In the recent years, HNT has come forth as a nonviral gene delivery agent because of properties like biocompatibility, negligible toxicity toward human and bacterial cells, and high mechanical strength.
A study was conducted, where an HNT-based gene delivery carrier was developed for the delivery of intracellular antisense oligodeoxynucleotides (ASONDs) for targeting survivin (a protein belonging to inhibitor of apoptosis family). APTES-functionalized HNT was used to show the better intracellular delivery and improved anticancer activity of ASONDs (bound to the outer surface of functionalized HNT), hence rendering a potential vector for gene delivery.20
Polyethyleneimine (PEI) modification of HNT has been reported to be efficient for gene delivery, such as short-interfering RNA (siRNA) and plasmid DNA (pDNA). Such modified HNTs were used for the intracellular delivery of therapeutic antisurvivin siRNA. The Western blot analysis showed that PEI-HNT-mediated siRNA delivery decreased the target proteins PANC-1 cells levels by efficiently knocking down gene expression of survivin, thus reinforcing its potential as cancer therapeutic agent.51 A recent study was conducted using PEI-grafted HNT (PEI-g-HNT) to bind green fluorescence protein labeled pDNA onto the surface of modified HNT. PEI-g-HNT showed less cytotoxicity than PEI and also had better transfection efficiency.19
Gene therapy holds great potential as a clinical treatment for cancer and genetic disorders. But to accomplish this, the gene carriers need to fulfill many delicate criteria, such as cytotoxicity and biocompatibility, which makes this route of treatment intricate. The use of HNT is still an ongoing exploration which has seen potentially viable results, hence opening up an imperative area of research in biomedicine.
Tissue engineering
The formation of nanocomposite as support matrix for controlled drug release, polymer nanocomposite, nanotemplating, and catalytic support has also been reported.25,52 HNT has been used as drug delivery agent for various kinds of targets as mentioned above, hence it also found its role as a delivery agent for tissue engineering. For instance, alkaline phosphatase (ALP) was incorporated into HNT for bone repair. HNT acted as a heat sink in the system which increased the thermal stability of the ALP. It also enhanced the activity of ALP highly, thus promoting the biomineralization process which was studied in vitro using a substrate, calcium glycerophosphate. This bioactive nanocomposite can also be incorporated into biomaterials used as scaffolds for tissue engineering.53
A crucial problem that needs to be attended to in tissue engineering is forming a scaffold, which is competent enough to support the three-dimensional tissue formation (Figure 5). Such scaffolds should meet basic specific requirements such as:
Figure 5.
Properties and types of scaffolds for tissue engineering.
High porosity with adequate pore size (to facilitate seeding and diffusion of nutrients).
Biodegradability and degradation rate (scaffold should be absorbed by tissue surrounding it along with new tissue formation).
Mechanical strength for strong support for growth of new tissue.
The microstructures of the scaffolds could be of various types like open pore structures, fibrous matrices, hydrogels, and other natural and synthetic materials.54 The use of HNT for tissue engineering is a relatively new venture; hence, there have been only few promising researches during the past decade. The core idea while using HNT is to couple it with various compounds which will make potential scaffolds for tissue and bone growth.
The chitosan-HNT nanocomposite scaffolds showed considerably improved compressive strength, compressive modulus, and thermal stability as compared to pure chitosan scaffold. Although HNT did not affect the porous structure and porosity of scaffolds that much, it also did not exert any cytotoxicity to cells. Also, it was observed that the cells attached and developed well on the scaffold.54 Chitosan-HNT was also produced by electrospinning with 0, 2, and 5 wt% HNT. The nanocomposite showed increasing Young’s modulus and maximum tensile strength with increasing HNT concentration. This is an ideal membrane with improved mechanical properties and thermal stability for bone tissue engineering.55
Another compound coupled with HNT was alginate to form a scaffold which showed high compressive strength, decreased water adsorption, and degradation rate. HNT enhanced the thermal stability of the alginate scaffold and also increased the cell attachments by increasing the surface roughness and biocompatibility.56 The physical properties of a sodium-alginate scaffold were improved by incorporating HNT and the composite was then cross-linked with calcium ions. At low HNT loading, the composite scaffold showed increased cell adhesion and proliferation in preosteoblast (MC3T3-E1) culture, which will be useful in bone tissue engineering.57
A study developed metronidazole-loaded HNT doped into poly(caprolactone)/gelatin microfibers by electrospinning as membranes with sustained drug delivery for guided tissue or bone regeneration. This inorganic–organic, anti-infective implant and drug delivery membrane can be used in various therapeutic applications requiring time extended functions, including pathologies demanding chronic drug treatments, wound healing, prevention of postsurgical adhesions, and tissue-engineering applications.58 Electrospun poly(l-lactic) acid nanofiber scaffolds have been reinforced with unidirectionally aligned HNT increasing the tensile strength, Young’s modulus, and fracture strain.59
A multilayered polylactic acid (PLA)/HNT porous membrane encapsulated with aminoglycoside antibiotic (gentamicin) was prepared as an antibacterial membrane for bone regeneration. It was found to have good antibacterial efficacy against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, hence was proposed as a potential use in prevention of infection in bone regeneration applications.60
An in vitro and in vivo study was performed which strengthened the fact that HNT-doped scaffolds are biocompatible. Freeze drying method was used to dope porous biopolymer hydrogels at 3–6 wt% with 50-nm diameter/0.8-μm HNT without any cross-linkers. A chitosan–gelatin–agarose hydrogel doped with HNT showed augmented mechanical strength, higher water uptake, and thermal properties. The scaffolds implanted in rats showed very good resorption at 6 weeks also. The newly formed connective tissue placed near the scaffold showed complete restoration of blood flow due to neovascularisation.61
Yet another study integrated HNT in gellan gum matrices to develop composite hydrogels with controllable physical features. It showed a good human dermal fibroblasts biocompatibility when the cells were seeded on the top of the gels or encapsulated within the polymeric matrix. Fibroblasts onto the hydrogels with HNT exhibited high metabolic activity due to enhanced mechanical and topographical features because of HNT. These scaffolds prove to be suitable for different soft tissue engineering applications (pancreas, liver, skin, and chondral regeneration).62
Even though there are only few studies that have researched the role of HNT as bionanocomposites for tissue engineering, the current trend of studies is promising of a huge role of HNT in tissue engineering.63 Wound healing
The tubular HNT has high mechanical strength, good biocompatibility, and hemostasis property which make them compatible for wound healing applications. HNTs were studied for their possible use as biocompatible nanocontainers for gradual and controlled release of antiseptics. Multiple studies have shown the use of the nanotubes for loading of antibacterial and antiseptic drugs for wound healing applications. Benzotriazole-copper coated HNT was loaded with brilliant green (antiseptic), which had shown sustained release (50–200 h) from the nanotube. The study also showcased controlled release of amoxicillin and iodine from the HNT.64
Another approach was to use HNT-based nanocomposite to make dressings to be used in wound healing. Three-dimensional, porous and flexible chitosan composite sponges were formed via the addition of HNT which had increased elastic modulus, compressive strength, and toughness. Also, it was noted that the HNT improved chitosan’s blood clotting ability. In vivo tests confirmed the composite sponges to be cytocompatible with enhanced wound healing properties.65 Better skin reepithelization and reorganization was demonstrated by chitosan oligosaccharides modified HNT, which were finer than that by HNT or chitosan separately. The wound healing by the composite is accelerated by enhancing the activity of inflammatory and repairing cells, which in turn is the resultant of the sustained release from HNT and chitosan oligosaccharides (homo- or heterooligomers of N-acetylglucosamine and d-glucosamine), making this nanocomposite a potential medical device for wound repairing.66
Likewise, HNTs were used as carriers of vancomycin in an alginate-based wound dressing. The demonstrated new dressing exhibited high stability and neutral character in relation to the living organisms applied in the bioassays. The results indicated that the designed potential wound dressing with optimized parameters can be an effective strategy in long-term treatment of wounds.67
CIP and polymyxin B sulfate-loaded HNT (HNT-B) were combined into a gelatin elastomer to form a double drug codelivery, elastic and antibacterial nanocomposite. The CIP with antibacterial activity was diffused into the nanocomposite matrix, whereas HNT-B was first loaded into the HNT and later dispersed in the matrix. The in vitro drug release behavior and the antibacterial properties improved significantly; this was attributed as the influence of CIP and HNT-B on the physical characteristics, cytotoxicity, fibroblast proliferation, and adhesion. This particular bionanomaterial has effective properties like high water absorbing quality, low cytotoxicity, adjustable biodegradability, and good elasticity, which are idyllic for wound healing application.68 HNT also strengthened an elastic nanofibrous material made of PCL and gelatin which can be used as wound dressings with sustained drug release.69 Cancer therapy and isolation of stem cells
The capability to capture rare circulating tumor cells from the blood of cancer patients provides a significant advance in cancer study, diagnosis, and treatment on a patient-to-patient basis.70 Chemotherapy has greatly made use of vehicles capable of targeted drug delivery; hence, naturally, HNT was also considered and studied upon for the same. Several approaches have been explored for such targeted drug delivery by HNT which have been discussed in Figure 6.
Figure 6.
Halloysite nanotube in cancer therapy.
Firstly, direct loading of the drug onto HNT and its effects on a cell line was assessed. HNT was loaded with resveratrol and its delivery to the cancer cells was analyzed. MTT assays were performed with a neoplastic cell lines model system (MCF-7) which showed that the resveratrol-loaded nanotubes strongly increased the cytotoxicity, thereby leading to cell apoptosis. These findings were correlated with the concentrations and the incubation times of the drug loaded HNT.71 Multicomponent HNT was examined as a vehicle to guide the delivery of anticancer drug DOX into cancer cells. The drug loaded FA-Fe3O4 HNT was found to be toxic to HeLa cell lines leading to apoptosis of cells.72
Further, a multicavity HNT–amphiphilic cyclodextrin hybrid was formed for codelivery of natural drugs (silibinin and quercetin) into thyroid cancer cells. The interaction between cells and the carrier revealed that the materials were uptaken into the cells surrounding the nuclei. Therefore, it was surmised that the multicavity systems could transport drugs into living cells safely.73
Curcumin is an antioxidant property which has been utilized for anticancer efficacy; several techniques have been used to load curcumin into HNT for the same. In one such method, positively charged HNTs were functionalized with trizolium salts. These HNT-based carriers of curcumin were used for drug delivery to various cell lines. The trizolium salts functionalized HNT loaded with curcumin were found to be active in many of the tumor cells.74
Also, biopolymer-grafted HNT was reported for the targeted delivery of anticancer drugs. In a study, chitosan-grafted HNT (HNT-g-CS) were studied for the delivery of curcumin to cancer cells. The HNT-g-CS loaded with curcumin demonstrated definite toxicity toward several cancer cell lines, such as HepG2, MCF-7, SV-HUC-1, EJ, Caski, and HeLa; out of which EJ cell line showed increased apoptosis. The content of reactive oxidative species (ROS) produced by curcumin loaded HNT-g-CS is more than by free curcumin, hence making a potential anticancer drug delivery vehicle.18 HNT grafted with chitosan oligosaccharide was also used for delivery of DOX for breast cancer using MCF-7 cell lines. The DOX-loaded HNT-g-CS entered MCF-7 cells and triggered mitochondrial damage and attacked nuclei.75
Another study was conducted on the cancer drug delivery efficacy of Chitosan modified HNT loaded with curcumin-gold hybrid nanoparticles. These HNT hybrid nanoparticles consisted of AuNP which have near-infrared (NIR) responsive property and pH-responsive curcumin release, hence making it a good candidate for targeted drug delivery of cancer cells with NIR imaging.76
In yet another study, nanotube-in-microsphere which is a fascinating new design that incorporated microfluidics, was implemented to make a drug delivery vehicle by encasing HNT in a pH-sensitive hydroxypropyl methylcellulose acetate succinate polymer. Atorvastatin and celecoxib were used as model drugs because they have dissimilar physicochemical properties and interactive effect on prevention and inhibition of colon cancer. The HNT/pH-responsive polymer composite prevented the premature release of the drugs at pH 6.5 while it allowed faster release and increased drug permeability at pH 7.4. These observations were made simultaneously with the occurrence of inhibition of proliferation of the colon cancer cells.77
Another fast, low-cost, and effective fabrication method was reported for fabricating large rough HNT coatings by thermal spraying of HNT ethanol dispersions. The enhanced surface communications between HNT coating and tumor cells led to effective capturing of the cancer cells as compared to the normal cells captured by the HNT coatings (except with HeLa cells). The HNT coatings were also effective in capturing tumor cells in artificial blood and blood samples from patients with metastatic breast cancer. This superior capture capability holds good potential for early diagnosis and examination of cancer. The high killing ability of the DOX-loaded HNT was also confirmed and hence it can be used as an implantable therapeutic device for preventing tumor metastasis.78
HNTs have been creatively used in isolation of stem cells as it was seen advantageous in capture of tumor cells previously. A research showed three-dimensionally printed PLA pattern functionalized with a polydopamine interlayer, this helped in firmly binding the HNT on the surfaces of the PLA pattern in order for guided cell orientation. The roughness and hydrophilicity of PLA pattern were improved significantly by HNT and the in vitro human mesenchymal stem cells culture analyses showed that PLA pattern with HNT exhibited different ability to induce cell orientation according to different stripwidths.79 Also, a multifunctional nanocomposite was made of supermagnetic HNT functionalized with chitosan (M-HNT) and was then adorned with the calcium phosphate 2-D nanoflakes (CaP). This was synthesized for increasing the osteogenic potency of human adipose tissue-derived mesenchymal stem cells.80
Anticancer therapy and stem cell study is an important and delicate field of research in biomedicine; therefore, the use of naturally occurring HNT with its myriad of properties for the same can be seen as an essential breakthrough with still a lot of potential for further exploration (Table 2). Table 2.
Open in a separate window
FA: folic acid; HNT: halloysite nanotube; AuNP: gold nanoparticle; LbL: layer-by-layer. Biosensing
Nanoparticles are receiving increasing attention as future contrast agents (CAs) for ultrasound molecular imaging, especially when decorated on its surface with biological recognition agents for targeted delivery and deposition of therapeutics. Nanoparticles or nanomaterials have several advantages which make them beneficial as CAs like their magnetic and optical properties can be adapted by manipulating their composition, structure, size, and shape. Additionally, their surfaces can be easily modified with desired materials (e.g. ligands) to make them targeted enhancement of desired site.81
A study showed the parametric assessment of the effectiveness of HNT as scatterers for safe ultrasound-based molecular imaging. The results demonstrated the possibility of employing a clinically available echograph to detect the ultrasonic backscatter produced by different concentration of HNT using both 5.7 and 7 MHz insonification frequencies.82
A hybrid nanocomposite was obtained by selectively modifying the outer surface of HNT to support AgNPs. The key to design highly sensitive enzymatic electrochemical biosensor is to achieve direct electron transfer between the enzyme and the electrode surface which in turn can be achieved by improved enzyme immobilization with high loading capacity. The modified HNT with AgNPs were observed to be a good support for the immobilization and electrical wiring of redox enzyme glucose oxidase (GOx). The glassy carbon electrode was the platform on which the GOx immobilized HNT/AgNPs was deposited and this setup was used for the bioelectrocatalyzed electrochemical detection of glucose.23
Another study used HNT-based composite for sensing vitamins by functionalization. A polyaniline (PANI) functionalized HNT composites were reported as highly sensitive ascorbic acid (AA) sensor. The sensitivity of the composite toward AA was about 826.53 μA mM−1 cm−2 within a linear range of 0.005–5.5 mM, and a low detection limit of 0.21 μM. This is a good sensing ability and could be credited to the porous structure which formed efficient sensing channels. This in turn enhanced the electron transport and interactions between the PANI and AA.83
Bioimaging is a less ventured application of HNT but is being probed on due to ease of selective and unambiguous modification of the nanotube. A study was reported in which HNTs were grafted with PEG amine and carbon dots were conjugated with the amino groups in PEG. The conjugation of biocompatible carbon dots into the PEG-NH2 branches of HNT-g-PEG nanoparticles enabled photoluminescent activity for cancer cells imaging.49
Despite the limited number of studies reported on the use of HNT in biosensing and bioimaging, the biocompatibility, bioavailability, selective modification and targeting, and nanosize among other characteristics allow the nanotube to have significant potential to be used in this field. Go to: Conclusion
HNTs are naturally occurring, economically feasible nanomaterial, which are finding applications in many major areas as discussed here. The advantageous tubular structure of HNT has given it numerous roles as drug delivery and gene delivery agents or nanocarriers. In the assortment of studies reviewed in this work, diverse types of modifications/functionalization of HNT have been discussed, hence it can be reasoned that ease of changing the properties of HNT is possible due to their nature of origin. This is so reasoned because there are certain types of HNT that show some limitations due to their length, lumen volume, and uniformity which hinders their applicability in certain applications.84
They have been firmly established as a superior nanocarrier with higher loading capacity and sustained release kinetics for various drugs as well as biological agents. They have been extensively studied and used in drug delivery and targeted drug delivery using variety of drugs. The positive results of such studies have given them therapeutic applications. HNTs have also been established as a gene delivery agent for siRNAs, oligonucleotides, DNA, hence making them significantly important for biomedicine. The studies pertaining to cancer cell-targeted drug delivery are of utmost importance for their significantly positive results compared to other existing methods of targeted delivery. The biocompatibility of the modified HNT is one of the major reasons for the same.
Since the past decade, more research is being focused on widening the range of applications of HNT, and the results are very promising. The present and known properties of HNT are being manipulated for them in different fields such as biosensing and bioimaging, anticancer therapy, tissue engineering, and wound healing. HNTs have also found pertinent roles in various other fields of life sciences and medicine in the way they had been used in bone implants, dental filings, and tissue scaffolds; these applications are possible especially because the modified HNT has low levels of cytotoxicity as verified on various cell lines and bioassays. Apart from the biological aspect, HNTs have relevant roles in environmental area, waste-water treatment (e.g. removal of auramine dye),85 catalytical applications, as glass coatings, in water purification, and in forensic sciences.86
The major hindrance in use of HNT is the absence of studies on humans for pharmaceutical applications. While HNT can serve as excipients for oral delivery of drugs and are not toxic in at least up to concentrations of 1000 μg mL−1,31 it, however, is still nonbiodegradable in blood and therefore is not preferred for direct injections into the living systems as it may lead to thrombosis.27 This opens up yet another potential area of research giving HNT wider range of applications which have been successfully tested on animal models, such as in topical cosmetic formulations, skin ointments, fighting lice, antimicrobial sprays, and traditional oral formulations, allowing for prolongation of the formulation of medical effectiveness.14
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