA, siRNA, and muramyl tripeptide. One study of the anticancer drug, doxorubicin (discussed previously), involves a liposome-based delivery system that can transport antisense oligonucleotides and the drug-to-cell nuclei where Bcl2 and MRP1 protein synthesis was inhibited and stimulated the apoptotic, caspase-dependent pathway in human cancer cells that is resistant to a variety of drugs [2,18]. Normally,

NANOMEDICINE IN CLINICAL SCIENCE

971

liposomes for this purpose vary between 30 nanometers and several micrometers in diameter, with their characteristics governed by their size, surface composition, and charge. The results suggest that some of the obstacles impeding the use of liposomes as drug-delivery agents, showing the potential for future use in drug and medical industries. Testing also is ongoing using liposomes to facilitate gene therapy protocols. In one such study, cell transfection rates in prostate and pancreatic cancer were increased by almost 40-fold [30]. Liposomes essentially are a sphere of phosopholipids, surrounding a hydrophilic (normally aqueous) center, with unique chemical and physical properties dependent on the surface features, such as type of lipids used, and way of synthesis [31,32]. Liposomes virtually are biodegradable, nontoxic, amphiphilic phospholipids that invoke minimal or no antigenic response because their constituency basically is the same as that for biomembranes. Liposomes can be small enough (with sizes as small as 30 nm) to carry their cargo across the cell membrane. The size is a critical factor for any nanoscale delivery system. Particles greater than 100 nanometers in size fall prey to opsonization by the mononuclear phagocytic system and must be smaller than the vascular pore range of 380 to 780 nanometers to extravasate and target specific tumors and other sites. If larger liposomes are desired, surface modifiers, such as dextrans [33]; gangliosides [34]; and hydrophilic polymers, such as PEG [35], poly-N-vinylpyrrolidones [36], and polyvinyl alcohol [37], have to be used. Several studies suggest that liposomes used as contrast agents or drug carriers can prolong circulation in the bloodstream, can be surface modified with antibodies or other ligands with affinity for specific targets, and can enhance the pharmacokinetics [38–40]. On the market, there are three pharmaceuticals involving the liposome encapsulation strategy: amphotericin B, daunorubicin, and doxorubiicin [8]. There are some hurdles that must be crossed, however, before liposomes and liposome-based delivery systems can be manufactured by pharmaceutical companies to deliver a variety of drugs confidently. Unmet challenges include poor or low rate of encapsulation recurrence, seepage of water-soluble agents in the blood stream, and lack of prolonged storage stability [41]. As an alternative, polymeric NPs within the size range of 10 to 100 nanometers can encapsulate drugs and couple to certain ligands to facilitate the solubility and receptor affinity. These new drug carriers are discussed briefly. Nanodrugs using micelles and nanoemulsions Although liposomes are excellent candidates for carrying countless drugs, micellar delivery systems normally are within the 100-nanometer and smaller size range that can facilitate the delivery of hydrophobic drugs that otherwise are insoluble in aqueous media. Exhibiting good pharmacokinetics and high distribution rates, micellular systems have a bright future. These micelles are not the typical surfactant type used commonly in

972

BREWER

et al

detergents; rather, they are formed using amphiphilic polymers. The hydrophobic core of the micelle can be loaded with contrast agents and drugs that become trapped within. Drugs, such as doxorubicin, cisplatin, and paclitaxel, are distributed with greater efficiency, as the circulation half-lives of micellular systems has proved longer than those of the free drugs. As a result, accumulation of these anticancer drugs in tumors is improved. Micelles used for drug delivery can be categorized into four groups: pluronic, poly(L-amino acid), polyester, and phospholipids. The phospholipids group is able to avoid opsonization by the MPS and demonstrates prolonged circulation time. As such, they are classified further as sterically stabilized micelles, which are demonstrated as able to solubilize several anticancer drugs successfully, including paclitaxel and camptothecin. Nanoemulsions are suspensions of oil and water into which drugs can be solubilized. By varying the weight ratios of water to oil or oil to water, hydrophobic and hydrophilic pharmaceuticals can be delivered. The dispersed oil or water particles tend to have sizes in the range of 5 to 14 nanometers. Preclinical trials using nanoemulsions to deliver anticancer drugs have proved able to reduce the toxicity in comparison with the free drug [2]. Nanodrugs using inorganic nanoparticles NPs can journey to targeted sites inside the body without sedimentation or occluding microvasculature, can penetrate tumors and other tissues with extravasation, and can be taken up at the cellular level by endocytosis. NPs already are in use to deliver imaging agents and therapeutics for the treatment of cancer. Other related applications are in experimental or clinical stages of development [2,9,42,43]. In general, the components of a nanodrug delivery system from the inside-out are the drug, a layer of material to serve as a casing around the drug (eg, dedrimers, liposomes, and polymeric materials), and functionalized appendages to facilitate solubility and targeting [2,3,9,43,44]. Once delivered to a site, the casing or encapsulating agent degrades over the time because of pH changes, heat, light, or simple erosion of the capsule. The drug also can be released by a slow diffusion of the drug through the casing. Polymers, such as polylactic acid and polylactic coglycolic acid, commonly are used to encapsulate drugs for slow dispensing [45]. One particular application is based on an insulin-dispensing platform (previously bound within a self-assembly of polyamino acids and encapsulated by a protein framework that disintegrates over time, slowly releasing the drug) with which a stable insulin level in blood can be maintained over a long time. Ranging from gene therapy to the delivery of conventionally used drugs, such as penicillin, and to thermal ablation of cancer tumors, this realm of nanodrug is with no doubt a revolution in the making [3,43,45,46]. NPs as carriers of therapeutics are showing remarkably swift progress toward introducing new pharmaceutical systems on the market as new

NANOMEDICINE IN CLINICAL SCIENCE

973

therapeutic choices for physicians. Nanodrug delivery systems can be tailored to combat solubility and chemical stability issues, to improve pharmacokinetics, and to increase efficacy of distribution to specified targets, which implies less distribution to healthy cells and tissues. Targets include inflamed sites where extravasation may occur, and tumors possessing a vasculature would be more permeable than that of healthy tissues. In some cases, the nanoscale carriers with appropriate biocompatibility are alternatives to existing carriers where hypersensitivity is a problem. There is a substantial reduction in toxicity because of smaller volumes of the nanocarriers. Diseases other than cancer (such as Alzheimer’s disease) potentially can be treated through the use of NPs. Amyloid b-derived diffusible ligands (ADDLs) appear in the cerebrospinal fluid (post mortem) of patients who have had Alzheimer’s disease [47]. Known to exist in excess as compared with same-age patients, ADDLs are considered toxic at these elevated levels. The correlation of cerebrospinal fluid ADDL level with Alzheimer’s disease is considered an advantageous test for the future acquisition of early-onset diagnosis of the disease. This process is termed, immuno–polymerase chain reaction (IPCR), which is similar to ELISA. Instead of a covalently bound enzyme to the secondary antibody, however, in IPCR, the antibody is bound to DNA. Through PCR amplification, the DNA is detected via either gel electrophoresis or a fluorescent probe. IPCR shows the promise of sensitivities similar to ELISA. The antibodies must be conjugated to DNA strands, however, undergoing PCR-like thermocycling followed by subsequent antibody amplification as in PCR. As an alternative approach, magnetic NPs laden with monoclonal antiADDL antibodies are exposed to a cerebrospinal fluid sample. A magnetic field then is applied and the magnetic NPs are separated and cleaned. A Au NP is bound to thiolated DNA and then a hybridized DNA attached as an identification tag, or ‘‘barcode.’’ The ADDL then is coupled to the barcodelike DNA on the Au NP. The unreacted antibody/DNA/Au NP complexes then are removed through the application of a subsequently applied magnetic field, resulting in the retainage of the ADDL/DNA/Au barcode conglomerates, where each Au nanosphere has more than 100 barcode DNA strands for the amplification [47]. Nanodrugs using polymeric nanoparticles There are several nanoscale drug-carrying systems currently on the market in Europe and in the United States. Polymeric NPs, however, are appealing to drug manufacturers. An early study of these NPs [48] revealed some important qualities that later researchers have substantiated further: (1) surface characteristics and size can be manipulated to allow for either passive or active transport; (2) sustained and controlled release is possible and can be altered; and (3) degradation of the drug in the presence of blood elements is minimized. It has been discovered that these highly desirable

974

BREWER

et al

characteristics can overcome some of the problems that have bothered pharmaceutical companies for years. Hence, one further classification must be made for polymeric NP systems, such as nanogels, albumin, polymer, and lipid-like systems, that all can serve in the transport process [2,4]. The polymeric NPs typically function to enclose or encapsulate a drug with the controlled release times and to direct the targeting. Ideally, polymeric NPs should be biodegradable, like those made of polylactic acid, polyalkylcyanoacrylate, polyglycolic acid, poly(3-hydroxybutanoic acid), or polyethylene oxide, among others [2,4,18]. Polymers or surfactant-covered nanocrystalline drug particles are being tested with drugs, such as amphotericin B, etoposide, paclitaxel, and camptothecin. The polymeric coverings alleviate particle aggregation. Polyesterand lipid-based NPs can be degraded into acidic metabolites. If this factor can be controlled, the NPs could be used as new carriers or distributors for such drugs as doxoribicin, camptothecin and tamoifen, cyclosporineA, and theophylline, respectively. Nanogels and dendrimers currently are in clinical trial as potential carriers for oiligonucleotides and antisense oligonucleotides. Nanodrugs using dendrimers Dendrimers show a propensity toward toxicity and immunogenecity because of their positively charged surfaces but are being studied in conjunction with indometacin and 5-fluorouracil. Albumin NPs have attained Food and Drug Administration approval to be used with paclitaxel for the treatment of metastatic breast cancer [2]. Dendrimers with the polymers (described previously) are being studied to determine the feasibility of their use in gene therapy, drug delivery of penicillin [49], and cancer therapy. Drug molecules can be attached by several different manners, through encapsulation within the dendrimer core, at the branches through the formation of networks of dendrimers and drugs, or through coupling the drug on the dendrimer surface [50]. Starburst dendrimers with regular branching structures simulating trees with diameters in the vicinity of a few nanometers have been synthesized. These are characterized by a central core surrounded by dendrimer modules. Each module can perform a designated function. The goal of these branched and diversely functioned systems is to perform multiple tasks of (1) disease detection at the cellular level, (2) diagnosis of disease, (3) delivery of therapeutics, (4) transmission of location, and (5) transmission of therapeutic results. If the modules can be made to work sequentially or in tandem and customized for particular diseases or conditions, such as cancer, dendrimers may attain a position of preeminence as nanoscale therapeutic NPs [43]. Already displaying potential to recognize approximately a half-dozen cancer cells, a PAMAM dendrimer consisting of an ethylenediamine core is armed with folic acid, fluorescein, and methotrxate for targeting imaging and

NANOMEDICINE IN CLINICAL SCIENCE

975

drug delivery, which has shown a 100% improvement in the efficacy of methotrexate treatments. Several studies using dendrimers are worthy of discussion. A PAMAM dendrimer can target folate receptors effectively in tumor cells. The dendrimer can be modified, and a folatemethotrexate-PAMAM version was synthesized. Folate receptors of cells derived from human epidermal carcinoma had a high affinity for the dendrimer complex, resulting in a cytotoxicity level that is 4 times greater than the free drug [50]. Also, in the research currently using dendrimers as drug-carrying systems is a new finding involving the anti-inflammatory drug, indomethacin. In arthritic rats, successful passive targeting of areas of inflammation was seen at a level 2.29 times greater than that of the free drug [51]. One challenge in controlled release is in the manufacturing of devices and device parts. Demand for new nanoscale sieves, needles, and pores poses manufacturing challenges while offering versatility in architecture and application in the way of drug delivery. The technology required to produce one nanodevice then can be applied to subsequent manufacturing of other devices. For example, a nanoneedle could be used to manufacture a nanopore in a material and many nanopores on a surface could form a nanosieve. Another porous silicon-based structure is porous hollow silica NPs. These NPs are spherical and hollow and contain drug molecules through a drying coalesce process of drying coalesce. Once again, the nature of the pore (shape, size, and so forth) determines the release rate of the drug. An interesting therapy involving silicon-based nanostrucures is delivery systems through porous silicon systems laden with platinum, serving as antitumor agents, antibiotic, DNA, and enzyme delivery. Of similar structure, hollow metal nanoshells of gold, silver, platinum, or palladium can trigger the drug release. After being implanted within a polymer drug carrier, the nanostructured shells can be stimulated to deliver the drug when exposed an alternating magnetic field or when exposed to IR radiation [4]. Nanodrugs using carbon nanostructures Controlled release naturally is related to targeting. Release in the blood stream in many cases is not desirable, whereas release at a specific site may enhance the therapeutic effects of a drug. Also, controlled release is a key to achieve the long blood circulation period in order for a drug to arrive finally at a target. Controlled release could be done via CNTs and clusters of carbon, such as C60 (known as buckyballs or fullerenes) [1,19,52]. The latter are under investigation for their antioxidant characteristics to treat Parkinson’s disease, multiple sclerosis, and other diseases that impute oxidative distress and damage. There is some ongoing research in this area in protecting from and repairing damage caused by prolonged exposure to radiation during

976

BREWER

et al

space travel and exploration. Future exploratory travel to Mars presents a medical challenge in continuous, long-term monitoring; prevention; treatment; and restoration of any medical condition. Radiation damage is inevitable on such a voyage, because astronauts will be exposed to radiation that is hard to block completely, thus defining a need for long-term monitoring of radiation exposure with in situ, continual, and autonomous systems to control and dispense antioxidant therapeutics [9,53]. Fullerenes seem small enough to cross the BBB, making them attractive in future treatment of neurodegenerative diseases. In anticancer and antioxidant therapeutics, fullerene derivatives also show promise [43]. As a derivative of the fullerenes, CNTs consist of graphite sheets rolled into tubes that are single walled (SW) or multiwalled (MW). Many CNTs already are in clinical trial stages of development and are known for good biocompatibility and low toxicity and show antiviral proclivity to the HIV virus most significantly, antiapoptosis agents in the treatment of Parkinson’s disease, and amyotrophic lateral sclerosis. They also can serve as needle-like nanostructures with the ability to cross cell walls without perturbation or disruption of the cell membrane and can situate into the cytosol and mitochondria. CNTs as an ideal delivery system for therapeutic agents has been made feasible because of the surface functionalization on the CNT not only to achieve a great solubility in aqueous environments but also to attain the desired biocompatibility. A study conducted recently tracked CNTs after the interaction with HeLa cell cultures and found that the CNT can access throughout cellular components, cytoplasm, and nucleus without significant cellular disturbance or damage. A new concern about the use of CNTs, however, is cell longevity after introduction. In another study, 50% of the cells died after 6 hours of incubation CNTs in a high concentration (5- to 10-mg/ mL solution) [21]. CNTs can be rendered water soluble for drug delivery in many ways, depending on the drug molecule or target site, and can be functionalized to allow them readily across the cell membrane, then reaching the specific cellular components. CNTs functionalized with peptides can serve as vaccine candidates. These peptides were conjugated to the CNTs to determine immunologic characteristics. The peptide was chosen to represent a viral protective and deactivating epitope. After injecting mice with the appropriate FMDV antibody, the CNTs increased the peptide responses to the virus without generating antibodies against the functionalized CNT. Furthermore, in comparison with the free peptide, the addition of the peptide funtionalized CNT provoked a stronger immune system response. This study shows potentiality of using CNT for vaccine delivery [21]. Nanoscale engineering is producing tools, bearings, and motors for use to fabricate other nanodevices, some of which have been in use for years with known protocols for their manufacture. Pharmacies on a chip in general are indicative of the new products soon to reach the market.

NANOMEDICINE IN CLINICAL SCIENCE

977

Nanodrugs using nanodevices Through the processes used commonly in the semiconductor industry, photolithography, etching, or various forms of deposition, silicon structures can be fabricated specifically for drug delivery. One structural form under investigation with promise is a system of nanopores generated on silicon, silica, or silicon dioxide (SiO2) substrates for making the nanodevices that resemble a sieve. With a control of the pore size and density, drug delivery rates can be regulated to ensure a programmed delivery pattern. Functionalizations on the surface of the carrier coupled with a carrier resistant to macrophages and dissolution in the bloodstream are issues that must be addressed to ensure or improve the controlled release. Microchips, mass produced since the 1980s, predominately are silicon or polymer based with potential for drug delivery after being integrated into a device to distribute drugs on demand in vivo. Although dispensing therapeutics is a somewhat new application, the incorporeal pharmacies are engineered by long-standing methods, such as photolithography, deposition, and etching, all of which are processes used commonly in semiconductor and MEMS manufacturing. Drug delivery rates in these systems are controlled by varying pore size and density, both of which are regulated at the manufacturing level. Micromachines, such as pumps, valves, and channels, are mounted on a microchip platform to deliver drugs on demand. After implantation, a long-term, pulsed drug administration can be realized. In 1999, researchers at the Massachusetts Institute of Technology successfully achieved a controlled release of a drug from microchips. Such systems are dubbed ‘‘pharmacies on a chip’’ [54]. The chips function via electrochemical mechanisms and control the release of drugs. The pharmaceuticals traverse along thin anode membranes connected to tiny reservoirs acting as drug carriers. Poly(L-lactic acid) microchip devices (1.2 cm in diameter and 500 mm in thickness) recently were manufactured to contain 36 drug reservoirs [4]. In future, these pharmacies on a chip will not be stocked with premade drugs; rather, they ideally will concoct, from a selection of molecular building blocks, the drug that the body needs at a particular time of chemical imbalance. Here then naturally comes a challenge: how to coordinate detection, feedback, execution of a process (in this case, drug synthesis), dispatch, and delivery to a target in vivo all in one nanostructured device.

Opportunities in regenerative nanomedicine Tissue regeneration and engineering is becoming a reality owing to the recent advancements in the field of nanostructure engineering. This interdisciplinary area of nanoresearch is centered on the growth and replacement of biologically important cell, tissue, and organ via developing alternatives to traditional modes of tissue and organ repair. Biomimetics engineering shows

978

BREWER

et al

some hope in creating biologic implants, such as blood vessels and joint replacements, and making possible the bottom-up or top-down fabrication of artificial extracellular matrices or scaffolds that are necessary to support and maintain cell growth and regeneration during tissue engineering [54,55]. Importance of extracellular matrices Reconstructive surgery or removal of severely damaged or irreversibly malfunctioning tissues followed by transplantation is the traditional practice in therapies. An alternative to these procedures, however, is to allow for selfregeneration of damaged tissues or organs. This takes advantage of the body’s own potential to heal itself. Heretofore, cell-by-cell reconstruction has been limited by the lack of cells, an extracellular matrix (ECM) that includes a scaffold, and growth factors to regulate the cell division [54–60]. Manufacture of a biomaterial-based scaffold alone poses many challenges. ECM scaffolds can be assembled using textile weaving technology already used to create artificial, polymeric cartilage, tendons, and blood vessels, to name a few. Nutrients and therapeutics must pass through the scaffolding framework, making the control over the pore (or void) structure inside the scaffolds a point of considerable research. Size, pattern, and shape of scaffolds can alter cell behavior and in some cases can dictate whether or not a cell lives or dies [54]. Computational fluid dynamics is providing the power to visualize, through the use of models, the growth factors that must be provided by a properly designed ECM scaffold. Such models have generated data related to oxygen profiles, system and cellular strains, vascularity, and angiogenetic factors that must be addressed within the proper scaffolding construct for viable regeneration of cells to the tissue stage of development [60–63]. On commencement of cell regeneration in the ECM, the newly developed cells begin to grow by their own within the ECM. The artificial ECM scaffolds can hinder subsequent morphogenesis once the cells initiate growth. Therefore, a successful removal or deterioration of the nanoscaffold, which served as the artificial ECM, is expedient and must be done without compromising the developing tissue. A ‘‘biodegradable’’ scaffolding system, then, is highly desirable. As the scaffold degrades, the matrix created naturally by the cells can support its own functions, such as angiogenesis, fluid and nutrient regulation, and cell migration [54,55]. The engineering via a bottom-up or topdown manufacturing method on substrates to serve as ECMs has been researched thoroughly within the past decade. Microlithography, microcontact printing (mCP), hydrolytic etching, and other techniques have been used in the development of suitable ECM scaffolding systems. Computer-assisted inkjet printing technology is being investigated as a feasible technique by which 3-D scaffolding and tissues can be constructed, which is helpful to the organ construction [64]. In tissue engineering, populations of cells are seeded on and proliferate within the 3-D scaffolding network, normally

NANOMEDICINE IN CLINICAL SCIENCE

979

fabricated from natural, synthetic, or partly synthetic biomaterials [54,55]. The scaffold is the determining factor of proliferation and shape of the new tissue and can be used alone in regenerative-type therapies. If required because of poor morphogenesis, growth factors must be introduced into the artificial scaffold. For the effective administration of growth factors, simple injection into the system fails because of rapid diffusion and excretion from the construction site. Drug delivery systems (discussed previously) can serve to specify delivery and ensure accumulation within the ECM. After injection of the growth factor and subsequent and successful morphogenesis, the scaffold should disintegrate [55]. Tissue regeneration at present can be classified into two primary types, in vitro and in vivo, and can be classified further according to the tissue type, hard or soft. New opportunities in stem cell nanobiotechnologies Using scaffolds to grow cells into tissues and organs requires a supply of healthy prototypical cells that can undergo morphogenesis, specialize, and become a specific tissue, such as kidney. Stem cells, because of their penchant to differentiate into almost any type of cells, are ideal candidates to occupy scaffolds and eventually become new tissue. There are two basic types of embryonic stem cells. Embryonic stem cells can differentiate to form brain, skin, or heart cells. The adult tissues can produce adult stem cells that are multifunctional. Therapeutic cloning, the act of removing the nucleus of a patient’s cells and transferring it into a stem cell whose nucleus has been removed, is an alternative manner of obtaining undifferentiated stem cells. Genetic specificity and genetic biocompatibility are advantages of cloning in this fashion. Eventually, stem cells can grow into tissues, soft and hard, and even into organs. Since the 1990s, soft and hard tissues have been grown using scaffolds and implanted successfully into animals of interest. One research group has grown arteries to seek the future potential of minimizing the expensive and lengthy heart-bypass surgery, thus eliminating the need to use vein, arteries, and grafts from other areas of a patient’s body. Artificial grafts of smooth muscle tissue resembling heart tissue was grown and implanted successfully in pigs. Much of the same research has been ongoing since the mid1990s and is continuing by research groups who are growing colon tissue, stomach tissue, and hard tissues, such as bone. Bone marrow transplants grown via human cells and skin tissue products are in use and commercially viable for patients who have cancer [60]. Further research with hard tissue implantation using engineered bone shows the same hardness of natural bone within 6 weeks after it was implanted. On x-ray analysis, researchers could not discern between the old and new bone. Furthermore, the new bone tissue grew blood vessels and was completely compatible when used in reconstructive surgery [54].

980

BREWER

et al

Growths of hard and soft tissues, including organs, are a medical dream that gradually is becoming a reality. In the future, organ transplants could take place without a long waiting period, as spare organs could be stored away, much like replacing automobile spare parts. Preventative maintenance using cellular and tissue growth could precede or even preclude the need for transplants, prostheses, pacemakers, and so forth; the list could go on and on in the future. Currently, there are many challenges in fields of the regenerative medicine. Understanding the cell biology within the environment of a scaffold, maintaining nutrient, waste transport, the proper biosensors to monitor growth and cellular processes, and the construction of adequate bioreactors to facilitate and promote cell growth are major challenges to nanoscience researchers. The development of microvascular networks to underpin 3-D tissue construction to provide waste metabolite removal and to regulate and supply nutrients, including oxygen, are challenges that have to be met for cell, tissue, and 3-D organ development. Those who present solutions to the challenges and problems will find themselves the avant garde, directing the future path of regenerative medicine, not to mention placing themselves in commercially strategic advantage [60,65,66]. Nanomaterial toxicology Researchers also are beginning to investigate the hazards associated with the use of biomedical nanomaterials. Toxicity could exist on a few fronts, including chemically and physically after inhalation exposure, dermal contact and penetration, or on contact via injection into the blood stream or via oral administration. Not only can chemical composition and reactions mediate resultant toxicologic responses in patients but also simply size alone can impart toxicity. Inhalation exposure may occur with particles from several nanometers to a few micrometers in size, with toxicity varying with surface area and activity, aggregation, or disaggregation of particles. End results of dermal exposure, whether or not intentional, through, for instance, the application of sunscreens containing titanium oxide (TiO2) or zinc oxide (ZnO), or accidental, during the manufacturing process, are uncertain. Because of its prolific use as a sunscreen in the cosmetics industry, however, studies with TiO2 on investigating dermal penetration are ambiguous. In 1997, penetration of TiO2 through the skin of rabbits was reported when applied as an oil suspension, agreeing with the results reported in 1996 that TiO2 can penetrate the skin of cadavers. Alternatively, a later study (2000–2003) concluded that TiO2 did not penetrate the skin of human volunteers. When applied in an oil-in-water suspension, the penetration occurred, especially in subjects who had hairy skin, suggesting that (1) hair follicles and pores were avenues of infiltration and in vitro studies and (2) TiO2 fluorescent particles of 0.5 to 1.0 mm could penetrate the epidermis and dermis. This is because these NPs are able to cross the BBB and

NANOMEDICINE IN CLINICAL SCIENCE

981

placenta membrane. To investigate the toxicologic effects, the National Toxicology Program on nanotechnology was begun in 2004 with a focus on fullerenes, TiO2, and QDs. Fullerenes can undergo translocation to lipid-laden tissues. Studies conducted in 2004 indicated that lipid peroxidation occurred in the brain tissue of juvenile largemouth bass, which could be caused by a transport mechanism between the olfactory nerve and the olfactory bulb. A follow-up study conducted in rodents bore a similar result after inhalation exposure, possibly through the same type of transport mechanism. Inhalation effects of CNTs, SW and MW, have been studied in vitro in several mammalian systems. In vivo inhalation studies conducted in rats suggest that SWCNTs produce granulomas in the lung. Further, MWCNTs are suspected of causing an inflammatory response and situate within cytoplasmic vacuoles with human epidermal keratinocytes. These studies were conducted using bare NPs, that is, particles without a biocompatible or otherwise functionalized surface coating. Research only has just begun for the development of comprehensive testing procedures and evaluation of the toxicologic effects of nanomaterials. Much is yet to be discovered regarding processes intricately related to uptake and distribution of NPs through the body, including metabolism, transport, size-dependent effects, and chemical toxicity. Other factors, many of which have not been explored for toxicologic impact, such as surface coating and general morphology, may determine toxicologic behaviors further because of their effects on the biologic activity. Nucleation and crystallization fundamentals The vision of nanomedicine begins on the chemist’s bench. Daily, new discoveries are bringing in new hopes and tools to researchers in materials science for fabricating new drug-delivery systems, biomimetic devices, tissue regeneration strategy, and so forth. Abounding with multifarious structures of biocompatible compositions and potential applications, TiO2, silicates, and ZnO structures play an important role in the future development of nanobiomaterials specifically postured for use in biomedical systems. Although size, morphology, and spatial organization of these metal oxide structures can dictate nanomaterials’ functions and the application potential, precise controls over them largely have remained a grand challenge in nanomaterials syntheses and fabrications, especially in the systems of 1-D nanomaterials (1DNMs). This mainly is because of the challenge in controlling the site-specific nucleation and directional growth of crystallites at the nanoscale. 1DNMs have generated steadily growing interest because of their anisotropic structure and properties. It is believed that potential novel applications could be induced from their nanoscale anisotropic structures. Many inorganic and organic 1DNMs have been fabricated successfully, such as CNTs (SW and MW) [67], inorganic elementary substances (Au, Ag, Pd,

982

BREWER

et al

Pt, Si, Ge, Se, Te, W, Pb, Fe, Cu, and Bi) [68] and compounds (ZnO, In2O3, Ga2O3, GeO2, MgO, ZnS, CdS, CdSe, Ag2Se, CdTe, GaN, GaAs, GaP, InP, InAs, SiC, WO3, SnO2, CuO, TiO2, MnO2, PbS, and so forth), organic small molecular 1DNWs [69], polymeric 1DNMs, and biomolecular one-dimensional nanostructured materials (1DNW) [70]. The 1DNMs can be produced by physical and chemical routes. Nonlithographic techniques, such as electron-beam [71], proximal-probe patterning [72], x-ray, or extreme-UV lithography [73], all have been used successfully in industrial scale. These techniques are limited, however, by high cost, low volume, and complexity in manufacture in comparison with the simple and inexpensive solution syntheses. Essentially, the formation of 1-D nanostructure is the process of crystallization. In chemical routes, the bottom-up approach usually was used to make the 1DNMs. Fig. 1 schematically illustrates some of these methods for controlling nucleation and crystal growth in the

Fig. 1. Schematic illustrations of six different strategies that have been demonstrated for achieving 1-D growth: (A) direction by the anisotropic crystallographic structure of a solid; (B) confinement by a liquid droplet as in the vapor-liquid-solid process; (C) direction through the use of a template; (D) kinetic control provided by a capping reagent; (E) self-assembly of 0-D nanostructures; and (F) size reduction of a 1-D microstructure. (From Xia YN, Yang PD, Sun YG, et al. One-dimensional nanostructures: synthesis, characterization, and applications. Adv Mater 2003;15:353–89; with permission.)

NANOMEDICINE IN CLINICAL SCIENCE

983

nanoscale: (1) a solid with anisotropic structure crystallographic was used for 1-D growth (see Fig. 1A); (2) the symmetry of a seed reduced by a liquid-solid interface (see Fig. 1B); (3) growing from a variety of 1-D templates (see Fig. 1C); (4) capping molecules introduced to control the growth rates of facets of a seed (see Fig. 1D); (5) 0DNMs self-assembled into 1DNMs (see Fig. 1E); and (6) size reduction of 1-D microstructures (see Fig. 1F). Although many kinds of 1DNMs have been fabricated, most of them were in a randomly oriented fashion. To make the highly oriented 1DNMs arrays useful in practical applications still is a big challenge. Two main methods, gas-phase synthesis and solution synthesis, are used to fabricate the oriented 1DNMs. The high temperature in the gas-phase synthesis makes the preparation of heat-sensitive nanomaterials (eg, biomaterials and organic materials) nearly impossible. Further, it is difficult to handle ceramic in the gas-phase synthesis because of the ceramic’s high melting point. The solution synthesis can avoid these problems. The composition, structure, and morphology can be controlled in the solution synthesis by adjusting the reaction parameters, such as precursors, pH, temperature, and so forth, properly. Mainly, the solution approach can be divided into the templating and templateless methods. Templating approach The template-based approach is used widely to fabricate nanotubes, NWs, and nanorods of metals, semiconductors, polymers, and biomolecules. Besides the desired pore or channel size, morphology, size distribution, and density of pores, template materials must meet the following criteria: (1) for the template electrochemistry synthesis, the template must be an electrical insulator; (2) the template should be chemically and thermally inert during the whole procedure; (3) the nanorods and NWs should be able to grow from one end to another end of the template, whereas nanotubes should grow from the pore wall and proceed inwardly; and (4) easy removal of these templates afterwards. There are limitations, however, in the template-based method. During the process of removing the templates, the orientation of nanostructures easily can be destroyed. In addition, these templates are difficult to be formed on textured surfaces or within a confined environment, such as a microfluidic channel. Thus, attention recently has been attracted to the templateless solution synthesis. Templateless approach Large-scale TiO2 nanotubes, ZnO nanorods, and polyanniline (PANI) NWs recently were fabricated successfully. Several important parameters, such as nucleation, nanocrystal growth orientations, 1DNM alignment on substrates, and interfacial chemistry, must be controlled well during the fabrications. According to the classic nucleation theory [74], demonstrated in

984

BREWER

et al

the equation, DG ¼ RTlnS þ scl þ (scs  ssl)Acs, four factors determine the free energy of forming stable nuclei on a substrate: the supersaturation degree (S), the interfacial energy (scl) between the particle (c) and the liquid (l), the interfacial energy (scs) between the particle and the substrate (s), and the interfacial energy (ssl) between the substrate and the liquid; A is the surface area of the particle. Fig. 2 shows the function between the number of nuclei (N) and the degree of supersaturation (S). There is a narrow region that is in favor of the formation of oriented nanostructures. The failure to obtain the oriented nanostructures is because of the too-high concentration used, which often results in the undesirable precipitatation. The following rules generally can be used to design and fabricate the 1DNMS from the solution synthesis according to the equation and function described previously [75]. 1. Usually, two critical parameters, low temperature and dilute concentration of precursors, need to be adjusted to control the supersaturation degree of solution, to inhibit the precipitation and at the same time ensure that nucleation and growth can proceed. The appearance of cloudiness is a sign of the formation of new materials, which can be monitored by light scattering or turbidity measurement. A rapid increase in cloudiness is usually is an indication of rapid precipitation, which should be avoided. 2. Try to decrease the interfacial energy between the particle and substrate. The conventional approach is to functionalize the substrate surface. A more feasible method is to grow the nuclei (seeds), which are same as the materials needed on the substrate surface, to minimize the energy barrier for the subsequent growth of 1DNMs. 3. Ensure the kinetic growth of oriented nanostructures is more favored over nonoriented structures. It is believed that in the very early stage, the nanostructures cannot be well aligned. As growth proceeds further, the space

Fig. 2. Idealized diagram for nucleation and growth. (From Bunker BC, Rieke PC, Tarasevich BJ, et al. Ceramic thin-film formation on functionalized interfaces through biomimetic processing. Science 1994;264:49; with permission.)

NANOMEDICINE IN CLINICAL SCIENCE

985

in-between these 1DNMs (eg, NWs or nanorods) can promote the oriented 1DNMs (growing along the normal direction of substrate) to grow rapidly. This kinetically controlled nucleation and growth strategy can result in the uniformly oriented 1DNMs on the substrates. This generalized approach can be applicable to preparing many kinds of highly oriented 1DNMs on a variety of solid substrates for many important applications, such as biosensors, drug delivery, electric devices, and so forth.

Systematically varying nanostructure morphology using selective surface adsorption Extended and oriented nanostructures show application potential in various fields. Currently, direct fabrication of complex nanostructures with controlled crystalline morphology, orientation, and surface architectures is, in general, a challenge for nanoscience researchers. Growing simple nanostructures Recently, Liu and colleagues [76] developed a simple solution synthesis, using sodium citrate as a structure-directing agent (surface modifier), to obtain complex and large arrays of oriented ZnO nanostructures with morphologies under a systematic control. The novel ZnO nanostructures are anticipated to have application potential in fields of piezoelectric transduction sensing in addition to the catalysis, optical emission, and so forth. Many organic and inorganic small molecules have the capability to control (promote or inhibit) crystal growth. Among them, sodium citrate is one that can strongly absorb on the surface of crystal surface to alter the surface properties and, thereafter, crystal growth behavior [77,78]. Long-oriented ZnO nanorods can be obtained easily under the help of the citrate (Fig. 3a). When more sodium citrate is added, the aspect ratio (height to width) of the ZnO rod is reduced greatly (see Fig. 3b), indicating that the citrate anions can slow down the growth of ZnO along the (001) orientation, probably because of the selective adsorption of the citrate on the (001) facet of the ZnO. On the primary ZnO rods (see Fig. 3b), the citrate can introduce thin nanoplatelets to grow sideways around the parent ZnO rod (see Fig. 3d). High-resolution scanning electron microscope (SEM) imaging shows that these nanoplatelets are uniform in thickness (7 nm on average) (see Fig. 3e). In a third growth, without using any citrate (7 nm on average), layered features mostly were ‘‘healed’’ and (001) growth restored (see Fig. 3f). It is among the first to show reversible in situ rod-to-plate morphology transitions in synthetic bioceramics growth. Such morphology transitions resemble those observed in abalone shells, where biomolecules are used as structure modifiers. Using multigrowth and high concentrations of citrate ions, large arrays of orientated helical ZnO columns composed of stacking nanoplates can be

986

BREWER

et al

Fig. 3. ZnO crystal morphology as a function of citrate concentrations. (A) ZnO with 0.2-mg sodium citrate in 30-mL solutions. (B) ZnO with 1.5-mg sodium citrate in 30-mL solutions. (C) Aspect ratios (height to width) as a function of citrate concentrations. (D) Secondary growth with citrate from (B). (E) High-magnification SEM image of 5- to 10-nm platelike nanofeatures on the (100) surfaces of the crystals. (F) ‘‘Healing’’ of the platelike nanofeatures after reaction without citrate ions. (From Tian ZR, Voigt JA, Liu J, et al. Complex and oriented ZnO nanostructures. Nat Mater 2003;2:821–6; with permission. Reprinted by permission from Macmillan Publishers Ltd: copyright Ó 2003.)

grown on the top of parent ZnO rods. The thickness of these platelets is uniform (approximately 15 nm) (Fig. 4a, b). This kind of structure resembles the microstructure of seashells (see Fig. 4c, d). This discovery gives valuable information regarding the synthesis of biomimetic materials. The columnto-plate transition also was achieved by using high-citrate concentrations (see Fig. 4e, f). This kind of transition can be used to fabricate the ZnO

NANOMEDICINE IN CLINICAL SCIENCE

987

Fig. 4. Oriented biomimetic ZnO nanostructures. (A) Oriented ZnO nanoplates. (B) Growth tip of oriented ZnO columns and plates. (C) Nacreous plate structure in red abalone. (D) Nacreous calcium carbonate columns and layers near the growth tip of a young abalone. (E) Platelike structures on top of ZnO bilayers. (F) Column-to-plate transition in the ZnO bilayers. (From Tian ZR, Voigt JA, Liu J, et al. Complex and oriented ZnO nanostructures. Nat Mater 2003;2:821–6; with permission. Reprinted by permission from Macmillan Publishers Ltd: copyright Ó 2003.)

bilayer structure composed of ZnO rods in the first layer and ZnO nanoplates in the second (see Fig. 4f). Photocatalytic degradation of volatile organic compound 4-chlorophenol (CLP) was performed using two kinds of ZnO samples. One is vertically oriented ZnO nanorods; the other is oriented

988

BREWER

et al

ZnO nanocolumns. These two samples were put vertically into the cells containing 30 ppm CLP. After 2.5 hours of sunlight irradiation, the concentration of CLP was decreased by 14% and 25%, measured by ultraviolet-visible spectroscopy (UV-vis). The enhanced photocatalytic property can be attributed to the high surface area or the specific active sites produced by these complex nanocolumns. In conclusion, organic surface modifier citrate ions play a critical role in systematically controlling the morphology of ZnO nanorods. Thereafter, the complex hierarchic nanomaterials can bring in some special properties, such as enhanced photocatalytic capability. Growing complex nanostructures Enormous effort currently is devoted to the controlled solution synthesis and fabrication of branched heteronanostructures of different sorts because of their unique application potential. Alivisatos’s and colleagues group has reported a process for controlled synthesis of branched (eg, tetrapods or dendritic heterostructures) and hyperbranched (eg, the ‘‘thorny balls,’’ treelike, or ‘‘spider-net’’) nanocrystals of CdTe and CdSe semiconductors [79–81]. The length and width of the branching arms and the degree of branching could be controlled by varying the amount and kind of organic surfactants (Fig. 5). These organic surfactants also act as the surface modifiers. Depending on the structure and the ratio of different surface modifiers used, hyperbranched structures (see Fig. 5e) with high (see Fig. 5d, f) and low branching (see Fig. 5a–c) degrees together with nanocrystal aggregation (see Fig. 5g–i) would be generated. The site-specific heterogeneous nucleation and the branch growth all are determined largely by surface modifiers [81]. Controlling morphology and spatial organization of nanostructures in nanometer–centimeter scales Controlling morphology, size, and structure is critical in the fabrication of nanomaterials because the nanomaterials’ properties can be changed by controlling these parameters. The effect of morphology on the properties of a nanomaterial has been well studied [82]. Controlling morphology of bioceramic zinc oxide nanostructures ZnO is a wide band-gap semiconductor with many useful properties [83,84]. In addition, ZnO is biosafe, biocompatible, and may be used for biomedical applications without coating. Thus, extensive efforts are focused on the fabrication of ZnO nanomaterials. In general, the fabrication methods of ZnO nanomaterials can be divided into gas-phase processes and solution syntheses. Various hierarchic morphologies, such as nanobelts, nanoheicals, nanorods, nanotubes, nanosprings, nanocombs, nanorings, nanotubes, nanocages, nanorotors, nanobriges, nanotrtrapods, and so forth, have been prepared by gas-phase processes (Fig. 6) [83].

NANOMEDICINE IN CLINICAL SCIENCE

989

Fig. 5. Effect of mono- and bifunctional phosphonic acids on particle morphology: (A–C) monofunctional phosphonic acid; (D, F) bifunctional carboxyethyl phosphonic acid; and (G–I) ethyl diphosphonic (EDPA). The ratios of TDPA to mono- or bifunctional phosphonic acid were varied, from 37:1 (A, D, G) to 11:1 (B, E, H) to 6.5:1 (C, F, I). All scale bars are 100 nm. The branched nanocrystal in (c) has a hexapod structure. (From Kanaras AG, Sonnichsen C, Liu H T, et al. Controlled synthesis of hyperbranched inorganic nanocrystals with rich three-dimensional structures. Nano Lett 2005;5:2164–7; with permission.)

Recently, attention has been attracted to the fabrication of ZnO nanostructures using the solution-based chemical route. Via the simple and cheap wet chemistry route, rod-, wire-, tube-, tower-, rotor-, doughnut-, and flowerlike ZnO nanostructures [85–88], together with oriented helical ZnO nanorod arrays, have been fabricated [89]. Precursors, pH, organic capping molecules,

990

BREWER

et al

Fig. 6. A collection of nanostructures of ZnO synthesized under controlled conditions by thermal evaporation of solid powders. Most of the structures presented can be produced with 100% purity. (From Wang ZL. Nanostructures of zinc oxide. Mater Today 2004;7:26–33; with permission.)

reaction temperature and time, concentrations of starting materials, pretreatments, and post-treatments are important in controlling the morphology of ZnO nanostructures prepared via the solution-based synthetic route. Liu’s group developed a more feasible approach that involves the sequential nucleation and growth for controlling the morphologies of ZnO [90]. First, ZnO nanorods were grown on a glass slide by a simple low-temperature, solution-based chemical route (Fig. 7a). Second, by adding bifunctional diaminoalkane molecules, tapered new crystals formed on the primary rods (see Fig. 7b). With a further growth, these small tapered crystals fused into a long hexagonal prismatic shape (see Fig. 7c). Wagon wheel– like crystals eventually were harvested from a likewise tertiary growth (see Fig. 7d). A variety of hierarchic ZnO nanostructures can be fabricated with this new multistep nucleation and growth (see Fig. 7e, f). It is believed that the diaminoalkane absorbed on the high-energy sites and reduced the surface energy, resulting in the heterogeneous nucleation. On that basis, a plethora of new complex nanostructures can be built via such rational design and hierarchic stepwise growths. SiO2 microspheres, for instance, can be used as the primary nanostructures to hierarchically fabricate oriented smart heteromicrostructures (Fig. 8) (see Tian and colleagues) [91]. Further, the biocompatible secondary ZnO rods readily could be grown on a magnetic iron oxide nanoparticle previously covered by a biocompatible

NANOMEDICINE IN CLINICAL SCIENCE

991

Fig. 7. Hierarchic ZnO crystals formed by multistep growth (SEM images): (A) primary ZnO crystals-bicrystals with the middle (0001) surface and the two flat (0001) terminal surfaces; (B) secondary crystals nucleated from the primary crystals; (C) second-order crystal structure with long secondary crystal branches; (D) tertiary crystal from renucleation on the secondary crystals in (C); (E) dense arrays of secondary structures; (F) tertiary crystal with long needles from three growth stages, using diaminobutane to nucleate branch crystals. The secondary crystal in (B) is observed at an early stage of growth (1 hour), and the crystals in (C–F) have had time (at least 6 hours in final growth step) to mature to equilibrium conditions. (From Sounart TL, Liu J, Voigt JA, et al. Sequential nucleation and growth of complex nanostructured films. Adv Funct Mater 2006;16(3):335–44; with permission.)

SiO2 shell. Such simple extensions in design and nanosynthesis would result in an unusual heteromicrostructure with integrated properties for potentially advancing nanomedical therapeutics, including clearing clogged arteries, breaking aggregated amyloids, and so forth. Controlling spatial organization of bioceramic zinc oxide nanostructures in nanometer–centimeter scales Fabricating large arrays of oriented nanostructures in nanometer–centimeter scales for industrial application is a challenge. In general, there are two approaches used in this type of nanofabricationdsolution-phase and gas-phase approaches. Solution-phase approach Vayssieres and colleagues developed a novel solution synthesis without templates and surfactants to fabricate metal oxides nanorods, NWs, and nanotubes in films on various solid substrates at large scale, low cost, and low temperatures (Fig. 9) [92–96]. A more feasible seeded growth process

992

BREWER

et al

Fig. 8. SEM images of the SiO2 microspheres and SiO2/ZnO heteromicrostructures: (A) SiO2 microspheres; (B) low-magnification SEM image of the heteromicrostructures; and (C–D) high-magnification SEM images of the heteromicrostructures. (From Zhang T, Dong W, Kasbohm J, et al. Design and hierarchial synthesis of branched heteromicrostructures. Smart Mater Struct 2006;15:N4; with permission.)

was reported by Tian and colleagues to fabricate oriented ZnO nanorods and TiO2 nanotubes in centimeter scales [76,89,97]. Thereafter, Yang’s group at Berkeley [98] successfully extended this method to produce homogenous and dense ZnO NWs on 4-inch silicon wafers and 2-inch plastic substrates for studying UV lasing (Fig. 10) properties. It is anticipated that with further improvements, these oriented ZnO NW

Fig. 9. SEM micrographs of ZnO-oriented microtube array chemically grown onto a transparent conducting oxide glass substrate. (From Vayssieres L, Keis K, Hagfeldt A, et al. Threedimensional array of highly oriented crystalline ZnO microtubes. Chem Mater 2001;13(12): 4395–8; with permission. Copyright Ó 2001, American Chemical Society.)

NANOMEDICINE IN CLINICAL SCIENCE

993

arrays can be applied to many electric devices, such as solar cells, UV lasers, and so forth. Recently, Liu’s group [99] found a novel solution synthesis, namely combining soft lithography and bioinspired crystal growth to make nanostructures of ZnO. The mCP 11-mercaptoundecanoic acid can self-assemble into a monolayer in the micropatterns on silver (Fig. 11). Here, the orientation and density controls are two important aspects in the spatial organization of nanostructure. Gas-phase approach Preparing oriented nanostructures in large scale and controlling them precisely at predetermined locations are important in practical applications, such as the nanolasing first reported by Yang’s group [100] demonstrateing that ZnO NWs can grow in the Au-coated areas (Fig. 12) in the gas-phase synthesis. The UV lasing was observed during the evolution of the emission spectra with increasing pump power. Further, growing large-area, hexagonally patterned, aligned ZnO nanorods was demonstrated by Wang and colleagues [101] with the help of microspheres’ self-assembled micropatterns. The ZnO nanorods have

Fig. 10. ZnO NW array on a 4-inch (approximately 10-cm) silicon wafer. At the center is a photograph of a coated wafer, surrounded by SEM images of the array at different locations and magnifications. These images are representative of the entire surface. Scale bars, clockwise from upper left: 2 mm, 1 mm, 500 nm, and 200 nm. (From Greene LE, Law M, Goldberger J, et al. Low- temperature wafer-scale production of ZnO nanowire arrays. Angewandte Chemie, International Edition 2003;42(26):3031–4; with permission.)

994

BREWER

et al

Fig. 11. Organized ZnO nanorod growth on Ag directed by SAM patterns. (A) Schematics of mCP SAM patterns on silver (Ag) surfaces (top) and resulting positioning of ZnO nanorods nucleating on the bare Ag regions that were delineated by the organic template (bottom). Xs in the top panel denote COOH endgroups of the SAM molecules, whereas Os in the bottom panel denote COO– HMT-Hþ complexes. (B) SEM images of ZnO nanorods organized in two rings. The surrounding regions are covered by HSC10H20COOH SAM molecules, where no ZnO was found. (C) Large-area patterns. ZnO nanorods appear white. (From Hsu JWP, Tian ZR, Simmons NC, et al. Directed spatial organization of zinc oxide nanorods. Nano Lett 2005; 5:83–6; with permission.)

uniform shape and length, align vertically on the substrate, and were distributed according to the pattern defined by the catalyst template (Fig. 13). Controlling morphologies of bioceramic titanium oxide–based 1-D nanostructures The development of nanoscale materials with novel morphologies is a focal point of nanomaterials research. Among the family of bioceramic nanomaterials, TiO2 and ZnO are studied extensively partially because of their usefulness in clinical science. Feng’s group, in 2004, reported an oriented organization of shape-controlled nanocrystalline TiO2, which is based on creating an interfacial interaction by chemical modification on the surfaces of NPs [102]. Nanorods or cubic nanocrystals can be organized in 2-D ordered arrays. Templated approach Because of the nanoscale anisotropic structures, there are challenges in controlling the morphology and spatial alignment of 1DNMs over large scales. The templates used most commonly or easily accessible for organizing 1DNMs are anodized alumina membrane [103], radiation track–etched polymer membranes [104], array glass [105], radiation track–etched mica [106], mesoporous materials [107], porous silicon by electrochemical etching of silicon wafer [108], zeolites [109], and CNTs [110,111].

NANOMEDICINE IN CLINICAL SCIENCE

995

Fig. 12. (A– E) SEM images of ZnO NW arrays grown on sapphire substrates. A top view of the well-faceted hexagonal NW tips is shown in (E). (F) High-resolution TEM image of an individual ZnO NW showing its !0001O growth direction. For the NW growth, clean (110) sapphire substrates were coated with a 1.0- to 3.5-nm thick layer of Au, with or without using TEM grids as shadow masks (mCP of thiols on Au followed by selective etching also has been used to create the Au pattern). Equal amounts of ZnO powder and graphite powder were ground and transferred to an alumina boat. The Au-coated sapphire substrates typically were placed 0.5 to 2.5 cm from the center of the boat. The starting materials and the substrates then were heated up to 880 C to 905 C in an Ar flow. Zn vapor is generated by carbothermal reduction of ZnO and transported to the substrates where ZnO NWs grow. The growth generally took place within 2 to 10 minutes. (From Huang MH, Mao S, Feick H, et al. Room-temperature ultraviolet nanowires nanolasers. Science 2001;292:1897; with permission.)

Alumina membranes with uniform and parallel porous structure are made by anodic oxidation of aluminum sheet in solutions of sulfuric, oxalic, or phosphoric acids [103,112]. The pores are arranged in a regular hexagonal array, with the densities as high as 1011 pores/cm2 [113] and pore size ranging from 10 nanometers to 100 micrometers [114]. In 1996, Patrick Hoyer [115] reported a preparation of TiO2 nanotube array from porous aluminum oxide mold. The tubular nanostructure was formed by electrochemical deposition in the mold. After dissolution of the template, an array of amorphous TiO2 nanotubes was formed with diameters ranging from 70 to 100 nanometers. The amorphous nanotube wall can turn into crystalline anatase after a heat treatment. In 1999, the direct preparation of anatase nanotubules in nanochannels of porous alumina membranes using a deposition technique from titanium tetrafluoride (TiF4) solutions was reported [116]. The tubes obtained had cylindric pores, which were found to consist of anatase NPs and contained mesoscale pores (shown in Fig. 14). Templateless approach In general, the template-based approach may encounter challenges when (1) removing the templates after the synthesis and (2) forming on textured

996

BREWER

et al

Fig. 13. (A) Low-magnification top-view SEM image of aligned ZnO nanorods grown onto a honeycomb catalyst pattern. (B) Side view of the aligned ZnO nanorods at an angle of 30 . (C, D) Top and a 30 view of aligned ZnO nanorods, where the hexagonal pattern is apparent. (E) Aligned ZnO nanorods at the edge of the growth pattern. (From Wang X, Summers CJ, Wang ZL. Large-scale hexagonal-patterned growth of aligned ZnO nanorods for nano-optoelectronics and nanosensor arrays. Nano Lett 2004;4:423–6; with permission.)

NANOMEDICINE IN CLINICAL SCIENCE

997

Fig. 14. SEM images of titania nanotubes deposited from a TiF4 solution (A–C) and a schematic model of deposition of anatase in a nanochannel and the bimodal pores of a nanotube (D). (From Imai H, Takei Y, Shimizu K, et al. Direct preparation of anatase TiO2 nanotubes in porous alumina membranes. J Mater Chemistry 1999;9(12):2971–2; with permission.)

surfaces or within a confined space, such as inside a microfluidic channel. Therefore, templateless solution synthesis of organized arrays of 1DNMs is sought. Recently, TiO2 1DNMs were synthesized without templates by Tian and colleagues (Fig. 15) [97].

Fig. 15. SEM images of large arrays of oriented TiO2-based nanotubes prepared at 150 C. (A) A low-magnification, face-on SEM image of the films after 6 hours of reaction. (B) A low-magnification SEM image of a 60 tilted sample after 6 hours. (C) A high-magnification SEM image of (B). (D) Curled foils (arrows) in transition to nanotubes after 3 hours. (From Tian ZR, Voigt JA, Liu J, et al. Complex and oriented ZnO nanostructures. J Am Chem Soc 2003;125:12384; with permission. Copyright Ó 2003, American Chemical Society.)

998

BREWER

et al

First, vertically aligned MW nanotubes readily are formed as a thin film on the substrate. On heating in a 10-mol/L sodium hydroxide solution for 3 hours, the TiO2 nanosized seeds on the substrate readily transform into a sheetlike nanostructure. A 6-hour hydrothermal heating can drive the nanosheets to start to fold into the nanotubes, and a 20-hour heating as such can lead to the formation of an oriented array of TiO2-based tubular 1DNMs on the substrate (see Fig. 15). This new alignment is believed driven by limited space in-between the close packed nanoseeds. Within the limited space, the nanotubes can grow nowhere but along the orientation perpendicular to the substrate surface. This sheet-folding process has highlighted a new synthetic progress involving an in situ structural transformation from nanosized bulk structures to sheetlike nanostructures and then to nanotubes organized vertically on the substrate surface. Unique structures in polyaniline nanowires for unique biosensing There are few reports on oriented organic nanomaterials, such as biomolecues [117] and polymers [118], because most of the fabrication techniques are difficult to apply to making polymeric 1DNMs. Oriented conducting polymer NWs, alternatively, have potential in important applications, including biologic sensing, artificial muscle fabrication, microelectronic devices and interconnects, energy conversion and storage, catalysis, optical emission, display, and data storage [119]. The favorable orientations would result in the large surface area, controllable diffusion, and high porosity, all of which are in high demand in development of clinic viable nanodevices. Templated approach PANI is unique among conducting polymers because of its attractive properties, such as low cost, good processability, unique chemical and physical properties, and excellent environmental stability [120–122]. Various methods are used to fabricate the PANI NW, including the template-directed synthesis and self-assembly growth. Martin [123] fabricated the PANI NW using polycarbonate and alumina membranes as templates. 3-D PANI NW networks were synthesized in a high yield by Yang’s group [124] using the ‘‘soft template’’ self-assembled by hexadecyltrimethylammonium bromide and oxalic acid. Depending on synthesis conditions or procedures, the networked PANI NWs are 35 to 100 nanometers in diameter. Yan’s group [125] reported a facile method to prepare helical PANI microwires or rods guided by polyacrylic acid. The average length and diameter of the helical strands were approximately 3.5 micrometers and 500 nanometers, respectively. The pitch distance was approximately 400 nanometers. The morphology of the helical PANI microwires was affected by the polyacrylic acid concentration. Then, a novel method for fabrication of highly oriented PANI NWs without removal of the template was developed by Wan’s group [126] via combining the self-assembly with the template techniques. By using a

NANOMEDICINE IN CLINICAL SCIENCE

999

self-assembly process under inhibition conditions, oriented arrays of PANI NWs growing out of the nanoporous template were obtained, with NW diameters ranging from 110 to 190 nanometers to lengths of several micrometers. Physical methods, such as electrospinnin [127,128] and mechanical stretching [129], also have been developed. There are some challenges in the templated fabrication of oriented arrays of the polymeric NWs. First, the random pore structures and misalignment of the polymers from the methods (described previously) are not ideal for high efficiency and fast kinetics [130], because the controlled orientation is critical for applications in light-emitting and microelectronic devices. Second, if using a membrane templating approach to grow oriented PANI NWs, the orientated PANI nanostructure is destroyed if the diameter of membrane supports is small [131]. In addition, the necessity to use a porous support membrane limits the applicability of this method to simple geometry or flat surfaces. Templateless approach Recently, Liu and colleagues [75] reported a new controlled nucleation and growth method for directly synthesizing large arrays of uniform and oriented PANI NWs on various smooth and textured substrates, such as platinum (Pt), silicone (Si), Au, carbon, or silica, without using a template. A large number of nuclei first were deposited on the substrate using a large current density during a stepwise electrochemical deposition process (Fig. 16). After the completion of the first step, the current density was reduced two times in order for the polymeric NWs to nucleate and then grow in the oriented fashion. Only high-density, short PANI NWs and thick-branched fibers that are hundreds of nanometers in diameter were formed from the nonstepwise experiments, showing that the stepwise control plays an important role in forming the long oriented PANI NWs. Not only are the polymer NWs well oriented but also the polymer molecules within the NWs are aligned based on the polarized IR spectroscopy. The SEM photographs depict oriented NWs uniform in length and diameter (Fig. 17). The diameters of the tips are from 50 to 70 nanometers and the length of the NWs approximately 800 nanometers. Oriented arrays of conductive polymers provide an ideal platform for a range of sensing applications. The reliable and sensitive amperometric detection of hydrogen peroxide (H2O2), for instance, is important in biosensing based on oxidase-type enzymes [132,133] and in ensuring the safety and quality of pharmaceutical and cosmetic formulations [134]. A chemical sensor for H2O2 was fabricated by using iron (III) hexacyanoferrate (FeHCF) NPs, which can function as the active ingredient for determination of H2O2 deposited on large oriented arrays of PANI NWs films. The presence of organic conducting polymers in a composite film can increase the stability of FeHCF [135–137]. The PANI/FeHCF sensor was

1000

BREWER

et al

Fig. 16. Schematic drawing of the steps for growing oriented PANI NWs. (A) Schematics of the reactions in the electrochemical cell. (B) Schematics of the nucleation and growth. (From Liu J, Lin YH, Liang L, et al. Templateless assembly of molecularly aligned conductive polymer nanowires: a new approach for oriented nanostructures. Chemistry 2003;9(3):605–11; with permission.)

fabricated electrochemically in two steps. First, oriented PANI NWs were deposited on the glassy electrode surface. Second, FeHCF was electrodeposited on the polymer-covered glassy electrode in an aqueous solution containing ferric choloride (FeCl3) and potassium ferrocyanide (K3Fe(CN)6). The almost linear response (catalytic reduction current versus H2O2 concentration) over a wide concentration range suggests that the PANI/FeHCF electrode is a useful configuration for H2O2 sensors (Fig. 18). Without FeHCF, the PANI glassy carbon electrode has a very low response. This sensor showed good stability and reliability. After six repetitive injections of 50 mL H2O2 solutions, the current has almost no change. Well-defined, reproducible peaks are observed at a low operation potential (0.10 V). The use of electrochemically polymerized PANI films to immobilize enzymes at electrode surfaces was reviewed; the interpretation and modeling of results from these studies are discussed by Bartlett and Cooper [138]. Biomimetically self-assembled nanoporous bioceramics Biomimetic silicates, such as seashells [139,140] and diatoms [141], display rich morphologies self-assembled precisely from nanometer to centimeter scales. Recently, attention has been paid to synthesis and self-assembly in this family of materials, including mesoporous oxides, patterned films, and hierarchic mesophase nanomaterials [142–146].

NANOMEDICINE IN CLINICAL SCIENCE

1001

Fig. 17. SEM micrographs of oriented PANI on a patient. (A) Low-magnification face-on. (B) High-magnification face-on. (C) Tilted view, low-magnification. (D) Tilted view, high-magnification. The image of the oriented NWs on Si substrate (inset). (From Liu J, Lin YH, Liang L, et al. Templateless assembly of molecularly aligned conductive polymer nanowires: a new approach for oriented nanostructures. Chemistry 2003;9(3):605–11; with permission.)

The first such self-assembled periodic silicates were prepared in 1992 from silicic acid and quaternary ammonium surfactants, in which surfactant liquid crystal structure serves as a template for the synthesis of the silicates. In this system, the silicate materials form inorganic walls between the ordered surfactant micelles, forming the ‘‘liquid-crystal templating’’ mechanism (Fig. 19). Burning off the templates afterwards can result in ordered nanopores ideal for drug loading and releasing. By controlling reaction conditions, such as the surfactant and auxiliary chemicals, the nanopore size can be controlled from 1.6 to 10 nanometers or more [147,148]. Since then, inorganic-organic hybrid mesoporous materials [149,150] and films [151,152] were prepared with silanes or bridged organic silsesquioxanes. To improve the applications of the organized nanopores, many synthetic methods are reported, including reactive and passive organic groups introduced in the porous solids via grafting and cocondensation strategies. Functional groups can be incorporated selectively on the surfaces of internal

1002

BREWER

et al

Fig. 18. Catalytic reduction currents of H2O2 at PANI/FeHCF-modified electrodes in a flow injection system. (A) Response of the sensor to H2O2 concentration change: (i) PANI/ FeHCF-modified glassy carbon electrode; (ii) PANI-modified glassy carbon electrode. Flow rate, 0.5 mL/min1; operating potential, þ0.10 V; carrier solution, (0.1M KCl þ 0.05 M acetate) buffer, pH 6.0. (B) Flow injection responses for repetitive injections of 50 ppm H2O2 at PANI/FeHCF-modified electrode. (From Liu J, Lin YH, Liang L, et al. Templateless assembly of molecularly aligned conductive polymer nanowires: a new approach for oriented nanostructures. Chemistry 2003;9(3):605–11; with permission.)

pore, for fine-tuning the surface adsorption/desorption properties of the nanoporous biosilicates. Recently, Inagaki’s group prepared well-defined hybrid nanoporous crystals using hexadecyltrimethylammonium chloride as a surfactant and 1,2-bis(trimethoxysilyl)ethane as a silica source [153]. In 2003, Tian and colleagues reported a similar synthetic work to prepare a new class of highly oriented superstacked crystallites hierarchically built from 5-mm sized building blocks of the self-assembled nanophase crystals through heterogeneous nucleation and growth [154]. High-order crystals were formed by repeated growths under the same conditions (Fig. 20). Under systematically varying experimental conditions (temperature, surfactant concentration, and reaction time), a wide range of new morphologies of the self-assembled mesophase crystals can be formed based on octahedron crystals’ edge-sharing stacking. As depicted in Fig. 21, the rich morphologies include triangular rosettes, round rosettes, flower-like patterns, stars, cubelike cages, and large rhombic dodecahedral structures.

NANOMEDICINE IN CLINICAL SCIENCE

1003

Fig. 19. Schematic illustration of the liquid-crystal templating mechanism. Hexagonal arrays of cylindric micelles form (possibly mediated by the presence of silicate ions), with the polar group of the surfactants (light gray) to the outside. Silicate species (dark gray) then occupy the spaces between the cylinders. The final calcinations step burns off the original organic material, leaving hollow cylinders of inorganic material. And transmission electron microscopy (TEM) image of the mesoporous silicate material (MCM-41). (From Kresge CT, Leonowicz ME, Roth WJ, et al. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature 1992;359:710; with permission. Reprinted by permission from Macmillan Publishers Ltd: copyright Ó 1992.)

Such unusual hierarchic structures and the complex morphologies would shed new light on self-assembly and crystal growth. This approach can be adapted for large-scale preparation on patterned substrates. The ability to form such ordered hierarchic structures spontaneously could lead to novel applications of self-assembled nanoporous materials for the development of new drug-delivery media. Tissue/organ regenerations using precisely engineered nanobiomaterials Gold long has been used in dentistry. Ivory, wood, and glass have been used for implant bones for long time. In 1937, the plastic, polymethyl methacrylate, first was used in dental prostheses and then in ophthalmology for intraocular lenses. Sutures, the first commercial biodegradable materials,

Fig. 20. Hierarchically ordered octahedral open crystal structures: (A) tertiary octahedral crystals; the insert shows small octahedral units nucleated on a secondary rosette crystal; (B) large open octahedral objects containing five primary octahedral units on each side; (C) large area view of high-order open structures; and (D) large open structures assembled from tertiary units. (From Tian ZR, Liu J, Voigt JA, et al. Hierarchical and self-similar growth of self-assembled crystals. Angew Chem Int Ed Engl 2003;42:413; with permission.)

1004

BREWER

et al

Fig. 21. Various morphologies of mesophase crystals formed on glass surfaces through polyhedral stacking: (A) triangular rosettes, (B) tilted triangular rosettes, (C) rounded rosettes, (D) tilted rounded rosettes, (E) flower-like patterns, (F) stars, (G) tilted stars, (H) cubelike cages by face-sharing growth, and (I) large rhombic dodecahedra by face-sharing growth. (From Tian ZR, Liu J, Voigt JA, et al. Hierarchical and self-similar growth of self-assembled crystals. Angew Chem Int Ed Engl 2003;42:413; with permission.)

were made from polyglycolic acid. In the 1980s, the application of biomaterials was focused mainly on bioactive materials. Recently, stem cells, combined with the development of new 3-D bioscaffolds, have been incorporated into tissue/organ regenerations, with a steadily growing interest in precisely engineered nanobiomaterials for applications in the regenerative medicine. Understanding the interactions between nanomaterials and tissue/organ may contribute significantly to the rational design of new-generation prostheses and postoperative patient management strategies [155,156]. In the body, in general, the engineered nanobiomaterials for tissue/organ regenerations should be either inert or passive, or they could interact with the body in specific and predictable ways. For mimicing natural underlying scaffolds of body tissues, artificial 3-D scaffolds can provide

NANOMEDICINE IN CLINICAL SCIENCE

1005

Fig. 22. The vision of regenerative medicine is to regenerate the soft and hard tissues, organs, and nerves responsible for major human disability. (From Stupp SI. Biomaterials for regenerative medicine. MRS Bull 2005;30:546; with permission.)

a similar environment for cell growth. Fig. 22 depicts a vision of the regenerative materials [57,157–164]. Regenerating hard tissues One of the focuses of tissue/organ materials in this area is on creating new 3-D scaffolds in which the cells can ‘‘talk’’ to each other in a precisely constructed porous environment [165]. For example, a spinal fusion product made by Medtronic works nicely for certain patients who have degenerative disk disease. It is a collagen sponge coated with bone morphogenetic protein inserted in a metal cage and placed in the spine for regenerative therapeutics (Fig. 23). Inside the bioscaffolds, the growth factor used can avoid the second surgery by harvesting bone tissue from a patient’s hip. In this practice, called ‘‘spinal fusion,’’ the metal cage repositions the spine and the collagen sponge with the growth factor serves to direct the growth of new bone tissues. Biocompatible scaffolds long have been used to induce the formation of bone in bone tissue engineering. The challenge is to fabricate the scaffold that can provide proper mechanical strength and a structural support. Calcium phosphate, a major component of natural bone, is main mineral phase of the bone [166,167]. Among these materials, the hydroxyapatite (HA), calcium hydroxyapatite (Ca10[PO4]6[OH]2), and b-tricalcium phosphate (b-TCP) popularly have been used for regeneration bone tissue [168–173].

1006

BREWER

et al

Fig. 23. (A–E) Scaffolds for tissue engineering: (A) porous polyurethane (magnification 30); (B) porous silicone (magnification 100; (C) expanded polytetrafluoroethane (magnification 600); (D) highly porous polyurethane (magnification 300); and (E) woven polyester with reinforcing wrap (magnification 66). (F) Modified expanded polytetrafluoroethane (magnification 400). (From Bergman RM. Innovations in biomaterials: achievements and opportunities. MRS Bull 2005;30:540; with permission.)

Up to now, many methods have been developed to prepare HA scaffolds with (1) large pores for the tissue cell to grow into and (2) enough mechanical strength to support body weight. For example, the replication of a polymer sponge, the incorporation of volatile organic particles in the HA powder, and gel casting of foams all are reported [174–179]. Lately, Zhang’s group improved the gel-polymer sponge process, which combines gel casting with polymer sponge methods [180]. HA nanofibers were prepared via a chemical precipitation process (Fig. 24). In this material, HA nanofibers in b-TCP matrix showed significantly improved mechanical properties of the porous scaffold. For example, the porous scaffold has 5% HA nanofibers with a porosity of 0.73%, a compressive

Fig. 24. (A) Transmission electron microscopy (TEM) micrograph of HA nanofibers prepared by a biomimetic method. (B) SEM micrograph of porous biphasic (TCP-HA) nanocomposite. (From Ramay HR, Zhang M. Preparation of porous hydroxyapatite scaffolds by combination of the gelcasting and polymer sponge methods. Biomaterials 2003;24:3293-302; with permission.) (C) SEM micrograph of sintered pore wall of biphasic (TCP-HA) nanocomposite scaffold.

NANOMEDICINE IN CLINICAL SCIENCE

1007

Fig. 25. (A–C) Bone regeneration therapy using marrow mesenchymal cells for bone graft applications for increased bioactivity: (A) bone marrow cells from a syringe are combined with porous HA (HAp [eg, coralline apatite]); (B) the marrow cell/HAp composite is cultured in an osteogenic medium; and (C) after 2–3 weeks of culture, the cultured bone graft/HAp hybrid is inserted into a bone defect. (D–F) Application of bone regeneration therapy method using marrow mesenchymal cells to a macrotextured orthopedic hip implant for increased bioactivity: (D) bone marrow cells are introduced into a joint prosthesis; (E) the prosthesis is cultured in an osteogenic medium; and (F) after 2–3 weeks of culture, the prosthesis is implanted in a total hip replacement. (From Yoshikawa T, Ohmura T, Sen Y, et al. In: Ben-Nissan, Sher D, Walsh W, editors. Bioceramics 15. Uetikon-Zurich: Trans Tech Publications, 2003. p. 383; with permission.)

strength of 9.8 MPa, and a toughness of 1.72 kN/m, comparable to the highend value of cancellous bone. Further, when cultured bone marrow cells are implanted into immunodeficient mice, these cells can combine with mineralized 3-D scaffolds to form highly vascularized bone tissue (Fig. 25). These cell/biomaterial nanocomposites can be used to regenerate new bone tissues with an excellent integration of the scaffold with bone for the good recovery [155,181]. Regenerating soft tissues An illustration of the structure of IKVAV and the self-assembly is shown in Fig. 26A. The SEM images of the self-assembled scaffolding nanostructures are shown in Fig. 26B. Fig. 26B shows 3-D networked scaffolds formed from the nanofibers. The nanofibers are 5 to 8 nanometers in diameter and hundreds of nanometers to a few micrometers in length. Furthermore, the scaffolds are a gel-like solid (see Fig. 26C–E). The transparent gel-like solid was obtained from mixing 1% peptide amphiphile aqueous solution with NPCs suspensions in media or physiologic

1008

BREWER

et al

Fig. 26. (A) Molecular graphics illustration of an IKVAV-containing peptide amphiphile molecule and its self-assembly into nanofibers. (B) Scanning electron micrograph of an IKVAV nanofiber network formed by adding cell media (DMEM) to a peptide amphiphile aqueous solution. The sample in the image was obtained by network dehydration and criticalpoint drying of samples caged in a metal grid to prevent network collapse (samples were sputtered with Au-palladium 0.l ms and imaged at 10 kV). (C, D) Micrographs of the gel formed by adding to IKVAV peptide amphiphile solutions (C) cell culture media and (D) cerebral spinal fluid. (E) Micrograph of an IKVAV nanofiber gel extracted surgically from an enucleated rat eye after intraocular injection of the peptide amphiphile solution. (From Silva GA, Czeisler C, Niece KL, et al. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004;303:1352; with permission.)

Fig. 27. Cell survival and morphology of NPCs encapsulated in IKVAV-PA gels or cultured on poly-(D-lysine) (PDL)-coated cover slips. Cell survival of encapsulated NPCs was determined by a fluorescent viability/cytotoxicity assay. Live cells fluoresce green due to the uptake and fluorescence of calcein in response to intracellular esterase activity; dead cells fluoresce red as a result of the entry of ethidium homodimer-1 through damaged cell membranes and subsequent binding to nucleic acids. Cell survival was determined at (A) 1 day, (B), 7 days, and (C) 22 days in vitro. (D) Transmission electron microscopy (TEM) of NPC encapsulated in an IKVAV-PA gel at 7 days. The cell has a normal ultrastructural morphology (N, nucleus; arrow, mitochondria). In addition, numerous processes can be seen in cross section (red asterisks) within the gel, surrounded by PA nanofibers (NF). (From Silva GA, Czeisler C, Niece KL, et al. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004;303:1352; with permission.)

NANOMEDICINE IN CLINICAL SCIENCE

1009

fluids in a 1:1 volume ratio. This solid-contained encapsulated dissociated NPCs or clusters of the cells are called neurospheres. The cells survived the self-assembly process and remained viable in 22 days (shown in Fig. 27A–C). The cells’ viability encapsulated in the nanofiber network (shown in Fig. 27D) was as good as the cells cultured on poly-D-lysine (PDL), a standard substrate used to culture many cell types. For the unique properties of the 3-D network, such as diffusion of nutrients, transition of oxygen and water, and other bioactive factors. The cells can survive for a long time. The TEM imagine of the scaffold with NPCs encapsulated showed a healthy and normal ultrastructural morphology. A recent article by Silva’s group reported another 3-D smart scaffold material in tissue repair. The scaffold was formed by the self-assembly of peptide amphiphile molecules and neural progenitor cells encapsulated in vitro. The self-assembly was triggered by mixing cell suspensions in media with dilute aqueous solutions of the molecules. The cells survive the growth of the nanofibers around them. The nanofibers gave the cells a neurite-promoting laminin epitope, IKVAV, at nearly van der Waals density. The nanofibers scaffold created a system for the fast differentiation of cells into neurons and limited the production of astrocytes [182].

References [1] National Science Foundation. National Nanotechnology Initiative: research and development FY 2002. Available at: www.nano.gov. [2] Koo OM, Rubinstein I, Onyuksel H. Role of nanotechnology in targeted drug delivery and imaging: a concise review. Nanomedicine 2005;1:193–212. [3] Prow TW, Salazar Jose H, et al. Nanomedicine-nanoparticles, molecular biosensors and targeted gene/drug delivery for combined single-cell diagnostics and therapeutics. Proc Soc Photo Opt Instrum Eng 2004;5318:1–11. [4] Gareth Hughes A. Nanostructure-mediated drug delivery. Nanomedicine 2005;1:22–30. [5] Kannan S, Kolhe P, Raykova V, et al. Dynamics of cellular entry and drug delivery by dendritic polymers into human lung epithelial carcinoma cells. J Biomater Sci Polym Ed 2004; 15:311–30. [6] Jevprasesphant R, Penny J, Attwood D, et al. Transport of dendrimer nanocarriers through epithelial cells via the transcellular route. J Control Release 2004;97:259–67. [7] Paleos CM, Tsiourvas D, Sideratou Z, et al. Acid- and salt- triggered multifunctional poly(propylene imine) dendrimer as a prospective drug delivery system. Biomacromolecules 2004;5:524–9. [8] Koo O, Rubinstein I, Onyuksel H. Camptothecin in sterically stabilized phopholipid micelles: a novel nanomedicine. Nanomedicine 2005;1:77–84. [9] Ye Hsin-Chi, Ho Yi-Ping, Wang Tza-Huei. Quantum dot-mediated biosensing assays for specific nucleic acid detection. Nanomedicine 2005;1:115–21. [10] Moghimi Moein S, Hunter Christy A, Murray Clifford J. Nanomedicine: current status and future prospects. FASEB J 2005;19:311–30. [11] Bulte Jeff WM. Magnetic nanoparticles as markers for cellular MR imaging. Journal of Magnetism and Magnetic materials 2005;289:423–7. [12] Yeh Hsin-chih, Ho Ping-Yi, Wang Tza-Huei. Quantum dot-mediated biosensiing asays for specific nucleic acid detection. Nanomedicine 2005;1:115–21.

1010

BREWER

et al

[13] Zhao W, Lin W, Tan W. Fluorenct Nanoparticles for Bactiria and DNA Detection in BioApplications of Nanoparticles. Toronto (Canada):University of Toronto; 2007. [14] Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination. Nat Biotechnol 1998;16:49–53. [15] Liang R, Qiu J, Cai P. A novel amperometric immunosensor based on three-dimensional sol-gel network and nanoparticle self-assemble technique. Analtica Chemic Acta 2005; 534:223–9. [16] Xu HX, Sha MY, Wong EY, et al. Multiplexed SNP genotyping using the Qbead Ô system: a quantum dot-encoded microsphere-based assay. Nucleic Acids Res 2003;31:e43. [17] Vaarno J, Ylikoski E, Meltola NJ, et al. New separation-free assay technique for SNPs using two-photon excitation fluorophore. Nucleic Acids Res 2004;32:e108. [18] Kawasaki Ernest S, Player A. Nanotechnology, nanomedicine, and the development of new, effective therapies for cancer? Nanomedicine 2005;1:101–9. [19] Agrawal A, Tripp Ralph A, Anderson LJ, et al. Real-time detectin of virus particles and viral protein expression with two-color nanoparticle probes. J Virol 2005;79(13):8625–8. [20] Ijiima S. Helical microtubules of graphitic carbon. Nature 1991;354:56. [21] Kostarelos K, Lacerda L, Partidos CD, et al. Carbon nanotube-mediated delivery of peptides and genes to cells: translating nanobiotechnology to therapeutics. J Drug Del Sci Tech 2005;15(1):41–7. [22] Cui DX, Tian FR, Kong Y, et al. Effects of single-walled carbon nanotubes on the polymerase chain reaction. Nanotechnology 2004;15:154–7. [23] Gooding JJ, Wibowo R, Liu J, et al. Protein electrochemistry using aligned carbon nanotube arrays. JACS 2003;125:9006–7. [24] Wang J, Liu G, Jan MR. Ultrasensitive electrical biosensing of proteins and DNA: carbonnanotube derived amplification of the recognition and transduction events. JACS 2004;126: 3010–1. [25] Callegari A, Cosnier S, Marcaccio M, et al. Functionalised single wall carbon nanotubes/ polypyrrole composites for the preparation of amperometriic glucose biosensors. J Mater Chem 2004;14:807–10. [26] Gavalas VG, Law SA, Ball JC, et al. Carbon nanotube aqueous sol-gel composites:enzymefriendly platforms for the development of stable biosensors. Anal Biochem 2004;329: 247–52. [27] Dai H, Hafner JH, Rinzler G, et al. Nanotubes as nanoprobes in scanning probe microscopy. Nature 1996;384:147. [28] De Heer WA, Chatelain A, Ugarte D. A carbon nanotube field-emission electron source. Science 1995;270:1179. [29] Teker K, Kousik S, Eric W, et al. Electronic sensing of antibodies using carbon nanotube devices. NSTI-Nanotech 2005;1:43–6. [30] Yu W, Pirollo KF, Yu B, et al. Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide. Nucleic Acids Res 2004;32:e48. [31] New RRC. Liposomes: a practical approach. Oxford: Oxford university Press; 1990. [32] Sdthi V, Onyuksel H, Rubinstein I. Liposomal vasoactive intestinal peptide. Meth Enzymol 2005;391:377–95. [33] Pain D, Das PK, Ghosh PC, et al. Increased circulatory half-life of liposomes after conjugation with dextran. J Biosci 1984;6:811–6. [34] Allen TM, Chonn A. Large unilamellar liposomes with low uptake in to the reticuloendothelial system. FEBS Lett 1987;223:42–6. [35] Lasic DD, Martin FJ, Gabizon A, et al. Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times. Biocheim Biophys Acta 1991;1070:187–92. [36] Torchilin VP, Levchenko TS, Whitemman KR, et al. Amphiphilic poly-N-vinylpyrrolidones: synthesis, properties and liposome surface modification. Biomaterials 2001;75: 3035–44.

NANOMEDICINE IN CLINICAL SCIENCE

1011

[37] Takeuchi H, Kojima H, Yamamoto H, et al. Evaluation of circulation profiles of liipososmes coated with hydrophilic polymers having different molecular weights in rats. J Control Release 2001;75:83–91. [38] Bally MB, Nayar R, Masin D, et al. Liposomes with entrapped doxorubicin exhibit extended blood residence times. Biochim Biophys Acta 1990;1023(1):133–9. [39] Bekersky I, Boswell GW, Hiles R, et al. Safety and toxicokinetics of intravenous liposomal amphotericin B (AmBisome) in beagle dogs. Pharmacol Res 1999;16(11):1694–701. [40] Salord J, Tarnus C, Muller CD, et al. Targeting of liposomes by covalent coupling with ecto-NADþ-glycohydrolase ligands. Biochim Biophys Acta 1995;1237(2):99–108. [41] Ahmad Farhan J, Khar Roop K. Nanotechnology: a revolution in the making. The Pharma Review 2005;4:49–56. [42] Berry CC, Curtis ASG. Functionalisation of magnetic nanoparticles for applications in biomedicine. J Phys D Appl Phys 2003;36:198–206. [43] Freitas RA. What is nanomedicine? Nanomedicine 2005;1:2–9. [44] Woo K, Hong J. Surface modification of hydrophobic iron oxide nanoparticles for clinical applications. IEEE Ttransactions On Magnetics 2005;41:4137–9. [45] Prasad Paras N. Emerging opportunities at the interface of photonics, nanotechnology and biotechnology. Mol Cryst Liq 2004;1:1–7. [46] Vladimir PZ, Kim Jin-Woo, Curiel DT, et al. Self-assembling nanoclusterrs in living systems: application for integrated phototherrmal nanodiagnostics and nanaotherapy. Nanomedicine 2005;1:326–45. [47] Keating CD. Nanoscience enables ultrasensitive detection of Alzheimer’s biomarker. PNAS, 102, (7), 2263–2264. [48] Couvreur P, Gruslain L, Lenaerts V, et al. Polymeric nanoparticles and microspheres. Boca Raton (FL): CRC Press; 1986. [49] Yang h, Lopina ST. Penicillin V-conjugated PEG-PAMAM star polymers. J Biomater Sci Polym Ed 2003;14:1043–56. [50] Quintana A, Raczka E, Piehler L, et al. Design and function of a dendrimer-based therapeutic nanodevice targeted to tumo cells through the folate receptor. Pharmacol Res 2002;19:1310–6. [51] Chauhan AS, Jain NK, Diwan PV, et al. Solubility enhancement of indomethacin with poly(amidoamine) dendrimers and targeting to inflammatory regions of arthritic rats. J Drug Target 2004;12:575–83. [52] Lavan DA, Lynn DM, Langer R. Moving smaller in drug discovery and delivery. Nat Rev Drug Discov 2002;1:77–84. [53] Donaldson K, Aitken R, Tran L, et al. Carbon nanotubes: a review of their properties in relation to pulmonary toxicology and workplace safety. Toxicolog Sci 2006;92(1):5–22. [54] Sargent T. The dance of molecules. New York: Thunder’s Mouth Press; 2006. [55] Tabata Y. Nanomaterials of drug delivery systems for tissue regeneration. Methods Mol Bio 2005;300:81–100. [56] Hardiingham T. Regional perspectives on tissue engineeringdview from a small island. Tissue Eng 2003;9:1063–4. [57] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6. [58] Langer RS, Vacanti JP. Tissue engineering: the challenges ahead. Sci Am 1999;280: 86–9. [59] Lysaght MJ, Hazelhurst AL. Tissue engineering: the end of the beginning. Tissue Eng 2004; 10:309–20. [60] Williams DJ, Sebastine IM. Tissue engineering and regenerative medicine: manufacturing challenges. IEE Proc Nanobiotechnol 2005;152(6):207–10. [61] Griffith LG, Naughton G. Tissue engineeringdcurrent challenges and expanding opportunities. Science 2002;295:1009. [62] Lavik E, Langer R. Tissue engineering: current state and perspectives. Appl Microbiol Biotechnol 2004;565:1–8.

1012

BREWER

et al

[63] Williams KA, Saini S, Wick TM. Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering. Biotechnol Prog 2002;18:951–63. [64] Roth EA, Xu T, Das M, et al. Inkjet printing for high-throughput cell patterning. Biomaterials 2004;25:3707–15. [65] Sipe JD. Tissue engineering and reparative medicine. Annals of the New York Academy of Sciences 2002;961:1–9. [66] Naughton GK. From lab bench to marketdcritical issues in tissue engineering. Ann N Y Acad Sci 2002;961:372–85. [67] Haddon RC. Carbon Nanotubes. Acc Chem Res 2002;36:997. [68] Xia YN, Yang PD, Sun YG, et al. One-dimensional nanostructures: synthesis, characterization, and applications. Adv Mater 2003;15(5):353–89. [69] Shimizu T, Masuda M, Minamikawa H. Supramolecular nanotube architectures based on amphiphilic molecules. Chem Rev 2005;105(4):1401–43. [70] Gao XY, Matsui H. Peptide-Based Nanotubes and their applications in bionanotechnology. Adv Mater 2005;17(17):2037–50. [71] F Cerrina, C Marrian, A path to nanolithography. MRS Bull, 1996;21:56. [72] Matsui S, Ochiai Y. Focused ion beam applications to solid state devices. Nanotechnology 1996;7:247. [73] Hong SH, Zhu J, Mirkin CA. Multiple ink nanolithography: toward a multiple-pen nanoplotter. Science 1999;286:523. [74] Bunker BC, Rieke PC, tarasevich BJ, et al. Ceramic thin-film formation on functionalized interfaces through biomimetic processing. Science 1994;264:48. [75] Liu J, Lin YH, Liang L, et al. Templateless assembly of molecularly aligned conductive polymer nanowires: a new approach for oriented nanostructures. Chemistry-A European Journal 2003;9(3):605–11. [76] Tian ZR, Voigt James A, Jun Liu, et al. Complex and oriented ZnO nanostructures. Nat Mater 2003;2:821–6. [77] Hidber PC, Graule TJ, Gauckler LJ. Citric acid: a dispersant for aqueous alumina suspensions. J Am Ceram Soc 1996;79:1857–67. [78] Biggs S, Scales PJ, Leong YK, et al. Effects of citrate adsorption on the interactions between zirconia surfaces. J Chem Soc Faraday Trans 1995;91:2921–8. [79] Manna L, Milliron DJ, Meisel A, et al. Controlled growth of tetrapod-branched inorganic nanocrystals. Nat Mater 2003;2:382–5. [80] Milliron DJ, Hughes SM, Cui Y, et al. Colloidal nanocrystals heterostructures with linear and branched topology. Nature 2004;430:190–5. [81] Kanaras AG, Sonnichsen C, Liu HT, et al. Controlled synthesis of hyperbranched inorganic nanocrystals with rich three-dimensional structures. Nano Lett 2005;5: 2164–7. [82] Hulliger J. Chemistry and crystal growth. Angew Chem Int Ed Engl 1994;33:143. [83] Wang ZL. Nanostructures of zinc oxide. Mater Today 2004;7:26–33. [84] Zhiyang Fan, Jia g lu, Zinc oxide Nanostructures: synthesis and properties. J Nanosci Nanotechnol 2005, 5, 1561–1573 [85] Vayssieres L. Growth of arrayed nanorods and nanowires of ZnO from aqueous solutions. Adv Mater 2003;15:464–6. [86] Wang Z, Qian XF, Yin J, et al. Large-scale fabrication of tower-like, flower-like, and tube-like ZnO arrays by a simple chemical solution route. Langmuir 2004;20:3441–8. [87] Gao XP, Zheng ZF, Zhu HY, et al. Rotor-like ZnO by epitaxial growth under hydrothermal conditions. Chem Commun 2004;12:1428–9. [88] Liang JB, Liu JW, Xie Q, et al. Hydrothermal growth and optical properties of doughnutshaped ZnO microparticles. J Phys Chem 2005;109:9463–7. [89] Tian ZR, Voigt JA, Liu J, et al. Biomimetic arrays of oriented helical ZnO nanorods and columns. J Am Chem Soc 2002;124:12954–5.

NANOMEDICINE IN CLINICAL SCIENCE

1013

[90] TL Sounart, Jun Liu, James A Voigt, et al. Sequential nucleation and growth of complex nanostructured films. Adv Funct Mater 2006;16(3):335–44. [91] Zhang T, Dong W, Jay Kasbohm, et al. Design and hierarchial synthesis of branched heteromicrostructures. Smart Mater Struct 2006;15:N46. [92] Vayssieres L, Beermann N, Lindquist S-E, et al. Controlled aqueous chemical growth of oriented three-dimensional crystalline nanorods arrays: application to iron (III) oxides. Chem Mater 2001;13(2):233–5. [93] Vayssieres L, Guo J, Nordgren J. Aqueous chemical growth of a-Fe2O3-a-Cr2O3 nanocomposite thin films. J Nanosci Nanotechnol 2001;1(4):385–8. [94] Vayssieres L, Keis K, Hagfeldt A, et al. Three-dimensional array of highly oriented crystalline ZnO microtubes. Chem Mater 2001;13(12):4395–8. [95] Vayssieres L. On the design of advanced metal oxide nanomaterials. Int J Nanotechnol 2004;1(1/2):1–41. [96] Vayssieres L, Graetzel M. Highly ordered SnO2 nanorod arrays from controlled aqueous growth. Angewandte Chemie, International Edition 2004;43(28):3666–70. [97] Tian ZR, Voigt JA, Liu J, et al. Large oriented arrays and continuous films of TiO2-based nanotubes. J Am Chem Soc 125(41):12384–5. [98] Greene LE, Law M, Goldberger J, et al. Low-temperature wafer-scale production of ZnO nanowire arrays. Angewandte Chemie, International Edition 2003;42(26):3031–4. [99] Julia WPH, Tian ZR, Neil CS, et al. Directed spatial organization of zinc oxide nanorods. Nano Lett 2005;5:83–6. [100] Huang MH, Mao S, Feick H, et al. Room-temperature ultraviolet nanowires nanolasers. Science 2001;292:1897–9. [101] Wang X, Summers CJ, Wang ZL. Large-scale hexagonal-patterned growth of aligned ZnO nanorods for nano-optoelectronics and nanosensor arrays. Nano Lett 2004;4:423–6. [102] Dong W, Pang G, Shi Z, et al. Oriented organization of shape-contolled nanocrystalline TiO2. Mater Res Bull 2004;39:433–8. [103] Furneaux RC, Rigby WR, Davidson AP. The formation of controlled-porosity membranes from anodically oxidized aluminium. Nature 1989;337:147. [104] Fleisher RL, Price PB, Walker RM. Nuclear tracks in solids. Berkeley (CA): University of California Press; 1975. [105] Tonucci RJ, Justus BL, Campillo AJ, et al. Nanochannel array glass. Science 1992;258:783. [106] Possin GE. A method for forming very small diameter wires. Rev Sci Instrum 1970;41:772. [107] Wu C, Bein T. Conducting polyaniline filaments in a mesoporous channel host. Science 1994;264:1757. [108] Fan S, Chapline MG, Franklin NR, et al. Self-oriented regular arrays of carbon nanotubes and their field emission properties. Science 1999;283:512. [109] Enzel P, Zoller JJ, Bein T. Intrazeolite assembly and pyrolysis of polyacrylonitrile. Chem Commun 1992;633. [110] Guerret-Piecourt C, Le Bouar Y, Loiseau A, et al. Relatin between metal electronic structure and morphology of metal compounds inside carbon nanotubes. Nature 1994;372:761. [111] Ajayan PM, Stephan O, Redlich P, et al. Carbon nanotubes as removable templates for metal oxide nanocomposites and nanostructures. Nature 1995;375:564. [112] Despic, Parkhuitik VP. Modern aspects of electrochemistry vol. 20. New York: Plenum; 1989. [113] Al Mawiawi D, Coombs N, Moskovits M. Magnetic properties of Fe deposited into anodic aluminum oxide pores as a function of particle size. J Appl Physiol 1991;70:4421. [114] Foss CA, Tierney MJ, Martin CR. Template synthesis of infrared-transparent metal microcylinders: comparison of optical properties with the predictions of effective medium theory. J Phys Chem 1992;96:9001. [115] Patrick H. Formation of titanium dioxide nanotube array. Langmuir 1996;12:1411–3. [116] Hiroaki Imai, Yuko Takei, Kazuhiko Shimizu. Direct preparation of anatase TiO2 nanotubes in porous alumina membranes. J Mater Chem 1999;9(12):2971–2.

1014

BREWER

et al

[117] Zhang SG. Fabrication of novel biomaterials through molecular self-assembly. Nat Biotechnol 2003;21(10):1171–8. [118] Liang L, Liu J, Windisch CF, et al. Angewandte Chemie-International Edition 2002;41(19): 3665–8. [119] Gangopadhyay R, De A. Conducting polymer nanocomposites: a brief overview. Chem Mater 2000;12(3):608–22. [120] MacDiarmid AG. Nobel Lecture: ‘‘Synthetic metals’’: A novel role for organic polymers. Rev Mod Phys 2001;73:701. [121] Doblhofer K, Rajeshwar K. Handbook of conducting polymers. New York: Marcel Dekker; 1998 [Chapter 20]. [122] Stejskal J, Gilbert RG. Polyaniline. Preparation of a conducting polymer. Pure Appl Chem 2002;74(5):857–67. [123] Martin CR. Nanomaterials: a membrane-based synthetic approach. Science 1994; 266(5193):1961–6. [124] Zhong W, Deng J, Yang Y, et al. Synthesis of large-area three-dimensional polyaniline nanowires networks using a ‘‘soft template’’. Macromolecular Rapid Communications 2005;26(5):395–400. [125] Lu X, Yu Y, Chen L, et al. Hollow nanometer-sixed polypyrrole capsules with controllable shell thickness synthesized in the presence of chitosan. Polymer 2005;46(14):5329–33. [126] Qiu HJ, Zhai J, Li SH, et al. Oriented growth of self-assembled polyaniline nanowire arrays using a novel method. Advanced Functional Materials 2003;13(12):925–8. [127] Doshi J, Reneker DH. Electrospinning process and applications of electrospun fibers. J Electrostatics 1995;35:151. [128] Renek DH, Yarin AL, Fong H, et al. Bending instability of electrically charged liquid jets of polymer solutions in electospinning. J Appl Physiol 2000;87:4531. [129] He HX, Li CZ, Tao N. Conductance of polymer nanowires fabricated by a combined eletrodeposition and mechanical break junction method. J Appl Phys Lett 2001;78:811–3. [130] Lee HS, Hong J. Chemical synthesis and characterization of polypyrrole coated on porous membranes and its electrochemical stability. Synth Met 2000;113:115. [131] Duchet J, Legras R, Demoustier-Champagne S. Chemical synthesis of polypyrrole: structure-properties relationship. Synth Met 1998;98:113. [132] Garjonyte R, Malinauskas A. Amperometric glucose biosensors based on Prussian blueand polyaniline-glucose oxidase modified electrodes. Biosens Bioelectron 2000;15:445. [133] Garjonyte R, Malinauskas A. Glucose biosensor based on glucose oxidase immobilized in electopolymerized polyprrole and poly(o-phenylenediamine) films on a prussian blue-modified electrode. Sens Actuators 2000;B63:122. [134] Wang J, Lin Y, Chen L. Organic-phase biosensors for monitoring phenol and hydrogen peroxide in pharmaceutical antibacterial products. Analyst 1993;118:277. [135] Ogura K, Endo N, Nakayama M, et al. Mediated activation and electroreduction of CO2 on modified electrodes with conducting polymer and inorganic conductor films. J Electrochem Soc 1995;142:4026. [136] Koncki R, Wolfbeis OS. Composite films of Prussian blue and n-substituted polypyrroles: fabricatin and application to optical determination of pH. Anal Chem 1998;70:2544. [137] Ikeda O, Yoneyama H. Polypyrrole film electrodes electrochemically doped with colloidal Prussian blue. J Electroanal Chem 1989;165:323. [138] Bartlett PN, Cooper JM. A review of the immobilization of enzymes in electropolymerized films. J Electroanal Chem 1993;362(1–2):1–12. [139] Jackson AP, Vincent JFV, Turner RM. The mechanical design of Nacre. Proc R Soc London Ser B Biol Sci 1998;234:415. [140] Belcher AM, Wu XH, Christensen RJ, et al. Control of crystal phase switching and orientation by soluble mollusc-shell proteins. Nature 1996;381:56. [141] Sumper M. A phase separation model for the nanopattering of diatom biosilica. Science 2002;295:2430.

NANOMEDICINE IN CLINICAL SCIENCE

1015

[142] Yang H, Coombs M, Ozin GA. Mesoporous silica with micrometer-scale designs. Adv Mater 1997;9:811. [143] Trau M, Yao N, Kim E, et al. Microscopic patterning of orientated mesoscopic silica through guided growth. Nature 1997;390:674. [144] Yang P, Deng T, Zhao D, et al. Hierarchially ordered oxides. Science 1998;282:2244. [145] Yang P, Wirnsberger G, Huang HC, et al. Mirrorless lasing from mesostructured waveguides patterned by soft lithography. Science 2000;287:465. [146] Doshi DA, Heusing NK, Lu M, et al. Optically defined multifunctional patterning of photosensitive thin-film silica mesophases. Science 2000;290:107. [147] Kresge CT, Leonowicz ME, Roth WJ, et al. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature 1992;359:710. [148] Beck JS, Vartuli JC, Roth WJ, et al. A new family of mesoporous molecular sieves prepared with liquid crystal templates. J Am Chem Soc 1992;114:10834. [149] Inagaki S, Guan S, Fukushima Y, et al. Novel mesoporous materials with a uniform distribution of organic groups and inorganic oxide in their frameworks. J Am Chem Soc 1999; 121:9611. [150] Lim MH, Stein A. Dual templating of macroporous silicates with zeolitic microporous frameworks. Chem Mater 1999;121(17):4308. [151] Dag O, Yoshina-Ishii C, Asefa T, et al. Oriented periodic mesoporous oranosilica (PMO) film with organic functionality inside the channel walls. Adv Funct Mater 2001;11:213. [152] Lu Y, Fan H, Doke N, et al. Evaporation-Induced Self-Assembly of Hybrid Bridged Silsesquioxane Film and Particulate Mesophases with Integral Organic Functionality. J Amer Chem Soc 2000;122(22):5258–61. [153] Guan S, Inagaki S, Ohsuna T, et al. Cubic hybrid organic-inorganic mesoporous crystal with a decaoctahedral shape. J Am Chem Soc 2000;122:5660. [154] Tian ZR, Liu J, Voigt JA, et al. Hierarchical and self-similar growth of self-assembled crystals. Angew Chem Int Ed Engl 2003;42:413. [155] Ben-Nissan B. Nanoceramics in biomedical applications. MRS bull 2004;29:28. [156] Samuel I. Biomaterials for regenerative medicine. MRS bull 2005;30:546. [157] Lendlein A, Langer R. Biodetgradable, elastic shape-memory polymers for potential biomedical applications. Science 2002;296:1673. [158] Teng YD, Lavik E, Qu X, et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Natl Acad Sci USA 2002;99:3024–89. [159] Niklason LE. Functional Arteries grown in vitro. Science 1999;284:489. [160] Wald HL, Georgios S, Lyman MD, et al. Cell seeding in porous transplantation devices. Biomaterials 1993;14:270. [161] Yannas IV, Burke JF, Orgill DP, et al. Wound tissue can utlize a polymeric template to synthesize a functional extension of skin. Science 1982;215:174. [162] Mooney DJ, Baldwin DF, Suh NP, et al. Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) with out the use of organic solvents. Biomaterials 1996; 17:1417. [163] Hing KA, Best SM, Bonfield W. Characterization of porous hydroxyapatite. J Mater Sci Mater Med 1999;10:135–45. [164] De Oliverira JF, De Aguiar PF, Rossi AM, et al. Effect of process parameters on the characteristics of porous calcium phosphate ceramics for bone tissue scaffolds. Int Soc Art Org 2003;27:406–11. [165] Bergman RM. Innovations in biomaterials: achievements and opportunities. MRS bull 2005; 30:540. [166] Bucholz RW, Carlton A, Holmes RE. Hydroxyapatite and tricalcium phosphate bone graft substitutes. Orthop Clin North Am 1987;18:323–34. [167] Hill PA. Bone remodeling. Br J Orthod 1998;25:101–7. [168] Martin RB. Bone as a ceramic composite material. Mater Sci Forum 1999;293:5–16.

1016

BREWER

et al

[169] Nunes CR, Simske SJ, Sachdeva R, et al. Long term ingrowth and appositions of porous hydroxyapatite implants. J Biomed Mater Res 1997;36:560–3. [170] Heise, Osborn JF, Duwe F. Hydroxyapatite ceramic as a bone substitute. Int Orthop 1990; 14:329–38. [171] De Groot K. Bioceramics consisting calcium phosphate slats. Biomaterials 1980;1:47–50. [172] Cerroni L, Filocamo R, Fabbri M, et al. Growth of osteoblast like cells on porous hydroxyapatite ceramics: an in vitro study. Biomol Eng 2002;19:119–24. [173] Sepulveda P. Gelcasting foams for porous ceramics. Am Ceram Soc Bull 1997;76:61–5. [174] Lyckfeldt O, Ferreira JMF. Processing of porous ceramics by a new direct consolidation technique. J Eur Ceram Soc 1998;18:131–40. [175] Woyansky JS, Scott CE, Minnear WP. Processing of porous ceramics. Am Ceram Soc Bull 1992;71:1674–81. [176] Sepulveda P, Binner JGP, Rogero SO, et al. Production of porous hydroxapatite by the gelcasting of foams and cytotoxic evaluation. J Biomed Mater Res 2000;50:27–34. [177] Chu TMG, Halloran JW, Hollister SJ, et al. Hydroxyapatite implants with designed internal architecture. J Mater Sci 2001;12:471–8. [178] Jingwang Li, Tian J. Calculated influences of starting materials composition on carbothermal nitridation synthesis of silicon nitried/silicon carbide composite powders. J Mater Sci 2001;36:3061–6. [179] Zhang Y, Zhang M. Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants. J Biomed Mater Res 2002;61:1–8. [180] Ramay HR, Zhang H. Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods. Biomaterials 2003;24:3293–302. [181] Paunesku T, Rajh T, Wiederrecht G, et al. Biology of TiO2 – oligonucleotide nanocomposites. Nat Mater 2003;2:343–6. [182] Gabriel AS, Catherine C, Krista LN, et al. Selective Differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004;303:1352–5.