Nanomaterials in medicine and pharmaceuticals: nanoscale materials developed with less toxicity and more efficacy

Optical imaging (OI)

OI is an important and challenging technique in the biomedical field due to its high temporal and spatial resolutions (6–8). In living organisms, OI employs a sensitive camera to detect fluorescence emitted from fluorophores (9). Conventional fluorophores, generally including organic fluorophores, quantum dots, fluorescent proteins and luminescent transition metal complexes, have been widely used (10–15). Among them, fluorophores with long emission at the NIR region are generally preferred in living tissue for their great properties such as the lower autofluorescence and greater penetration depth (16). It was reported that lanthanide (Ln)-doped upconversion nanoparticles (UCNPs) had tremendous advantages over conventional fluorophores, such as low cytotoxicity, non-photobleaching, non-photoblinking and high spatial resolution etc (17–25). Chatterjee et al. reported that UCNPs could enhance penetration depth into tissues upon NIR excitation (26). In addition, there is a significant decrease of auto-fluorescence from surrounding tissues (25, 27). Due to the low vibration energy, fluoride-based UCNPs have also been identified as one of the most efficient upconversion fluorescent nanoparticles nowadays (28). Chatterjee et al. have further demonstrated that the luminescence from Ln-doped UCNPs could be still clearly observed upon NIR excitation when the nanoparticles were located ~10 mm beneath the skin in vivo animal imaging (26). In summary, Ln-doped UCNPs hold great potential as novel fluorophores in biomedical field (29, 30).

Another class of versatile tool for in vivo fluorescence imaging is semiconductor nanocrystals or quantum dots (QDs) (31–34). QDs typically have a core/shell structure of 2–8 nm in diameter, and are comprised of atoms of elements from group II to VI or III to V in the periodic table (35, 36). QDs are attractive for tumor imaging, which possess a broad excitation bands and narrow emission bands (37, 38). In addition, they also have other unique optical properties for in vivo optical imaging such as superior fluorescence, resistance to photo bleaching, and long blood circulation time (39, 40). After QDs conjugated with poly-N-isopropyla-crylamide (PNIPAM) were injected intravenously via the tail vein in mice bearing JHU-31 prostate cancer tumors for 3 h, tissues were cryosectioned for imaging. It was found that the QD-PNIPAM was localized at the periphery of the tissue sections while unmodified QDs were not found in the cancer tissue (41). Following by targeting of the prostate tumor cells, QDs conjugated with antibodies to the prostate-specific membrane antigen (PSMA) accumulated at the tumor xenograft in live nude mice (34). Tail vein injection showed that untargeting or passive targeting of QD probes demonstrated weak or no signals, while antibody-conjugated QDs showed intense signals inside the tumor. Because the receptor of folic acid was overexpressed on the surface of many cancer cells, including ovarian, lung, breast, endometrial, renal, and colon cancer cells, folate-QD conjugates were specifically detected in cancer cells compared to normal cells. Now CdSe QDs conjugated with folate receptors were employed for targeting mouse lymphoma cells in vivo (42). Now NIR emitting QDs were developed in order to minimize light absorption by blood and water and improve tissue penetration depth. Diagaradjane et al. conjugated QD800 (emission peak at 800 nm) with anti epidermal growth factor receptor (EGFR) antibody in order to specifically target EGFR overexpressed at tumor sites (43). Cai et al. reported the use of QD705 conjugated to the arginine-glycine-aspartic acid (RGD) tri-peptide receptors following tail vein injection in mice bearing human glioblastoma xenografts (44). Hu et al. employed NIR emitting QDs conjugated with cyclic RGD peptide for targeting in vivo tumor vasculature (45). In addition, other groups have also successfully developed procedures for multiplexed fluorescent labeling of tissue specimens by QDs (46–50).

With the increasing availability of fluorescent probes, OI is gaining momentum as an important translational tool between basic research and clinical application. Although traditional small molecule NIR dyes continue to be used, the development of fluorescent organic, biological, and inorganic nanoparticles for in vivo OI offers powerful tools for some exciting applications. The continued evolution of these new technologies will allow more molecular targets and diseases to be interrogated in vivo.

Magnetic resonance imaging (MRI)

MRI is a non-invasive imaging technique, which uses a strong magnet and radiofrequency waves to produce images of internal organs. Colloidal iron oxide nanoparticles (IONPs) generally produce enhanced proton relaxation rates at significantly lower doses than paramagnetic ions due to their larger magnetic moment, and provide negative contrast by enhancing T₂ relaxivity of water protons. So, colloidal IONPs, such as superparamagnetic iron oxides (SPIOs) and ultra superparamagnetic iron oxides (USPIO), have been widely explored in MRI field.

Several SPIO agents with a variety of surface coatings have been extensively studied for the detection and diagnosis of solid tumors (51). It was found that SPIOs were mostly taken by macrophages in the liver and spleen and subsequently metabolized over several days after intravenous injections. Active targeting of SPIOs can lead to increased contrast enhancement of tumors over non-targeted tissues. SPIOs functionalized with antibodies have been used to image rectal carcinoma and breast cancer (52, 53). Although passive targeting of iron oxide NPs to tumors can be achieved through the EPR effect, a range of molecules have been attached on their surface to improve tumor targeting for MRI applications, including proteins, antibodies, peptides, and oligosaccharides (54). Researchers have recently shown preferential accumulation of SPIOs functionalized with a targeting peptide overexpressed in MMP-2 glioma tumors (55). Folic acid, a vitamin, is another ligand, which has been extensively used as targeting agent (56, 57). Iridium complexes have been loaded into magnetic NPs for dual-modal luminescent and magnetic resonance imaging.

USPIO with diameters <40 nm have also been clinically investigated as contrast agents, which can accumulate at the margins of human brain tumors, improve their delineation on MRI (56). Researchers have compared the utility of USPIOs with Gd-chelates for brain-tumor MRI and found that USPIOs could offer prolonged delineation due to lower diffusion from tumor sites and increased internalization by tumor cells (58).

In addition, it was also reported that some nanomaterials might be used in ultrasound imaging (UI) and radionuclide imaging (RI) fields (59–62). Recent advances in nanotechnology and biotechnology have contributed to the development of multifunctional nanoparticles as representative nanomedicine. Because each imaging modality has its pros and cons, the integration of several imaging agents with different properties into multifunctional nanoparticles allows precise and fast diagnosis of disease through synergetic multimodal imaging. The multifunctional probes have already appeared from iron-oxide and dendrimer-based dual MRI fluorescence imaging contrast agents to a fused X-ray CT fluorescence imaging system (63, 64). Nanoscale multimodal imaging probes which carry more than two imaging agents have the potential to overcome the limitations of a single imaging modality and provide more detailed information of the target site through targeted delivery (8, 65, 66).

In summary, this is an active area in the future because clinicians need a contrast agent that can be specifically targeted to disease tissue allowing for more accurate diagnosis of various stages of diseases.

Photodynamic therapy (PDT)

Photodynamic therapy is known as a noninvasive medical technology to treat cancer. In the process of PDT, the photosensitizer (PS) molecule transfers the photon energy to surrounding oxygen molecules, which will generate reactive oxygen species (ROS) to kill cancer cells under the irradiation of light with appropriate wavelengths (72, 73). Compared to traditional chemotherapies and radiotherapies, PDT cancer treatment shows remarkably reduced side effects and improved selectivity since only the lesion is exposed to the light, while other tissues in the dark are not affected. An important benefit of PDT is that it can be used repetitively without any immunosuppressive or myelosupressive effects. It can also be used after surgery, radiotherapy, and chemotherapy (74). Because most of the PS employed in PDT are hydrophobic and show poor water solubility, this makes them difficult to administer these drugs for in vivo applications. To increase the water solubility of PS molecules and improve their delivery into cancer cells, various nanomaterials have been developed (75).

Upconversion luminescence (UCL) refers to a non- linear optical process that converts two or more lower-energy pump photons from the NIR spectral region to a higher-energy output photon with a shorter wavelength (76, 77). In recent years, UCNPs that exhibit UCL emission have been developed. Comparing to traditional fluorescent probes, they have shown many benefits including the free of auto-fluorescence background, higher photochemical stability and tissue penetration depth (78–81). UCNPs have the ability to convert NIR light to visible photons, which can activate PS absorbed on nanoparticles to generate ROS to kill cancer cells. Wang et al. loaded Chlorin e6 (Ce6) on polymer-coated UNCPs to form an UCNP-Ce6 complex that can produce singlet oxygen and further kill cancer cells under NIR light, and excellent PDT efficiency is also achieved in tumor-bearing mice (Figure 2) (82).

Figure 2

A schematic drawing showing NIR-induced PDT using UCNP-Ce6 (82).

Another biggest success in PDT is the use of Au nanoparticles. Considering that Au nanoparticles can maintain the stability and activity of hydrophobic PS in aqueous environments, there have been considerable efforts to develop Au nanoparticles as PS delivery vehicles to overcome the shortcomings of PS, for example, poor solubility and limited selectivity towards cancerous tissue (83). Gamaleia et al. have synthesized a conjugate of hematoporphrin with Au nanoparticles and evaluated its photodynamic activity. The results show that its PDT activity is much higher than that of the original PS in vitro (84).

It was reported that QD-PS complexes had better advantages over PS alone due to the high photoluminescence quantum efficiency, tunable optical properties and imaging capabilities (85, 86). The conjugation of QD to PS provides flexibility and further activates the PS molecule by utilizing variable excitation wavelength since the QDs can exhibit broad absorption spectra. Peptide-coated QDs can form extremely stable conjugates with PS, which may be used as targeted multifunctional probes for live cell targeting, imaging and PDT (73).

In addition, other kinds of PS-loaded nanoparticles, such as ceramic, liposome, and silica-based nanoparticles have been reviewed by Chatterjee et al. (87).

Photothermal therapy (PTT)

Photothermal therapy is an experimental use of electromagnetic radiation that is proposed to treat various diseases, including cancer. This activation brings the sensitizer to an excited state where it releases vibrational energy. The heat is the actual method of therapy that kills the targeted cells (88). Unlike PDT, PTT does not require oxygen to interact with the target cells or tissues (89). Current studies also show that PTT is able to use longer wavelength light, which is less energetic and therefore less harmful to other cells and tissues.

Carbon nanotubes (CNTs) are able to absorb light in the NIR region and produce heat (90). This exclusive property has been exploited as a method to kill cancer cells via thermal effects (91). A number of earlier studies have shown the promise of carbon nanotubes for photothermal ablation of cancer cells in vitro (92, 93). Recently, it was reported that intra-tumor injections of multi-walled carbon nanotubes (MWNTs) and single walled carbon nanotubes (SWNTs) followed by NIR laser irradiations at powers of 2.5 W/cm² and 3.8 W/cm², were able to destroy tumors grown on mice (94, 95). In a study conducted by Liu et al., SWNTs functionalized by PEGylation achieved an optimal blood circulation half-life at 12–13 h, afforded relatively low reticuloendothelial systems (RES) accumulation, high tumor uptake and low skin retention in mice, these results are ideal for cancer treatment in vivo (96).

As the sister of CNTs, nanographene can be also utilized in PTT due to the ability of NIR optical absorption (97–99). In the recent work, Yang et al. studied the in vivo behaviors of nanographene sheets (NGS) with PEG coating by a fluorescent labeling method. The results showed high tumor uptake of NGS in several xenograft tumor mouse models. Compared to PEGylated CNTs, PEGylated NGS shows some advantages in vivo behaviors including highly efficient tumor passive targeting and relatively low retention in RES (100).

Au NPs include gold nanorods, gold nanoshells and hollow Au NPs and so on, they show largely red-shifted properties which may have great potential in PTT (100–107). Similar to scatter counterpart, Au NPs absorb light millions of times stronger than the organic dye molecules. Nearly 100% absorbed light is converted to heat via the nonradiative properties (108). These features make them a new cohort photothermal contrast agents for PTT (109). It was reported that gold nanoshells demonstrated highly efficient NIR hyperthermia ability due to its strong NIR absorption (102, 110). Huang et al. reported gold nanorods as NIR hyperthermia agents for photothermal cancer therapy (111). Because the surface chemistry is a key factor for the cellular uptake and tissue penetration of nanomaterias (112), it can affect thermal therapy efficiency. Jin et al. have synthesized Au nanorods with three different polymer coatings: cetyltrimethylammonium bromide (CTAB), polystyrene sulfonate (PSS) and poly (diallyldimethylammonium chloride) (PDDAC). Then multicellular tumor spheroid (MCTS) was treated by these different nanorods, after radiated under the NIR laser for 4 min, the thermal therapy efficiency of each kind of Au nanorods is very different. In the PSS group, the compact structure of the MCTSs was broken by the Au nanorods thermal therapy, but this phenomenon in the other two groups was not observed. The result indicated that the difference in thermal therapy efficacy was due to the effective heat generated by the Au nanorods with different surface coatings, which were affected by different Au nanorods distribution patterns in the tumor (113).

Magnetic hyperthermia (MH)

Magnetic hyperthermia is based on the fact that magnetic particles, when subjected to an alternating magnetic field (AMF), produce heat. As a consequence, if magnetic particles are put inside a tumor and the whole patient is placed in an alternating magnetic field of well-chosen amplitude and frequency, the temperature in the tumor would rise and further kill cells (114).

Magnetic nanoparticles like magnetite have shown an intriguing ability to mediate a high rate of heat induction at low concentrations (115). The conversion of electromagnetic energy into heat by magnetic nanoparticles has great potential as a non-invasive and powerful therapy technique. For example, magnetic fluid hyperthermia (MFH) can be used to generate localized heating for tumor repression (116). By targeting the magnetic nanoparticles at the tumor site with an applied AMF, the temperature at tumor sites can be increased to 42–46°C, which largely reduces the viability of cancer cells (114, 117, 118). Previous studies have focused on designing and optimizing the size, composition and structure of magnetic nanoparticles to maximize the specific absorption rate (SAR) of MFH (119, 120). The size distribution of nanoparticles has a crucial influence on the SAR, where monodispersed magnetic nanoparticles show a larger SAR than polydispersed magnetite nanoparticles (121). Ma et al. found that magnetite nanoparticles with a size of 46 nm exhibited the highest SAR, whereas Hergt and Dutz reported that 30 nm magnetite nanoparticle suspensions had the highest SAR (114, 122). Other than the effect from the intrinsic nature of the inorganic component, a useful approach to improve the performance of magnetic nanoparticles is the optimization of the surface coating (117). Recently, the study on surface coating on magnetic nanoparticles has made a great progress. Liu et al. have successfully synthesized a highly monodispersed magnetite nanopaticles which are coated by mPEG. These nanoparticles show a good thermal conductivity and a high SAR, this may provide a chance for high performance hyperthermia agents (116).

Sonodynamic therapy (SDT)

Sonodynamic therapy is a recently developed anticancer treatment where ultrasound is used to activate the cytotoxic effect of chemical compounds, known as sonosensitizers. SDT diverges from PDT, as drug activation is achieved through ultrasound rather than light. Moreover, SDT is able to overcome the main drawback of PDT. Whether pulsed or continuous ultrasound is considered to be safer and more readily accepted by the patient than either radiotherapy or chemotherapy (123). In addition, ultrasound may be used as an external remote to control over time, dose, and location of drug release, provide tailor-made and localized cancer therapy (124).

Nanomaterials have some features that make them particularly suitable for SDT. For example, TiO₂ NPs are useful for SDT owing to their special properties (125). Wang et al. reported that various ROS could be generated when the nanosized TiO₂ powder was irradiated by ultrasound (126). Harada et al. have introduced ultrasound therapy combining with TiO₂ NPs on melanoma cells. The results demonstrated that the cell viability was significantly decreased after ultrasound irradiation in the presence of TiO₂ NPs (127).

Cryosurgery (CS)

Cryosurgery is the application of extreme cold to destroy abnormal or diseased tissue. It is appropriate for treating localized diseases, and solid tumors larger than 1 cm, but the tiny or diffuse metastases are usually not affected by cryosurgery. Generally, all tumors that can be reached by the cryoprobes during an operation are treatable (128–130). However, in many clinical cases, it has been found that freezing alone could not completely destroy the targeted tumor, moreover, there is always a high recurrence rate with follow-up surveys (131, 132), the major reason concerns the freezing rate, which does not produce a massive ice nucleation in tumor cells, especially at the edge of the tumor, thus the procedure cannot guarantee complete lethality to all of the tumor cells. Consequently, a major concern in conventional cryosurgery is to avoid insufficient freezing between multiple cryoprobes and maximize the freezing efficiency.

To establish an always-efficient freezing treatment for tumors, nanoparticles were introduced to significantly mediate various freezing procedures, which finally led to a generalized tumor-treatment concept called nanocryosurgery (Figure 3) (133). According to theoretical interpretation and experimental measurements, intentional loading of nanoparticles with high thermal conductivity into the target tissues can lower the final temperature appreciably, increase the maximum freezing rate, and enlarge the ice volume. Functional solutions with specific nanoparticles were injected into the targeted tissues to improve the diffusion process, regulate the freezing scale, modify iceball formation location, and prevent the surrounding healthy tissues from freeze damage. Di et al. introduced MgO NPs as a biodegradable nontoxic agent to innovate nanocryosurgery. The result shows that administering MgO NPs-mediated cryosurgery, either by direct tumor injection or intravenous delivery of nanoparticles, is possible (134). In addition, the new modality also offers many chances to ablate tumors better. For example, a minimally invasive needle with both freezing and heating capability can be designed for a combined surgery. The nanoparticles can be first injected into the targeted tumor for nanocryosurgery, which can then be followed up by a nano-hyperthermia, or vice versa.

Figure 3

Schematic illustration of computational domain loaded with nanoparticles during cryosurgery (not to scale) (133).

In addition, nanomaterials cannot only play the role of adjuvant therapy, but also they may become a therapy agent. In 2005, Chen et al. have reported that gadolinium metallofullerenol [Gd@C₈₂(OH)₂₂]_n nanoparticles (f-NPs) have the ability of antitumor (135). In recent years, numbers studies have indicated that f-NPs exhibit strong inhibitory activity against various tumor, such as MCF-7 cells in vivo . In addition, f-Nps have the ability of antimetastasis. The anticancer mechanism of f-NPs was also studied. The results show that the anticancer mechanism of f-NPs may be related to immune enhancement (135), G0/G1 phase arrest (138), angiogenesis inhibition (136), and scavenging of ROS (139). Liang et al. reported f-NPs could reactivate the defective endocytosis of cisplatin in the cisplatin-resisitant human prostate cancer cells and cause accumulation of intracellular cisplatin, thus tumor resistance to cisplatin was circumvented by treatment with a combination of f-NPs with cisplatin both in vitro and in vivo (140). Based on the above description about f-NPs unique properties, we believe that f-NPs may become a novel antineoplastic chemotherapy agent with high efficiency and low toxicity.

Drug targeting

One of the challenges in developing effective drug delivery nanoparticles is to rationally design them so that they can perform two critical functions: evading the body’s normal immune response and reaching their intended targets. Based on the ability to locate and deliver a drug to the interest area in the body, NP delivery systems can be classified as passive and active target (Figure 4).

Figure 4

Passive (Right) vs. active targeting (Left).

The defective vascular architecture was created due to the rapid vascularization, which was necessary to serve fast-growing cancers (143, 144). Passive targeting can be achieved by the EPR effect. As a consequence of EPR effects, nanoparticles that encapsulated drugs, proteins or other therapies can accumulate in the tumor at a concentration five to ten times higher than that in normal tissue within 1–2 days (145). For instance, Abraxane^®, an albumin-bound paclitaxel nanoparticles for the treatment of metastatic breast cancer and Doxil^®, a poly (ethylene glycol)-coated (PEGylated) liposomal system as doxorubicin (DOX) delivery for cancer therapy have been approved by the FDA. These agents circulate in the body with a half-life about 100 times longer than that of free anticancer drugs while simultaneously reducing systemic toxicity (146–150).

Because of different degree of vascularization and porosity of tumor vessels, targeting cancer cells by the EPR effect is not feasible in all tumors, this limits the passive targeting (151, 152). Attaching target molecule to the nanoparticle surface is one approach to overcome this limitation, which is called active targeting. Active targeting, which was performed by equipping the delivery system with a “homing device” that was capable of guiding the carrier to the intended target (145). These “homing device” are special in that they can recognize and bind to complementary molecules, or receptors found on the surface of tumor cells. After such molecules are added to the drug delivery nanoparticles, they can target to the tumor cells, this will increase the efficacy of the treatment and reduce toxic effects on surrounding normal tissue. For example, Kumar et al. have synthesized a novel class of Au nanoparticles, which was functionalized with the therapeutic peptide, PMI (p12), and a targeted peptide, CRGDK for selective binding to neuropilin-1 receptors which were overexpressed on the cancer cells and regulated the process of membrane receptor-mediated internalization. In an in vitro studies, AuNPs functionalized with CRGDK resulted in maximal binding interaction between the CRGDK peptide and targeted Nrp-1 receptor overexpressed on MDA-MB-321 cell surface, which improved the delivery of therapeutic P12 peptide inside targeted cells (153).

At present, several targeted delivery systems are under clinical trials, Table 1 lists the nanoparticle-based drugs that are approved or under clinical development. Although ligand-mediated targeting drug carriers have not yet made a considerable clinical impact on human health, it will soon be feasible to develop targeted nanoparticle candidates for clinical translation.

Controlled drug release

An ideal drug carrier should not only have the nature with high load of drug, but also be able to have the ability of controlled drug release. When some drugs are early released in the blood circulation, they will damage normal tissues and produce serious side effects. Nowadays, with the developing of nanotechnology, nanomaterials as a drug delivery can overcome these side effects.

Several biodegradable materials such as liposomes, polymeric nanoparticles and dendrimers have been used as “smart” carriers that can controllably release pharmaceutical drugs in aqueous solution upon the structural degradation of the carriers triggered by various chemical factors, such as pH. Several exciting DDS have been prepared following this approach (160). Though structurally unstable “soft” materials are beneficial for the drug delivery, it is difficult to achieve “zero” premature release of drugs. In many cases, the matrix-entrapped drug molecules would start to release from the biodegradable carrier as soon as the system was introduced in vivo. The premature release problem not only limits the usage of these biodegradable DDS materials for effective cancer treatment, but also presents a major challenge for the site-selective delivery of protein and nucleotide-based drugs via oral administration. The precious pharmaceutical or nutriceutical cargo of enzymes, DNAs, and RNAs would decompose in the highly acidic environment of stomach if the carriers could not offer the necessary protection (161).

Recent research has focused on developing structurally stable drug delivery systems that are able to deliver a relatively large amount of drug molecules without any premature release problem to targeted tissues or even intracellular organelles. In 2003, Lai et al. first designed a mesoporous silica nanosphere (MSN)-based stimuli-responsive system using a concept of gatekeeping (162). This system has the advantage of a variety of chemical entities as “gatekeepers” to regulate the encapsulation and release of drug molecules (Figure 5). Kim et al. reported a unique methodology for controlled-release delivery systems (163, 164). Cyclodextrin gatekeepers on the surface of MSNs can be hydrolyzed by amylase to release guest molecules from the porous reservoir. The ester linkage in the stalk part of the cyclodextrin gatekeeper can also be cleaved by lipase to release guests from the channel. Other groups also explored some control release systems that employed different gate based on MSNs, such as Au nanoparticles, silver nanoparticles, dendrimer and other gatekeepers (165–168). Among structurally stable materials that have been investigated for drug delivery, Bio-MSN becomes the best candidate, because biodegradable MSN have many advantages for intracellular delivery, such as large surface area, tunable pore sizes and volumes, and encapsulation of drugs, proteins and biogenic molecules (169).

Figure 5

Representation of an MSN loaded with guest molecule and end-capped with a general gatekeeper (162).

In addition, to achieve the goal of drug controlled release, nanocarriers have been designed as an environment sensitive delivery. Recently, by self-assembly approach, Hu et al. fabricated a pH/thermo-responsive nanocarriers with dually responsive poly(N-isopropylacrylamide) (PNiPAM)-co-acrylic acid (AA) hydrogel core enclosed in silica shell. Comparing with common MSNs, these core-shell particles have a more uniform size and better dispersity. In vitro cell culture studies, it is more biocompatible and less cytotxic than MSNs. In addition, the drug release results indicate that PNiPAM/AA@SiO₂ particles have pH responsive characteristics and higher releasing efficiency than MSN particles, which are very attractive for cancer treatment (170).

Multi-drug resistance

In the treatment of disease, especially cancer, drug-sensitive cells were killed, but a proportion of drug-resistant cells are left. This is so called multi-drug resistance (MDR) (171). One mechanism of MDR is increasing drug efflux (172). The increasing drug efflux is caused by the integral membrane proteins, known as MDR transporters. The transporters such as p-glycoprotein (MDR1), multidrug resistance associated proteins (MRPs) can actively pump chemotherapeutic drugs out of the cell and reduce the intracellular drug dose below lethal threshold level (173). The onset of nanomaterials has endowed the ability to provide solution to this limitation by bypassing the MDR transporters (174, 175). For example, SP1049C, a novel P-glycoprotein targeting micellar formulation of DOX exhibits greater efficacy than DOX against a variety of drug-resistance tumor cell lines due to the ability of circumventing p-glycoprotein-mediated drug resistance (176). Other studies have addressed the challenge of MDR using polymeric nanoparticles (177), lipid nanocapsules (178), and micelles (179) within cell lines or in mouse tumor models. Although those nanocarriers can bypass the transporters to an extent, it is not sufficient for overcoming drug resistance. Taking into account the mechanism of drug resistance, the overexpression of transporters is one of the major mechanisms of drug resistance in cancer cells, knockdown of those gene expressions by siRNA could help to restore the intracellular drug levels to the concentrations requiring for induction of apoptosis and cytotxicity. So, the nanobased drug co-delivery systems have been designed for targeting the transporters concurrently delivering drugs for increasing intracellular drug concentration (175). Meng et al. have reported a new approach to overcome MDR by utilizing siRNA to silence the expression of efflux transporter. In their studies, MSNs were functionalized to effectively deliver DOX as well as p-glycoprotein siRNA to a drug-resistant cancer cell line in an additive or synergistic fashion (180).

Another problem in MDR is activation of anti-apoptotic pathways. Cells have developed strategies to avoid death by the anti-apoptotic pathways (181). It often involves the genetic and epigenetic alterations of cancer cells including overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptosis regulator protein and mutations in tumor suppressor p53 gene. The nanocarrier is also possible to co-delivery cytotoxic drugs and gene targeting apoptosis regulator protein including Bcl-2 (182), transcription factor NF-κB and hypoxia-inducible factor alpha (HIF-1α) (146). With the synergistic effect, the intracellular drug accumulation can increase therapy efficacy. Several groups have designed DDS based on nanoparticles or liposomes for co-delivery of anticancer drugs and siRNA. For example, Chen et al. obtained CaCO₃/DNA/DOX nanoparticles by CaCO₃ co-precipitation technique for co-delivery of p53 gene and DOX (183). These nanoparticles could induce a higher cell inhibition and promote tumor cell apoptosis more effectively.

Multifunctional nanocarrier

By a synergistic effect, multifunctional nanocarriers as delivery systems are capable of delivering therapeutic payloads and/or image contrast enhancement agents to targeted sites in the body. Kim et al. designed a tumor-homing chitosan-based nanoparticles (CNPs) containing a near-infrared fluorescent (Cy5.5) and anticancer drug (paclitaxel) for cancer treatment. The CNPs have many advantages such as stability in serum, deformability, and rapid uptake by tumor cells. It presents a new strategy in cancer treatment, in which early stage cancer diagnosis, drug delivery, and real-time non-invasive monitoring for therapeutic efficacy are carried out simultaneously (184). Yang et al. have developed a multifunctional nanosystem combining magnetic nanocrystals with therapeutic antibodies and the chemotherapeutic drug DOX, this nanosystem has the ability of imaging, targeting and treating (185). Multifunctional nanomaterials have also been used for in vivo imaging, siRNA delivery and silencing in tumors. Medarova et al. have obtained a nanosystem, which is consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model target. This nanosystem is easily translocated to cytosol and has high gene transduction efficiency (186). Guo et al. designed and prepared a kind of multifunctional nanoparticles by graft-degradable amphiphilic cationic copolymer, poly(ɛ-caprolactone)-graft-poly(2-(N,N-dimethylamino)ethyl methacrylate) (PCL-g-PDMAEMA). It was found that PCL-g-PDMAEMA nanoparticles were able to simultaneously entrap hydrophobic paclitaxel and load DNA. These nanoparticles had pH dependent sensitive ability, drug could be released faster in an acidic environment than in a neutral environment. In addition, the PCL-g-PDMAEMA nanoparticles showed high gene transfection efficiency than Lipofectamine™ 2000. The most interesting thing is that these nanoparticles can escape from the endosome and release the payloads effectively in cytoplasm. All of those advantages suggest that PCL-g-PDMAEMA has great potential for achieving the synergistic effect of drug and gene therapies in vivo (187).

The application of nanomaterials as drug delivery has made a great success. Current pharmaceutical nanocarriers, such as liposomes, micelles, biodegradable MSN, demonstrate a broad variety of useful properties, such as longevity in the blood, enhanced intracellular penetration, specific targeting. With continuous advances in new biomarkers and targeting ligands, it will be increasingly feasible to develop optimized nanocarriers as promising candidates for clinical translation.

Bone

As a type of dense connective tissue, bone provides a frame to support and protect internal organs. Due to the crucial roles of the skeleton for the human body, rehabilitation of bone tissue is certain to be imperative when it is damaged by trauma, severe infection and other disorders. Recently, several nanomaterials have been applied in tissue engineering.

As a major component and an essential ingredient of normal bone and teeth, the size of natural hydroxyapatite(HAp) particles is demonstrated to be nano-scale shaped like fibers, 70% of HAp particles have 2–5 nm in width and 20–80 nm in length (188). Besides their mechanical functions, natural HAp particles can modulate activity of bone cells. Hence, artificial nanomaterials substituted for HAp are required not only to possess good mechanical behavior, but also to benefit bone cells. Nowadays, the novel ways to modify nano-HAp are found continuously to better fulfill the mechanical and biological requirements. Kong et al. synthesized nano-HAp/chitosan scaffolds with high porosity, the scaffolds showed better biocompatibility than pure chitosan scaffolds (189). Zandi et al. prepared rod-like nano-HAp/gelatin scaffolds coated with nano-HAp particles. The HAp particles on the scaffold wall easily enter the microscopic fractures, and increase the mechanical strength. The incorporation of rod-like nano-HAp and scaffolds coated with nano-HAp particles make the scaffolds possess desirable biocompatibility, high bioactivity, and sufficient mechanical strength in comparison with noncoated HAp samples (190). Liao et al. developed a nano-HAp/collagen/PLA three-dimensional porous scaffold by biomimetic synthesis. This scaffold mimics the microstructure of cancellous bone and shows some features of natural bone both in main composition and hierarchical microstructure. Cell culture and animal model tests showed that the scaffold was bioactive (191). Another three-dimensional scaffold consisting of porous nonwoven silk fibroin net/nano-apatite composite was prepared by Zhao et al. The nano-sized HAp crystals were needle like, with a length of approximately 100–300 nm and a diameter of approximately 20–60 nm. The results demonstrated that the nonwoven silk fibroin net/nano-apatite composite showed excellent cytocompatibility for the growth of osteoblasts and had the capability to improve the viability of osteoblasts (192).

Titanium and its alloys have been widely applied to implantation materials in orthopedic and dental surgery. In contrast to pure Ti metal, TiO₂ imparts stronger bioactivity and chemical bonding to bone (193). Great progress in nanotechnology makes further improvement of TiO₂ as a bioactive material for tissue engineering. Nanophase titania/PLGA composites were designed and synthesized by combining the advantages of degradable polymers with nano-size ceramic grains to optimize physical and biological properties for bone regeneration. Liu et al. found that the PLGA composites with nanophase titania could enhance osteoblast functions. The study also revealed that the dispersion of titania in PLGA was enhanced by increasing the intensity of sonication and osteoblast adhesion was correlated with the dispersion of nanophase titania (194). To obtain more excellent biomechanical compatibility and bioactivity, Li et al. prepared bioactive nano-titania ceramics. The data showed that the mechanical properties of nano-titania ceramics were analogous to that of the human bone. Moreover, HAp additive plays an important role in adjusting the grain/particle size of nano-titania ceramics, which has a great effect on the osteoblast proliferation (195). Wang et al. prepared PLGA/nano-TiO₂-particle composite microspheres. The introduction of TiO₂ component has been proven to be capable of largely enhancing mechanical properties of PLGA/TiO₂ microsphere-sintered scaffold. In addition, nano-TiO₂ additives are also capable of increasing the proliferation, calcium secretion and alkaline phosphatase (ALP) activity of osteoblasts (196).

In addition, we previously reported the effects of AuNPs on the differentiation of MSC and the associated molecular mechanisms (197). Cellular assay results demonstrated that AuNPs promoted the differentiation of MSCs toward osteoblasts over adipocytes with increasing ALP activity and mineralization of the extra-cellular matrix. The results at the molecular level showed that the interaction of MSCs with AuNPs may lead to the activation of the p38 mitogen-activated protein kinase pathway (MAPK) signaling pathway and the up-regulation of osteogenic genes and the down-regulation of adipogenesis specific genes.

Cartilage

Cartilage is a flexible connective tissue composed of chondroblasts and a large amount of extracellular matrix (ECM). Several diseases are correlated closely with cartilage damage, which is difficult to heal because of disability of chondrocytes to migrate and shortage of blood supply in chondral tissue. Nanomaterials for tissue engineering have emerged as an interesting therapeutic option for the treatment of articular chondral defects with good and potentially durable results. To mimic the native ECM of cartilage for tissue repair and regeneration, many types of 3D tissue engineering scaffolds were fabricated as a collagen-like nano-fiber. Wimpenny et al. improved the nano-scaffolds using poly(L, D-lactide) (PLDLA) nano-fiber coating on microfibers or films. The nanostructure promoted the expression of chondrogenic markers, such as collagen type IIaI and aggrecan. The lower wettability of polymeric nanofibers favored the maintenance of rounded chondrocyte morphology (198). Compared to the above-mentioned nano-scaffolds, some bioactive peptide-linked nanomaterials may exhibit a more potent therapeutic effect for cartilage damage. Sethi et al. prepared a nanostructure combining vasoactive intestinal peptide (VIP) with sterically stabilized micelles (SSMs). It was found that the nano-sized SSM-VIP protected the bioactive peptide from degradation or inactivation in biological fluids, and had superior actions to traditional medications. Kon et al. reported that a 46-year-old athletic patient was successfully treated with a closing-wedge high tibial osteotomy and the implant of a newly developed biomimetic nanostructured osteochondral bioactive scaffold. After 1 year of follow-up, the patient was pain-free, had full knee range of motion, and returned to his pre-operation level of athletic activity (199).

Nerve

The repair of damaged nerves is believed to be a tough clinical problem. For example, one of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that can mimic the ECM and assist in nerve regeneration. It was reported that nanotopography or orientation of the fibers within the scaffolds greatly influenced the nerve cell morphology and outgrowth, and the alignment of the fibers ensured better contact guidance of the cells (200).

The initial experimental results indicated that nanostructures might lead to suppress nerve regeneration because of hindering nutrient and oxygen supply, which are essential for nerve growth. In order to avoid to this problem, Oh et al. developed the nanomaterials for repairment of nerve. Nerve guide conduits (NGCs) with selective permeability and hydrophilicity were fabricated using PLGA and pluronic F127. The inner surface of the tube has 50 nm nano-size pores, which can effectively prevent from fibrous tissue infiltration but permeate nutrients and retain neurotrophic factors, while the outer surface has micro-size pores, which can allow vascular ingrowth for effective supply of nutrients and oxygen into the tube. From the investigations of mechanical property, water absorbability, and model nutrient permeability of the tubes, the hydrophilized PLGA/F127 (3 wt%) tube seems to be a good candidate as a NGC for the effective permeation of nutrients as well as the good mechanical strength to maintain a stable support structure for the nerve regeneration (201).

The chitosan nanofiber mesh tube may be also a promising substitute for autogenous nerve graft. Wang et al. developed bilayered, deacetylation rate and oriented chitosan tube (202–204). Bilayered chitosan tube that comprises an outer layer of chitosan film and an inner layer of chitosan nonwoven nano/microfiber mesh. Nerve regeneration in chitosan tubes, on which the CGGGGGGYIGSR peptide was immobilized, exhibited efficacy similar to that of the isograft and represented a promising candidate for promoting peripheral nerve repair (202). It was reported that the chitosan nano/microfiber mesh tubes with a deacetylation rate of 93% had sufficient mechanical properties to preserve tube space, provided a better scaffold for cell migration and attachment, and facilitated humoral permeation to enhance nerve regeneration. As a result of fiber orientation, the tensile strength along the axis of the sheet increased (203). Because Schwann cells aligned along the nanofibers, oriented fibrous sheets could exhibited a Schwann cell column, thus functional and electrophysiological recovery occurred in time in the oriented group as well as in the bilayered group. Furthermore, histological analysis revealed that the sprouting of myelinated axons occurred vigorously followed by axonal maturation in the isograft, oriented, and bilayered group in the order (204).

In addition, the applications of nanomaterials in bladder and blood vessel tissue engineering have been reported (205, 206). In summary, at present, although there are many problems, we believe that nanomaterials are very important in tissue engineering fields in the future.