An insight on electrospun-nanofibers-inspired modern drug delivery system in the treatment of deadly cancers

Abstract

In spite of ample researches and admirable achievements, there are still a significant number of deaths happening every year due to cancer. Furthermore, the number of new cases recorded is also not reduced despite the advent of various preventive measures. Though current clinical approaches yield commendable results, they elicit severe systemic side-effects and also fail to avoid the recurrence of the disease. To address these issues, nanotechnology-empowered modern drug delivery systems showcase excellent properties for the targeting and controlled delivery of biomolecules over a period of time. In the past decade, the materials-based cancer research field has witnessed the exploration of several attractive drug delivery approaches for the administration of synthetic drugs to genetic materials. Among those, the electrospinning-based nanofibrous mesh has attracted several works on treating common deadly cancers such as those of the lung, breast and colon. The capability of nanofibers to enable increased drug loading, maintenance of significant bioactivity, excellent drug encapsulation, controlled and targeted delivery, has helped the researchers to achieve successful administration of a variety of anti-cancer agents. This review gives an insight into the process of electrospinning, its essential parameters, the types of drug incorporation and the works reported on common deadly cancers. Moreover, the future direction of this effective alternative is also delineated, making electrospun nanofibers as a suitable vehicle for delivering drugs to the cancer sites.

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1. Introduction

The concept of drug delivery systems was formulated in the early 1970s and the first reliable system to be demonstrated was polymer-based (lactic acid). Even though it was introduced decades ago, extensive researches have been reported in the last five to ten years, especially after the popularization of nanotechnology. Modern drug delivery systems are exploited to carry drugs, growth factors, genes and biomolecules for treating cancer, promoting tissue regeneration and curing several clinical ailments.¹ However, the notion was solely intended to address cancer, a major health problem in all parts of the world. Typically, the functions and process of cells are controlled by biomolecules synthesized with the help of various types of DNA and RNA, the ultimate workhorses. When these prime-molecules undergo mutation they generate faulty substances which in turn make the cells behave and proliferate anomalously, resulting in the development of cancerous tissue or neoplasm. By mimicking normal cells, these escape the attack of the immune system, start invading neighbouring tissues and eventually spread throughout the body. Factors responsible for this deadly mutation remain diversified, but they mostly include occupational hazards, environmental pollution, genetic disorders, microbial attacks and a certain lifestyles. There are over 200 types of cancer, which can befall at 60 different organs, implying that cancer can develop in virtually any part of our body. Unfortunately, irrespective of age and sex, all people are affected by cancer, though the older population is at greater risk.^2,3

Recent statistics show that a total of 1 658 370 new cases and 589 430 deaths will be reported due to cancer by the end of 2015 in the United States alone. If it continues in this fashion, global deaths because of cancer will rise to over 11 million annually by 2030.^4,5 Besides booming mortality, the economic loss owed to cancer is also increasing drastically. According to the American Cancer Society, the economic loss caused due to cancer is high among all types of non-communicable diseases, accounting for about 895 billion US dollars (excluding direct medical costs) globally in 2008 and it is expected to rise above $1500 billion within 2020.⁶ Among all types, lung, breast and colorectal cancers are considered more deadly since they account for more than 40% of total cancer deaths. Further, the chances of developing metastasis is also relatively high in the above three cases, whose targets include various important organs like bone, brain, liver, the adrenal gland, etc.^7,8 Hence, in this review we have concentrated only on lung, breast and colon cancers.

An ideal therapeutic window to address this serious issue, particularly when it diagnosed in the later stages, is still under research. Because of its varied pattern, the method of treatment is generally chosen by physicians based on the type, location, the extent of spread, patient age, health status, etc. Commonly, the selection will be from any one or combination of the following approaches: surgery; chemotherapy; radiation; hormone therapy; bone-marrow transplantation; and immunotherapy. The main motive of these methods are to halt cancerous proliferation, spreading and symptoms, and to promote apoptosis or self-destruction. However, most of the therapeutic options currently available for clinical usage are accompanied by a series of systemic side-effects and recurrent problems. In general, treatments like chemotherapy will target rapidly growing cells (tumour cells) but also destroy normal rapidly growing cells in that process. This is mainly because of undirected or unspecified administration. Hence, during chemotherapy, the patient will experience abnormalities like heavy hair loss, unbearable body pain, accumulation of fluid, drastic changes in body weight, damage to the kidney, nausea and metabolic imbalances. Meanwhile, options like surgery fail to avoid the recurrence of the disease, especially when it has already spread to nearby tissues.⁹ Therefore, in most cases the drawbacks associated with available treatments have reported a worsening of the condition of the victim rather than curing.

However, modern drug delivery system serve as a compelling option to eliminate most of the complications existing in current clinical approaches for cancer. They comprise a carrier for drugs, targeting agents and shielding materials which offer excellent drug loading efficiency, targeted delivery, controlled release and also high bioavailability. Ample researches have explored various novel delivery systems; but those inspired by nanotechnology and biodegradable polymers have got immense interest.^10,11 Nanotechnology, popularly termed as the “technology of the future world”, has significantly influenced and updated several fields of research. As for the pharmaceutical field, scientists have already demonstrated their skills in controlling the pharmacokinetic profiles of drug delivery systems and the possibility of intracellular delivery. Nanotechnology, along with biodegradable polymers has given rise to smart or intelligent drug delivery systems which safely release the drug only at the tumour population and can maintain bioavailability for a long period. Moreover, with this sensible system, we can also reduce the drug quantity needed to achieve the appropriate therapeutic effect, and thus treatment cost, attaining the anticipated mode of delivery as well as sparing healthy cells.¹² Therefore by the end of 2015, the total market value of nanotechnology-based drug delivery systems is expected to reach $220 billion, which is a 37% increase when compared to 2012 statistics. By 2021, this sector is expected to cover more than 15% of the global nanotechnology market.^13,14

Nanoparticles, micelles, liposomes, micro/nanofibers, nanotubes, microspheres, hydrogels, dendrimers and quantum dots^15,16 are some of the popular delivery vehicles which can be administrated through oral, transdermal, topical, nasal, transvenous or implantable methods. With these systems, scientists have successfully reported safe delivery of a spectrum of natural and synthetic biomolecules such as proteins,¹⁷ antibodies,¹⁸ antioxidants, polyphenols, anticancer drugs,^19,20 etc. In addition, recent researches have also reported the delivery of several genetic materials like RNA,²¹ miRNA, siRNA,²² DNA^23,24 to address cancer at the molecular level which is one of the future perspectives of modern drug delivery approaches. Moreover, advanced technologies available nowadays have simplified the synthesis of these intelligent structures and the loading of desired substances. More interestingly, each structure has been illustrated to possess unique features. For instance, liposomes can carry both hydrophilic and hydrophobic drugs concurrently and dendrimers can hold multiple drugs, whereas some systems can invade cancer cells themselves and deliver the drugs intracellularly.^15,25,26

However, all types of modern systems mostly exploit either active or passive targeting to reach and deliver drugs at the pre-determined site. Generally, the carcinogenic tissues are characterized by high vascular density and active angiogenesis to support their rapid proliferation and spreading, but with poor lymphatic drainage. This develops a condition called enhanced permeation and retention (EPR). In passive targeting, the delivery system reaches the tumour site by tracking this leaky vascular structure and accumulating drugs. In contrast, the active targeting system uses specific ligands attached at the surface to recognize pathological cells.²⁷ Though the modern drug delivery approach sounds like an ideal option, the complicated tumour microenvironment, the presence of P-glycoprotein barriers, enzymolysis, endosomal or lysosomal degradation, and hepatic and renal clearance remain as great challenges for researchers to achieve reliable in vivo outcomes.^28,29

Nevertheless, drug delivery systems like liposomes, nanoparticles, micelles, microspheres etc., exhibit several advantages, most of them suffer from problems such as an instant burst of drugs, poor loading efficiency or renal clearance, and fail to retain the preferred drug concentration for long periods.^30,31 For instance, in case of colon cancer treatment, the projected delivery system is supposed to remain constricted for long periods, but the problem of an undesirable initial burst may damage normal tissues in the gastrointestinal track before reaching the target. Furthermore, this undesired burst also disables the ability of the intended delivery system to accomplish the goal of sustained release. Similarly, while treating solid tumours it is vital to maintain an suitable drug concentration at the appropriate site. However, scientists successfully rectified the above problems by utilizing the unique properties of non-woven electrospun micro/nanofibers. Furthermore, because of their high surface area, ability to enclose a variety of biomolecules, significant loading capacity, and excellent encapsulation efficiency, researchers are also able to establish extensive targeted drug release and achieve multiple therapeutic approaches as well.³¹ This effective alternative can offer intensive protection to conjugated biomolecules; meanwhile it also facilitates oral, transdermal and direct implantation of the drug delivery system at the tumour site itself. Despite this, they can also be cut into required dimensions, which make them a reliable choice for several clinical applications.

Inspired by these capabilities, in this review the potential of electrospun micro/nanofibers, one of the most attractive and upcoming modern drug delivery systems, in fighting the top three deadliest cancers (i.e. lung, breast and colon cancers) are summarised.⁴ Meanwhile, in vitro and in vivo works have been conducted utilizing several natural and synthetic biomolecules, and the capability of electrospun nanofibers to safely deliver active substances to the tumour site is discussed in detail. Moreover a brief overview of the electrospinning process, the essential parameters involved and techniques available to couple drug molecules into electrospun fibers is also presented.

2. Electrospun nanofibers

The technique of electrostatic spinning (electrospinning) is not a new notion, its utility in textile industries can be tracked back to the 19th century. It uses a high voltage to convert the liquid polymer solution into solid fibers of diameter ranging from micrometres down to tens of nanometres. A simple electrospinning unit comprises a syringe provided with a control pump, power supply and a grounded target to collect the fibers. Initially, the polymer solution loaded into desired spinneret will be subjected to high voltage, which makes the pendant droplet elongate due to repulsive forces between like charges in solution and the attractive force exerted by the collector. Increasing the electric field at this point will enable the electrostatic force to overcome the surface tension of the solution which forms a Taylor cone. If the voltage increases beyond this point, it will lead to the formation of solid fibers followed by the evaporation of solvents before it reaches the grounded collector, as shown in Fig. 1. Through the electrospinning technique, we can synthesize natural, synthetic, biodegradable or non-degradable fibrous mats or tubes and it also afford control over the geometry of fibers generated.^31,32 But, the efficacy of this electrospun fibers is greatly influenced by the processing parameters, governing variables and the type of spinneret used. Hence, it is essential to know about these vital parameters to design a reliable drug delivery system with required fiber diameter, porosity, orientation, bulk properties etc.

Fig. 1 Systematic representation of electrospinning process.

2.1 Essential parameters and their influence on nanofiber geometry

In order to produce fibers with better pharmacokinetic properties, notable care should be taken to study the parameters involved in the electrospinning process. According to extensive researches and published reports, issues like solution concentration, viscosity, conductivity, solvent volatility, applied voltage, flow rate, environmental factors, collector shape and capillary distance play a vital role in determining the geometry of the nanofibers (Fig. 2). Depending on its range, nanofiber properties such as diameter, porosity, mechanical strength, structure, surface area, etc., are observed to differ significantly.³³ In general, most of the parameters are interconnected and its optimal range varies for different polymers. First and foremost, the concentration and viscosity of polymer solution determines the spinnability of the preferred biomolecules, i.e. the possibility of synthesizing fibers or not. To obtain low bead-formation, smooth fibers with a minimum diameter and a commendable mechanical strength, the concentration of the electrospun solution has to be low. But if the concentration and viscosity is too low it will form broken fibers with poor structure and beads. Meanwhile, at a super-high concentration it is difficult to form fibers and control the processing parameters.^34,35

Fig. 2 Essential parameters involves in electrospinning process.

It is well known that electrospinning technique is largely dependent on the achievement of electrostatic interactions; hence, the loaded solution should possess high conductivity to make the most of the applied electric field. A solution with a high charge carrying capacity will subject the fiber jet to a greater tensile force, which yields non-beaded fibers with decreased diameter in contrast to resistive solutions.³⁶ Solvent volatility is one of the vital factors, especially when the fibers are proposed for drug delivery applications. In general, to prepare an electrospinning solution a broad variety of solvents like chloroform, Teflon, water, etc., are used. However, the used solvent is expected to disappear during the transit of the solid fibers to the collector. This will ensure the bio-integrity of the nanofibers, furthermore the highly volatile nature of the solvent is greatly significant for the production of a porous mesh with increased surface area as well. On the other hand, solvents with poor volatility are reported to form nanofibers with increased pore size, which may affect the shielding of coupled active molecules from the biological environment.³⁷ In addition, ambient factors like temperature and humidity are also noted to play a significant role in determining the fiber diameter and pore size, respectively.³⁸ Polymers like polystyrene (PS), polycarbonate (PC) and poly(methylmethacrylate) (PMMA) were found to attain interesting sub-micron features when they were electrospun in a humid atmosphere.^33,37,39 For instance, the electrospinning of PS under atmospheric humidity less than 25% is inferred to yield smooth defectless fibers. However, to gain pore formation the humidity range of 30% is found to be ideal and further increase leads to an increase in pore numbers and diameter, respectively. On the other hand, at an extreme humidity range it was difficult to obtain continuous fibers because this affects the rhythm of spinning unit.⁴⁰ Accordingly, the temperature range may also influence fiber morphology, as beadless fibers with decreased diameter are reported to be produced at relatively high temperatures. This is mainly due to the lower viscosity of the polymer solution with the same concentration at high temperatures than in room temperature.⁴¹ Meanwhile, elevated temperature levels also increase the probability of electrospinning of concentrated solutions. Demir et al. reported that the highest polymer concentration of polyurethane which could be electrospun at room temperature was only 12.8 wt%. In contrast, at high temperatures, a stunning concentration of 21.2 wt% could be electrospun into fine fibers with the desired morphology.⁴² Again, these ranges differ significantly based on the polymer and the resultant electrospinning solution, as described in the reviews.^39,41

Besides solution parameters and ambient factors, the applied voltage, flow rate, collector distance and design also add some favourable characteristics. The strength of the electric field will chiefly decide the diameter of fibers. At optimal voltage, non-beaded smooth fibers with minimum size were formed, meanwhile if the field strength is very high or too low, fibers with poor geometry and beads were recorded.^31,43 On the other hand, the optimum flow rate serves as an essential factor to maintain continuous production of solid fibers without any breakage. However at high flow rates, undesired effects like beads, poor fibrous morphology and increased pore size were noted mainly because of partial drying.⁴⁴ In addition, the drying of fibers and solvent vaporization depend on the space between the spinneret and the collector unit. If they are separated by a proper distance, we can obtain beadless, porous nanofibers with the required diameter range. This also ensures complete drying of solvent before the solid fibers reach the collector of choice. Moreover, by changing the morphology of the collector, such as a flat, rotating drum, parallel electrodes, a rotating wire drum, a rotating tube collector with knife-edge electrodes, a disc collector, an array of counter electrodes, a water bath or a conductive or non-conductive target, we can obtain randomly oriented or highly aligned porous nanofibers in both 2D and 3D forms.^33,45,46 To gain detailed information about the processing parameters readers may refer following reviews: ref. 31, 32, 38, 39 & 45–47.

Therefore, to maintain integrity of the delivery system and to protect the coupled biomolecules against the dynamism of the biological environment special efforts should be taken to explore an appropriate range of parameters for the prepared electrospun mixture.

2.2 Techniques employed for loading drugs into electrospun fibers

Electrospinning not only offers control over the geometry and bulk properties of nanofibers but also afford various methods for loading drugs and biomolecules based on the requirements. By varying the nozzle configuration and exploiting the surface modification technique, scientists successfully reported the incorporation of active substances through co-electrospinning, side-by-side, multi-jet, multi-layer, co-axial, emulsion electrospinning and surface immobilization as shown in Fig. 3. And each method has reported exhibiting unique characteristics, whose selection depends on requirements like, high drug loading, increased initial burst, sustained release, prolong circulation, etc.^32,33,48 The method of co-electrospinning is the simple and cost-effective technique in which the polymer solution and the drug/biomolecule to be incorporated is mixed prior to electrospinning. It offers high drug loading, homogenous spreading of drug molecules and an increased initial burst. However, the direct exposure of drugs to the electric field involved in this method may affect their bio-activity.

Fig. 3 Techniques for loading drug molecules into electrospun nanofibers.

Since the nanofiber surface is predominantly occupied by loaded substances, the total drug release is reported within few days due to diffusion and polymer degradation. In a few cases, it fails to shield the conjugated biomolecules from the attack of bodily fluids, which may result in un-desired release.³² If the drug/biomolecule of interest is not soluble in a common solvent, it still can be spun into nanofibers using a side-by-side approach. In this method, the carrier/polymer solution and biomolecules are loaded in a separate spinneret. While applying the electric field, fibers with a distinct upper and lower layer are deposited on a common target. This approach can be used to delay the initial burst of the drug molecules.^33,49

Most cancer treatment involves the exploitation of multiple medications especially in case of addressing solid tumours; this type of treatment method is popularly termed combination therapy. Through electrospinning, it is possible to load different biomolecules into a single system by using either a multi-jet or multi-layer method. In the multi-jet method, the carrier and multiple proposed drugs will be loaded into different spinnerets and will be drawn into the nanofibers simultaneously.^50,51 On the other hand, the multi-layer method involves the sandwiching of drugs in between the carrier and the targeting layer. Furthermore, multi-layer nanofibers can be achieved using a basic electrospinning unit and will provide better protection for the underlying biomolecule to be delivered. Meanwhile the targeting layer can be replaced with another drug which intended to disperse instantly, followed with the sustained release of the second drug.^52,53 By utilizing the porous nature and high surface area of the nanofiber system, the immobilization of drugs by physical and chemical methods also reported. The main motive of this mechanism is to preserve the bio-activity of laden molecules from the attack of electric field or solvent utilized. During physical immobilization, the drug molecules are reported to engage on the nanofiber surface by electrostatic, hydrophobic or van der Waals interactions. Meanwhile, in the chemical method, the nanofiber surface will be modified with amine, carboxyl, hydroxyl or thiol groups to hold drug molecules. In addition, the chemical immobilization has been reported to allow better control over the amount of drugs loaded when compared with the physical method.^32,54,55 Nonetheless, the co-axial and emulsion electrospinning techniques have been gaining great attention nowadays because of their excellent ability to shield biologically active substances and to reduce the complication of the initial burst. In the co-axial method, the drug particles will be sealed in the centre core surrounded by the polymer/targeting shell. Here, the electrospinning solution will be loaded into a specially designed spinneret that has separate compartments for loading the polymer and drug solution respectively. Thus, the resultant nanofiber will exhibit a core–shell structure and the drug molecules are completely isolated from the electric field since the outer surface of the Taylor cone is formed by the polymer solution. Moreover, in co-axial spinning the preferred carrier and drug will come in contact only at the point of synthesis of the nanofiber. Significantly, this avoids undesired reactions reported to occur between the polymer and biomolecules prior to electrospinning.^56–58 Later, this core–shell structure was reported to be generated using a single nozzle electrospinning unit using emulsion input, popularly termed emulsion spinning. However, the formation of a perfect core–shell structure is not observed in most cases due to the effect of increased emulsion.⁵⁹ Apart from this, electrospun-nanofibers-based hybrid systems powered by micro and nano-carriers have been extensively studied in the past five to six years. This technique allows the incorporation of other drug delivery vehicles like nanoparticles, nanospheres, microspheres, liposomes, micelles etc., into electrospun nanofibers. Through this novel strategy, we not only achieve better pharmacokinetics properties, but also ensure the intracellular delivery of drugs.^60–62

3. Electrospun nanofibrous drug delivery system for treating lung cancer

In this section, studies reported on lung cancer cells are discussed alone and for better understanding it is divided into subdivisions of DOX loaded nanofibers, synthetic implantable nanofibers, stimuli-responsive and natural-extract-loaded nanofibers (Fig. 4), respectively, based on the works available. Before that, a brief introduction to lung cancer is presented.

Fig. 4 Schematic representation of electrospun fibers for lung cancer treatment.

3.1 Lung cancer

Lung cancer is one of the best examples of clinical ailments originating from poor lifestyle choices. Since the past decade, lung cancer has accounted for higher deaths in both men and women than any other cancers. By the end of 2015, a total of 221 200 new cases and 158 040 deaths are expected because of lung cancer in the United States alone.⁴ It is reported to retain this position for the next 30 years by affecting twice as many people in 2040 than in 2010. Major risk factors associated with this mammoth issue are smoking (almost 90%), family history, radiation, diet, HIV infection, emphysema, scarring of the lungs and environmental pollution. This deadly disease mostly arises in the cell linings of bronchi, bronchioles, alveoli or other parts of the lungs as a result of fatal mutations. However, this clinical condition will not generate tumour tissue all-of-a-sudden, it will take months of time for the mutated cells to develop into an outgrowth which is visible in imaging modalities such as X-ray etc.

Therefore, the early prognosis of lung cancer is difficult and by the time it can be made out it is already in an advanced stage. Medical conditions such as a persistent cough, severe chest pain while laughing or coughing, hoarseness, weight loss, shortness of breath and bloody phlegm are few possible indicators of lung cancer. In general, it is distinguished into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The SCLC is least reported among two types, by accounting for only 10% to 15% of total lung cancers. This small-sized cancer growth is mostly observed in the bronchi, located near to the chest, and is likely to spread throughout the body in advanced stages. In contrast, the NSCLC is diagnosed in 85% of lung cancer patients and it is mainly caused by smoking. Based on the location and cell type, NSCLC is classified into adenocarcinoma, squamous cell carcinoma and large cell carcinoma. In spite of decades of research and exploration of advanced medical techniques, the one-year survival rate after diagnosis is 43%, whereas for five years it is only 17% percentage. This strongly emphasises the demand for reliable and promising modes of treatment.⁶³

3.2 DOX-loaded nanofibers

Nevertheless, electrospun nanofibers loaded with robust natural and synthetic anticancer agents have expressed positive signs in the development of trustworthy therapeutic alternatives. Recently, Behnaz et al. proposed doxorubicin (DOX) integrated polyethylene oxide (PEO), chitosan (CS) and graphene oxide (GO) based electrospun scaffolds to avoid side-effects while using free DOX for lung cancer treatment.⁶⁴

DOX is a powerful anticancer drug which has been in clinical use for more than 40 years, serving as an effective option to treat broad spectrum of cancers.^65,66 Its mechanism involves in the formation of a ternary complex with topoisomerase II (TOP-2) and DNA. The isoform Top2α, which is overexpressed in tumour cells is the primary target of DOX-induced cell death. As mentioned, the undefined delivery of DOX is frequently reported to elicit the onset of cardiomyopathy and other heart-related ailments, indicating the need for proper delivery.⁶⁷ The chitosan exploited in this research, is a widely preferred natural polymers for applications ranging from tissue regeneration to drug delivery systems. Furthermore, it also couples efficiently with common biodegradable polymers utilized for drug delivery purpose such as poly(vinyl alcohol) (PVA), cellulose, poly(lactic acid) (PLA), poly(caprolactone) (PCL), polyethylene glycol (PEG) and various biomolecules of interest.⁶⁸ Likewise, GO is well known for its biocompatibility, degradable properties and excellent stability among its fellow graphenes.⁶⁹ To providing a sustained release, DOX was initially coupled with GO and added to the PEO/CS mixture to form the electrospinning solution. Afterward, the prepared composite was spun into fine nanofibers using a typical electrospinning system. The successful loading of the drug into GO and establishment of a chemical reaction between GO/CS was inferred from the FTIR fingerprints. Nanofibers with a minimum diameter were obtained at the lower concentration of GO (0.5%) due to the increase in the conductivity of the prepared solution. In contrast, nanofibers with a beaded morphology were noted at the higher concentration of GO due to the increasing viscosity.

Meanwhile, TEM images confirmed, the successful incorporation of drug-loaded GO into the nanofibrous system. As with nanofiber morphology, the drug loading capacity of the PEO/CS nanofiber was also affected by increasing GO concentration, due to the occurrence of π–π stacking between DOX and GO. Additinally, the loading efficiency was also inferred to change significantly at different pH levels. As expected, the extensive mechanical properties of GO played a crucial role in delaying drug release from the PEO/CS/GO/DOX nanofibers when compared to controls. Further, the release kinetics were also found to depend on the pH level and maximum release was recorded at pH 5.3, which clearly exposed the pH sensitivity of the proposed combination. Finally the in vitro cytotoxicity of PEO/CS/GO/DOX nanofibers against A549 lung cancer cells was noted to be robust when compared with both GO/DOX and free DOX. More importantly, the prepared nanofibers were able to maintain anti-tumour activity for a long period, which permitted the sustained release of DOX.⁶⁴

In a different study, Nadia and colleagues reported another effective drug delivery system to safely deliver DOX at the site of cancerous tissue. Here, the drug was loaded into a PLA/PEG/multi-walled carbon nanotube (MWCNT) composite and more importance was given to the selection of electrospinning parameters. Initially, the mixture to be electrospun was prepared through a pre-designed protocol by using common solvents like acetic acid, chloroform and DMF. Then, the electrospinning process was carried out by changing the concentration (C), applied voltage (AV), flow rate (FR) and collector distance (CD) to get an ideal model. As discussed, the processing parameters have an immense ability to influence the overall efficacy of projected nanofibers, however, practical investigation involving optimisation of each and every parameter is a time-consuming procedure. Hence, they used the Box–Behnken design (BBD) method, one of the statistical experiment design techniques available for gaining optimised values. A series of mathematical and statistical breakdowns yields augmented values for four parameters, C, AV, FR and CD, as 8.15%, 0.2 mL h⁻¹, 18.50 kV and 13.0 cm, respectively.

Meanwhile, the minimum fiber diameter (225 nm) inferred through SEM analysis corroborated significantly with the value (228 nm) predicted by BBD analysis in optimised range. This clearly expresses the precision of BBD analysis and also the importance of processing parameters. Furthermore, the incorporated MWCNTs showed improved drug loading and the efficiency of prepared nanofibers was retained. In the interim, the in vitro drug release studies showed that the degradation behaviour varies depending on the percentage of PEG, MWCNT and DOX used. From their report, it can be concluded that the PLA/PEG 20%/MWCNT 5%/DOX 10% combination possesses appropriate release behaviour with a minimised initial burst. Like th previous study, the in vitro cytotoxicity was expressed using A549 lung cancer cells; this shows that the prepared delivery system possesses significant anti-tumour activity and killed 65–92% of cultured cancer cells. When compared with its counterpart (PLA/PEG/MWCNT), DOX-loaded PLA/PEG/MWCNT nanofibers maintained their cytotoxic effect for an extended period, far better than free DOX which has a half-life of only 16–18 h.⁷⁰

3.3 Implantable, synthetic-drug loaded nanofibers

Chen and his research team projected a controlled drug delivery system for administrating titanocene dichloride (TDC) to overcome the problems of solubility and short lifetime reported in its phase II clinical trials.⁷¹ TDC is an effective anti-neoplastic agent, whose exploration was largely inspired by metal-containing complexes like cisplatin, etc. The main target of TDC is observed to be DNA, furthermore the cytostatic and cytotoxic effects depend on its ability to arrest the cell cycle in the late S/early G2 phase by causing severe DNA damage. It reported to form DNA cross-linking, DNA adducts and accumulation of p53 which eventually leads to cell apoptosis.^72–74 Here, poly-L-lactic acid (PLLA) polymer was used as the carrier system to offer targeted and controlled release of TDC. The drug was directly loaded into PLLA/dichloromethane solution and fabricated into nanofibers. SEM micrographs and XRD results depict significant blending of the drug with PLLA nanofibers and the formation of a smooth bead-less morphology. However, beads were observed while increasing concentration of the drug to more than 15%. Hence, it is concluded that this is the maximum TDC quantity which can be loaded without affecting nanofiber morphology. Likewise, the tensile strength also decreased with increasing TDC percentage, though it was reported to be in adequate level for an implantable drug delivery system. The drug release behaviour was recorded by dissolving nanofibers in three different solutions namely: PBS with 50 mg L⁻¹; PBS with 10 mg L⁻¹ proteinase K; and PBS without proteinase K. In all cases, the initial burst was observed only in the presence of the proteinase K enzyme due to rapid degradation, but in virgin PBS the PLLA/TDC fibrous system exhibited excellent release kinetics. Furthermore, the cytotoxicity was scrutinized using human lung tumour spc-a-1 cells; an MTT assay revealed that both free TDC and TDC-coupled PLLA nanofibers possess an equal inhibition rate at different concentrations. However, the PLLA/TDC exhibited decreased IC₅₀ values when compared with a free TDC to produce the same effect mainly because of the sustained release. Therefore, they proved that this nano-formulation can be possibly exploited as an implantable delivery system to administrate TDC for lung cancer treatment without reported complications.⁷¹

Later, Yong et al. fabricated an electrospinning-inspired combination- or multiple-drug-delivery system to manage the release of the anti-cancer drugs paclitaxel and cisplatin.⁷⁵ Paclitaxel is also one of the most highly reputed anticancer drugs in clinical usage, which is obtained from the barks of the Pacific yew tree.⁷⁶ It has a complex chemical structure formed by taxane ring with a four-membered oxetane ring and an ester side chain at the C-13 position. It elicits a unique mechanism of action by targeting cell tubulin. Researchers have reported that paclitaxel-treated cells have difficulty with spindle assembly, cell division and also chromosome segregation, mainly due to the action of the drug against the cell microtubules.^77–79 On the other hand, the drug cisplatin has been used for the treatment of several cancers which mainly include bladder, head and neck, lung, ovarian and testicular tumours. The attacking nature of cisplatin against cancer cells is linked with its ability to crosslink with the purine base of DNA, interfering with DNA repair mechanisms, causing DNA damage, and subsequently inducing apoptosis.⁸⁰ So, the simultaneous administration of these two effective drugs will produce promising results. They used polypropylene carbonate polymer to integrate both drugs, and the electrospinning technique was used to produce drug-loaded microfibers. Through in vitro studies performed on A549 lung cancer cells, it was shown that polypropylene carbonate/paclitaxel/cisplatin microfibers possess synergetic effect and excellent cytotoxicity compared to controls, the free drugs and fibers incorporated with single drug.⁷⁵

3.4 Stimuli-responsive nanofibers

In addition to targeted and site-specific distribution, nowadays drug delivery systems are also expected to act based on bodily environmental conditions, virtually like a smart-robot system. Such artificial intelligence or stimuli-responsive nanofibers were first reported by Salehi et al.; they used poly(N-isopropylacrylamide) (PNIPAAm) to offer predetermined delivery of DOX. PNIPAAm has the ability to behave differently in response to temperature change, but the problem of higher water solubility hinders its application, especially in drug delivery systems. To overcome this, PNIPAAm was cross-linked with hydrophilic polymers so as to yield water-insoluble fibers. After performing a series of procedures, a freeze-dried powder form of PNIPAAm was obtained and it was dissolved along with DOX to form a homogenous electrospinning solution. Several characterization, release kinetics and in vitro cytotoxicity studies were performed to investigate the effects of electrospinning parameters to influence nanofiber efficacy using the response surface methodology (RSM) and BBD method. The mathematical minimum fiber diameter value (175.90 nm) obtained when using an optimised range of C (7.65 mg L⁻¹), AV (21.57 kV), FR (0.54 mL h⁻¹) and CD (9.75 cm) was confirmed by the practical value (176.57 nm) inferred from SEM analysis. Meanwhile, SEM images also displayed the formation of smooth fibers, which indicates the efficient assimilation of drugs, and the fiber diameter was noted to increase with DOX concentration.

Then, the retention of DOX molecular structure after the electrospinning process was confirmed by UV-Vis investigation; furthermore, the FTIR spectrum reveals the presence of desired chemical substances by exposing characteristic peaks. The DOX loaded PNIPAAm nanofibers exhibited a linear relationship between the drug release percentage and time (approximately) at different concentrations of the drug studied. It implies zero-order release kinetics of the drug and also guarantees steady discharge of active molecules over an extended period of time. However, a high initial burst was noted for a shorter period of time; later the discharge was levelled-off, indicating a greater amount of drug encapsulated inside the nanofibers than on its surface. Interestingly, the stability of drug release was observed for more than 30 days, which confirms the excellent encapsulation and release kinetics profile of the proposed nanofiber system. This behaviour is also recorded in an in vitro cytotoxicity study against A549 cells. The DOX-integrated PNIPAAm nanofibers can retain the same level of toxicity for 72 h due to the sustained release and greater eradication of cancer cells when compared to controls. Hence, they suggests that these intelligent stimuli-responsive nanofibers can be employed plausibly as an auspicious drug delivery system to treat lung cancer.⁸¹

3.5 Natural-molecules-coupled nanofibers

Beyond synthetic drugs, various natural-agents-incorporated nanofibers are also significantly utilized in the fight against lung cancer cells. Sridhar and his colleagues synthesized a PCL-based nanofibrous drug-eluting device to deliver extracts of curcumin (CU), aloe vera (AV) and neem (NE). The natural ingredients employed are a rich source of proven antioxidants, anti-cancer and anti-tumour biological molecules. To suggest a synergistic and efficient combination, different mixtures were electrospun to yield PCL, PCL/CU, PCL/AV, PCL/NE, PCL/CU/AV and PCL/CU/NE nanofibrous systems. Initially, the presence of all active substances was confirmed through FTIR analysis. The ability of the presented nanofiber mesh to maintain its integrity and stability was illustrated using the uniaxial testing machine. They observed that the addition of AV improved tensile strength, Young’s modulus and elasticity in contrast to CU and NE because of fiber-producing substances in AV. Meanwhile, SEM investigation displayed that fiber diameter decreased with the addition of AV and NE when compared to CU. However, the incorporation of CU was inferred to advance both the encapsulation and release behaviour of natural extracts. Lastly, PCL/AV, PCL/CU, PCL/CU/NE and PCL/CU/AV meshes expressed 70%, 65%, 23% and 18% cytotoxicity against A459 cells, respectively. In particular, the 1% AV and 5% CU coupled PCL nanofibers were observed to be 15% more cytotoxic when compared with a 1% commercial drug (cisplatin)-loaded PCL nanofiber.⁸²

Subsequently, in another work, Shao et al. reported the anti-cancer property of PCL/MWCNT/green tea polyphenol (GTP) electrospun nanofibers.⁸³ GTP is a natural storehouse of potential antioxidant, anti-inflammatory and cancer preventing active agents, whose capability in addressing lung, skin, oesophagus, duodenum, liver and stomach cancer has been reported previously. However, the problem of instability and the lack of site specificity hinders its utilization.^84,85 To solve this, the GTP was loaded in MWCNTs to obtain sustained release and PCL/MWCNT/GTP nanofibers were spun with different concentrations of GTP. Then, the occurrence of non-covalent interaction between GTP and MWCNTs was illustrated using UV-visible spectrophotometry; further FTIR studies were also carried out to confirm the presence of desired components. The excellent conductive property of MWCNTs yields fibers with a minimum diameter; however, it increases with the concentration of GTP due to the influence of elevated viscosity. Meanwhile, the well-oriented distribution of MWCNTs is expressed by TEM images, and laser scanning confocal microscope (LSCM) images disclosed homogenous spreading of GTP along the nanofiber surface. Interestingly, the fluorescence intensity was higher in PCL/MWCNT/GTP nanofibers when compared to PCL which once again confirmed the non-covalent interaction between MWCNTs and GTP which leads to higher loading. As expected, the addition and appropriate orientation of MWCNTs significantly improved the mechanical properties of PCL.

Nevertheless, with increasing concentration of GTP the Young’s modulus, maximum tensile strength and elongation at break of the nanofibers were noted to reduce gradually due to its plasticizing effects. The prepared meshes can sustain the displayed fibrous structure, even after 4 weeks, and swelling was observed only after the 12th week. Moreover, sudden initial bursts of hydrophilic GTP were inferred in the absence of MWCNTs, since the active components are dispersed on the fiber surface. This clarifies the role of MWCNTs and non-covalent interaction in delivering controlled and sustained release. So, the release behaviour can be tailored by adjusting the MWCNT content based on these requirements. In the interim, the Alamar blue assay was carried out using osteoblasts and human lung epithelial cells (A549) to determine the toxicity of the hypothesised delivery system against normal and cancerous cells. Surprisingly, the cytotoxicity of PCL/GTP and PCL/MWCNTs/GTP remained in a similar range for first two days, but on the fifth day the cytotoxicity of the projected nanofiber was significantly higher towards the cultured malignant cells. This pattern was also reflected in fluorescence microscope images of lung cancer cells cultured on PCL/MWCNTs/GTP fibers. Hence, they concluded that GTP incorporated PCL/MWCNTs have commendable properties to destroy lung cancer cells in a controlled manner with only traceable cytotoxicity against normal cells.⁸³

From the above researches, it can be clearly inferred that the resultant nanofiber geometry and properties heavily depend on the selectivity of the carrier (polymer), the concentration of the drug molecules and the processing parameters. In particular, the intended active substance is observed to affect fiber morphology, mechanical characters and release kinetics. So, extensive care should be taken when determining the quantity of drugs to be incorporated in favour of both nanofiber capabilities and its anti-cancer activity. Meanwhile, for optimising electrospinning parameters, it will be more appropriate to adapt available statistical experiment design techniques rather than going for time-consuming practical analysis. Successively, their significant precision with practical values are illustrated by several studies.

Overall, the works listed above evidently expose the aptitude of electrospun micro/nanofibers to achieve the required pharmacokinetics and improved cytotoxicity effects against lung cancer cells when compared with free drugs.

4. Electrospun nanofibrous drug-delivery system for breast cancer treatment

Several interesting works have been published on the utilization of electrospun fibers for treating as well as preventing the reappearance of breast cancer. This Section will present the works carried out on micro/nanofibers based DNA silencing, combination therapy, implantable drug loaded scaffolds and bio-inspired delivery systems, as illustrated in Fig. 5. Moreover, in all works the in vitro efficacy of the proposed prototype was demonstrated using breast cancer cell lines.

Fig. 5 Electrospinning inspired fibrous mesh for breast cancer treatment.

4.1 Breast cancer

Breast cancer is the second most fatal cancer reported globally, however it is considered to be the most frequently recorded cancers among the female population. This non-skin carcinoma is noted in nearly one out of every four diagnosed cancer cases, with more than 2.8 million survivors who have either past or current history reported in the US alone. The economic loss due to breast cancer treatment is the highest amongst all other cancers and to settle this serious health issue National Cancer Institute (NSI) is spending more than half (559.2 million dollars in 2013) of its total budget exclusively on breast cancer research.⁸⁶ Lethal, malignant tumours arise due to troublesome molecular changes that occur at cells present in three vital parts of the female breast, namely the lobules, ducts and stroma. In breast cancer, the complication of metastasis, i.e. spread of cancer cells to the bloodstream is significant because of the higher concentration of lymph nodes in the breast. Clinically, it is classified into non-invasive ductal carcinoma in situ (DCIS), invasive lobular carcinoma in situ (ILC) and invasive ductal carcinoma in situ (IDC).

IDC is the most frequently reported breast cancer type, accounting for about 50–80% of total breast cancer cases and one in eight women in the US is expected to develop IDC during their lifetime. This invasive carcinoma arises in the milk ducts; as the tumour tissue develops it will break through duct walls and reache the fatty tissue. By the end of 2015, a total of 231 840 new cases of IDC are expected to be diagnosed among US women. Furthermore, people affected by IDC have a higher chance of developing metastasis. Unlike IDC, the non-invasive ductal carcinoma does not spread to surrounding breast tissue from its origin, i.e. the ducts, therefore it is comparatively less lethal. Nevertheless, ILC is another invasive type of carcinoma develops in the milk producing glands of the breast, known as lobules. In addition, medullary carcinoma, mucinous/colloid carcinoma, tubular carcinoma, inflammatory breast cancer, Paget’s disease of the nipple, adenocystic carcinoma, and phyllodes tumours are some other rarely recorded breast cancer types. Though it sounds like a gender-oriented disease, the male population is also reported to develop breast cancer. Irrespective of age and sex, the five-year survival rate of victims with localized breast tumours is 98.3%, while that for those diagnosed with metastasis is stated to be only 23%.⁸⁷

4.2 Nanofiber-assisted DNA-delivery for silencing expressive breast cancer genes

At present, cancer research has attained significant evolution in the search for promising and persuasive treatment modalities beyond the horizon of chemotherapy. Treating cancer by using its properties, i.e. administrating chemical/genetic materials specifically to suppress the oncogenic expression, thereby disturbing it’s cell cycle or inducing apoptosis is one of those advanced techniques. Regrettably, vital requirements like site-specific delivery and an ability to maintain the bioactivity of genetic materials have been a great challenge for scientists to establish this notion. However, by making use of recent innovations and advancements, researchers have reported the successful delivery of DNA,²³ RNAi, microRNA (miRNA), small interfering RNA (siRNA),^21,22 plasmids for tissue regeneration and cancer treatment. Favourable properties like high surface area, porous fiber morphology and pre-determined release allowed electrospun micro/nanofibers to maintain appreciable bioactivity of the coupled molecules when compared to other nanostructures.^88–91 Inspired by this capability, Achille et al. tailored PCL electrospun fibers for the delivery of DNA plasmids to suppress targeted oncogenic gene expression to halt cell division and eradicate breast cancer cells. Essentially the purified DNA plasmid (800 mg) was added directly to the PCL solution and three different scaffolds were framed namely, pristine PCL, PCL/pKD-Cdk2-v5 plasmid (Cdk2i) and PCL/pKD-EGFP-v1 plasmid (EGFPi). About 85% of the PCL/DNA solution was measured to be electrospun with approximately 2.125 mg cm⁻² of DNA plasmid in each scaffold. From the SEM images it can be inferred that in pristine PCL the fibers exhibited smooth and stacked morphology, whereas in both DNA-plasmid-loaded mats, tangled fibers with a grooved structure were noted. Meanwhile, the addition of plasmid increased the fiber diameter from sub-micron in pristine PCL to 10 μm. Subsequently, EGFPi- and Cdk2i-loaded scaffolds expressed a similar pattern of degradation with a sustained release of DNA plasmid over three weeks, following a maximum burst in the first 24 h. These observations were confirmed by gel electrophoresis studies; moreover both scaffolds were able to maintain the integrity of fibers for 21 days – after that severe cracks were formed, indicating the degradation of the microfibrous system. Besides, to verify the achievement of this research objective, i.e. delivering DNA encoding for stimulating shRNA to disrupt the cell cycle of breast cancer cells by silencing Cdk2 genes, several tests were conducted.

Firstly, quantitative real time PCR (Q-PCR) showed that the suppression of Cdk2 genes was higher in cells treated with the pKD-Cdk2-v5 plasmid than others. Similarly, visual monitoring through the microscope exposed decreased cell viability in the presence of the Cdk2 shRNA encoded plasmid. Interestingly, they were able to produce the same result when culturing human breast cancer cells (MCF-7) on synthesised scaffolds in in vitro conditions; this implies that expression of Cdk2 genes was lower in cancer cells present on PCL/pKD-Cdk2-v5 plasmid scaffolds. Through a LIVE/DEAD assay they also confirmed that the cell death observed is due to silencing of oncogenic expression. This research serves as a “proof of concept” that electrospun fibers have the capability to administrate genetic materials with desired bioactivity.⁹²

4.3 Nanofiber-based combination therapy for breast cancer

Often, the administration of a single chemotherapeutic agent is not sufficient to address solid or advanced tumours. Accordingly, a blend of drugs is preferred during this clinical condition. Milena et al. designed a multiple drug delivery system to provide combination therapy for breast tumours. They used poly(L-lactide-co-D,L-lactide) (coPLA) as a carrier to deliver natural quaternized chitosan (QCh) and the popular synthetic drug DOX.⁹³ QCh reported in this research, is one of the derivatives of chitosan which exhibits better antibacterial, antimycotic and in vitro anticancer capabilities.^94–96 To extend its application to breast cancer research, it was tested against MCF-7 cells by combining with a synthetic anticancer agent. Active molecules were directly added to a coPLA solution then nanofibers with single and multiple anti-cancer agents were electrospun. The addition of QCh and DOX were reported to decrease the fiber diameter by increasing the conductivity and decreasing the viscosity of the electrospinning solution. Because of this, a maximum drug loading percentage of 96 and 98 was achieved in coupled DOX and QCh/coPLA/DOX meshes, respectively. An MTT assay delineated the excellent cytotoxicity of the synthesized nanofiber mats QCh/coPLA, coPLA/DOX and QCh/coPLA/DOX against the cultured MCF-7 cancer cells. Furthermore, the maximum reduction in cell viability was observed after 72 h of incubation in coPLA/DOX (18.8 ± 1.0%), QCh/coPLA/DOX (18.8 ± 1.6%), and free DOX (19.1 ± 1.2%) respectively.

Meanwhile, the morphological changes occurring in MCF-7 tumour cells observed through SEM revealed the better adherence of cancer cells on pristine coPLA nanofibers; these expressed normal bilateral symmetric morphology with numerous microvilli on the surface. However, on QCh/coPLA and DOX/coPLA nanofibers, the transformation of bilateral morphology to round holes in the cell wall and cytoplasmic extrusions were observed. Meanwhile, on the combined nanofiber system, i.e. QCh/coPLA/DOX, breast cancer cells were severely beaten by late apoptosis, cell shrinkage, surface blebs and formation of apoptotic bodies. This observation confirmed the higher viability of multiple-drug-loaded nanofibers compared to individual drug delivery systems. These effects were also confirmed through intravital staining of cells incubated on nanofibers for 24 h. The colour changes expressed in fluorescence micrographs of the different nanofiber surfaces depict the appropriate state of breast cancer cells, such as normal morphology, the formation of apoptotic bodies, etc. Once again, the highest apoptotic percentage (100%) was reported on QCh/coPLA/DOX mats due to the combined anti-tumour activity of the biomolecules utilized. So, by using this integrated drug delivery system we not only avoid the side-effects of using free DOX, such as cardiotoxicity, myelosuppression and cytotoxicity to normal mitotic cells, but also deliver intense therapeutic benefit.⁹³

4.4 Implantable drug-loaded electrospun scaffold

Yuan and his research group developed an implantable electrospun scaffold for treating breast tumours exclusively in postoperative conditions to prevent the recurrence of the disease. DOX was used as the anti-cancer agent and it was dissolved along with PLLA to form the electrospun solution. However, the hydrophobic nature of PLLA may greatly hinder the release kinetics of DOX. In order to solve this, the electrospun fibers were surface modified with polydopamine (pDA) to improve their hydrophilic properties.⁹⁷ Polydopamine is basically an adhesive protein, which can act as a feasible agent for surface functionalization of materials without affecting its geometry. pDA is reported to improve the wettability of materials by forming a thin nano-film on the surface, meanwhile it will encourage the integration of several biomolecules by serving as a coupling agent.^98–100 To make use of this excellent chemical, electrospun PLLA and DOX loaded PLLA fibers were immersed in a pDA solution to form PLLA/pDA and PLLA-DOX/pDA fibers. The diameter of the electrospun fibers was measured to be on the order of micrometres and the elevated nitrogen peak noted in the XPS graph indicates possible grafting of pDA on the microfiber surface.

After functionalization, the PLLA microfibrous mat exhibited excellent wettability but the drug encapsulation efficiency dropped slightly from 98.4% in PLLA-DOX to 90.1% in PLLA-DOX/pDA fibers. Likewise, the percentage of DOX released from the PLLA-DOX/pDA system (90.6%) was observed to be higher than in un-functionalized PLLA (82.1%) over the course of 40 days. However, this minimal change is reported not to affect the efficacy of the pDA grafted PLLA-DOX drug delivery system, these changes evidently depict successful embedding of pDA. In addition, extensive in vitro and in vivo pathological studies were also performed to prove the effectiveness of the PLLA-DOX/pDA drug delivery system. The expected in vitro cytotoxicity of PLLA-DOX/pDA microfiber against human breast cancer cell lines (MDA-MB-231) was illustrated by the MTT assay. In vivo studies were carried out using 5–6 weeks old healthy Balb/c mice. Tumour growth was induced by subcutaneous injection of MDA-MB-231 cells and the histopathological assay was performed after the tumour size reached 100 mm³. The mice were divided into four groups then pristine PLLA, PLLA-DOX, PLLA-DOX/pDA fibers were implanted at the tumour site followed by removal of a small section. A biopsy performed on the third day after implantation showed that the tumour tissue has grown rigorously in the control, but with no necrosis on both PLLA and PLLA-DOX group, respectively. But in PLLA-DOX/pDA group, a small area of necrotic tissue was inferred as a sign of the cytotoxicity activity of the projected drug delivery system. Likewise, on the 7th day, except for the PLLA-DOX/pDA group, all other expressed invasion of the tumour tissues, hyperplasia and mesenchymal inflammation. Interestingly, in the PLLA-DOX/pDA group, massive necroses of tumours were recorded. Moreover, a TUNEL and caspase-3 activity assay illustrated increased apoptotic activity and cancer cell dysfunction, respectively, on a PLLA-DOX/pDA fiber implanted tumour. Finally, qRT-PCR analysis confirms increased levels of mRNA expression of Bax (which induces apoptosis) and decreased Bcl-2 (which has anti-apoptotic activity) in PLLA-DOX/pDA implanted tumour cells. Therefore, from the above it can be concluded that these pDA-functionalized PLLA-DOX microfibers can be plausibly exploited as implantable drug delivery systems to fight against breast cancer.⁹⁷

In another study Laiva et al. developed a drug delivery system for the safe delivery of TDC by rectifying the complications reported during clinical trials. Initially, pristine PCL, PCL/SF and PCL/SF/TDC nanofibers were fabricated from an homogenous electrospinning solution. The addition of SF and TDC increased the conductivity and hence smooth fibers with decreased diameters were obtained. The SEM micrographs confirmed the formation of highly porous beadless nanofibers and those with a minimum diameter were noted in PCL/SF/TDC system. Furthermore, improved wettability after the addition of SF was confirmed by contact angle studies, meanwhile the FTIR spectrum reveals the presence of appropriate chemical groups. Excitingly, the average fiber diameter is reported to affect the stress–strain relationship, and a maximum tensile stress value was measured in PCL/SF nanofibers with a high percentage of TDC. Besides, the in vitro drug release shows that the proposed nanofibers maintained sustained release for a maximum of six days, and a PCL/SF system with a higher concentration of TDC is described to release more drug after an incubation of 144 h. These results imply the excellent stabilization of drugs within the nanofibers, which offers organised release with a minimum or negligible initial burst. On the other hand, the MTT assay exposed the excellent cell viability of MCF-7 cells cultured on a highly concentrated TDC-coupled PCL/SF nanofiber system when compared with both the pristine and free drug at the end of the third day. It is reported to increase significantly with respect to time, which reflects the outcomes of in vitro drug release kinetics. The optical microscopy and SEM images expressed adverse changes in the morphology of breast cancer cells cultured on drug loaded nanofibers which evidently confirms the apoptotic capability of TDC.¹⁰¹

Sundar and Sangeetha fabricated stimuli-responsive drug releasing scaffolds to provide post-operative chemotherapy for breast cancer patients. The electrospun scaffolds were drawn from the polymeric solution of collagen (CG)/PNIPAA/CS loaded with the widely used anticancer drug 5-fluorouracil (5-FU) and the concentration of CS was varied to yield different nanofiber mats. Collagen is the chief structural protein of all vertebrates which constitutes over 90% of the extracellular proteins of the tendon, bone and ECM, and it is divided into several types. Furthermore, the drug 5-FU is basically an antimetabolite agent which has been in clinical usage for the past 20 years. Typically, it is an analogue of uracil with a fluorine atom at the C-5 position in place of hydrogen. It rapidly enters the cell and forms several intracellular active metabolites such as fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), etc. These metabolites disrupt DNA formation, RNA synthesis and also inhibit thymidylate synthase (TS) which eventually leads to cell death.^102–104 The nanofiber geometry was highly influenced by the concentration of chitosan but not by the loaded drug. At higher concentrations, nanofibers with uneven and beaded morphologies were obtained, mainly due to the increase in the viscosity of the electrospinning solution. Furthermore, a greater percentage of the drug was deposited on the nanofiber surface in the form of aggregates and only a small amount was assimilated inside. Because of this, the initial burst was observed in all mats, however increasing the concentration of CS was observed to slow down the degradation and diffusion of 5-FU molecules. In addition, the release kinetics also varied in response to the pH of buffer solution, having an increased cumulative drug dispersion at low pH and vice versa. This variation is inferred due to the swelling and protonation of integrated natural polymers. As anticipated, the in vitro cell viability of cultured MCF-7 cells decreased with increasing drug concentration. Meanwhile, normal L929 cells cultured on CG/PNIPAA/CS/5 FU nanofibers exhibited a typical cell morphology which proves the retention of bioactivity and the oncogenic cell dependent anti-cancer activity of the incorporated chemotherapeutic agent.¹⁰⁵

4.5 Natural-extract-loaded nanofiber scaffolds

Polyphenols are bioactive substances which are found abundantly in common ingredients used to make our regular diet; popularly called dietary polyphenols. They are highly essential for maintaining good cardiac health, normal cell viability, scavenging free radicals, etc. An exciting piece of research reported the capability of these familiar chemicals to induce cancer cell death, mainly because of their rich antioxidant content. However, the same effect was difficult to reproduce at in vivo/clinical conditions mainly because of poor selectivity and reduced bioavailability at the tumour site.^106,107 However, with the modern drug delivery options available the above deficiencies can be easily rectified. Priya et al. synthesized a poly(D,L-lactide-co-glycolide) PLGA/PEO nanofiber carrier system for site-specific delivery of ferulic acid (FA).¹⁰⁸ FA is a polyphenolic phytonutrient commonly present in rice, wheat, oats, coffee seeds, peanut, apple, artichoke and orange. It inferred to act severely against free radical and reactive oxygen species (ROS), substances associated with DNA damage or mutation. Nevertheless, it is documented to induce pro-apoptotic effects, especially in breast and liver cancer cells.^109–111 In this research, the FA was added directly to the PLGA/PEO polymer blends, then a basic electrospinning setup was used to fabricate free FA and PLGA/PEO/FA nanofibers. FESEM characterization studies show that at lower concentrations of FA bead, free, smooth and cylindrical nanofibers were formed. However, at higher concentrations of FA, particularly between 6–8 wt%, electrospraying of the polymer solution is reported rather than nanofiber formation. Therefore, to produce smooth fibers without crystallization, the maximum percentage of that FA can be loaded was suggested as 2 wt%. Moreover, the fluorescent image revealed the presence of a core–shell arrangement of FA inside the PLGA/PEO polymer blend. On the other hand, XRD patterns exposed the amorphous state of the encapsulated FA in composite nanofibers and TGA graphs presented the decomposition of FA-loaded PLGA/PEO nanofibers at two different temperatures.

In addition, the drug encapsulation efficiency of the projected drug delivery system was measured as 66 ± 1.34%. The release kinetics studies revealed that more than 50% of FA was released within the first 24 h, followed with controlled release for the next 10 days. Accordingly, the MTT assay displayed significant cytotoxicity of the PLGA/PEO/FA (2 wt%) nanofibers against MCF-7 cells when matched with controls, meanwhile it achieved a cell inhibition rate of 51.4 and 67%. On the other hand, the fluorescence microscopy images depict FA-induced apoptosis signs of breast cancer cells, such as chromatin condensation, cell wrinkles and cytoplasmic remnants on the PLGA/PEO/FA nanofibers. These observations clearly confirm the retention of the anti-cancer characteristics of loaded polyphenol even after the electrospinning process.¹⁰⁸ In another study, Sridhar et al. synthesized a PCL-based nanofibrous drug eluting device to deliver extracts of curcumin (CU), aloe vera (AV) and neem (NE) for breast cancer treatment. The in vitro cell viability studies showed PCL/CU/AV/NE nanofibers were able to kill a greater number of MCF-7 cells in culture when compared to the other individual systems; PCL/CU, PCL/AV, PCL/NE/CU and PCL/AU/CU. This observed effect is inferred due to the synergistic action of exploited natural extracts.⁸²

4.6 Functionalization of hydrophobic polymers with natural materials

Though there are a variety of polymers available in the market, only a few of them can be electrospun into nanofibers. To be fabricated into nanofibers, the selected polymer should be dissolved in common volatile solvents to form a homogeneous solution. However, through melt spinning even a non-degradable polymer can also be made into nanofibers by applying high temperature. Since it is difficult to control the processing parameters, melt-spinning often yields fibers with poor morphology and porosity.^112,113 In order to solve this problem and to cogently utilize poor or non-hydrophilic polymers for drug delivery applications, Thangaraju and colleagues reported an innovative solution. They demonstrated their notion with the help of a poorly hydrophilic poly L-lactide (PLLA) polymer; furthermore the drug delivery application of modified PLLA nanofibers was also demonstrated using curcumin (CU). To make PLLA more hydrophilic, they initially used a water-bath method to collect the synthesized fibers. In another method, PLLA was coupled with natural hydrophilic polymers like cellulose acetate (CA) and silk fibroin (SF). Among the described schemes, the water-bath approach yields microfibers in the range 2 to 2.5 μm, whereas nanofibers with different diameters were reported in the second method. SEM analysis implies that nanofiber diameter variation depends greatly on the concentration of CA, SF and C; meanwhile the clear dispersion of curcumin in the synthesized fibers was displayed by TEM images. All three electrospun fibers presented a diversified distribution of pores, and contact angle studies disclosed the superhydrophilic character of the PLLA/SF nanofibers compared to its counterparts. The in vitro drug-release kinetics of C-PLLA microfibers and C-PLLA-CA & C-PLLA-SF nanofibers unveiled no significant changes among different systems and excitingly the initial burst release was absent in all cases. When compared to the microfiber system, C-PLLA-CA and C-PLLA-SF nanofibers exhibited better sustained-release of hydrophobic curcumin for 11 and 18 days, respectively. The proposed systems reduced significantly the cell viability of MCF 7 breast cancer cells through the controlled the delivery of curcumin. The achieved effect was concluded to be due to curcumin-induced cell cycle arrest by targeting highly expressive signalling molecules, an increase in p53 levels and caspase-dependent apoptosis. In the interim, the modified curcumin loaded nanofibers expressed approximately zero percent cytotoxicity against normal NIH/3T3 fibroblast cells. Hence, this suggests that the modified superhydrophilic nanofibers are preferable for the delivery of hydrophobic drugs intended for breast cancer treatment.¹¹⁴

The successful cytotoxicity of various natural and synthetic nanofibers against different human breast cancer cell lines were reported in the studies discussed above. More excitingly, the in vitro results were also reproduced in animal models, which indicates the excellent retention of the nanofibers’ potential under in vivo conditions as well. Each and every model possess unique features, and the specific use of nanofibers to delivery gene therapy for cancer is one of the most plausible therapeutic options under research. In contrast to influencing the nanofiber morphology, the addition of preferred biomolecules is reported to enhance the geometry of electrospun nanofibers in a few cases. Therefore, by exploiting above appropriate drug delivery models we can offer combination therapy, natural therapy, gene therapy or site-specific chemotherapy to control and eradicate breast cancer cells.

5. Electrospun nanofibrous drug-delivery system for colorectal cancer treatment

Besides lung and breast cancers, the ability of electrospun fibers to eradicate colon cancer has also been reported in several studies. Researchers have proposed implantable scaffolds, drug-eluting stents, magnetic nanofibers and electrospinning empowered chemotherapy to successfully deliver drugs to inaccessible organs of the gastrointestinal tract (Fig. 6). Moreover, their cytotoxicity was illustrated using different colon cancer cells.

Fig. 6 Nanofibers for eradicating colon cancer cells.

5.1 Colon cancer

The colon is the largest part of the digestive system, it starts from the lower right abdomen and extends up to the rectum. The main function of the colon is to adsorb water, minerals, vitamins, electrolytes, maintain fluid balance, process fibers in indigestible food material and to propagate waste to the rectum which eventually expelled through the anus. Colon cancer is reported to develop in the caecum, the right lower part of the abdomen and it will progress through the right-ascending colon, transverse colon, left-descending colon and sigmoid colon. It is traditionally referred to as colorectal cancer, since cancer develops in the colon and rectum shares many common features. It usually arises in the form of non-cancerous polyp growths on the lining of the colon/rectum and it slowly develops over years to form a mass of cancerous tissue. Though all polyps do not advance into cancer, of the two main types the adenomatous polyp is alone observed to form oncogenic tissue. Since the colon is deep inside the abdominal cavity, it is difficult to detect the cancerous growth in its early stages. Symptoms such as blood in the stool, change in bowel moments, constipation and persistent abdominal distress are chiefly the indication of disease maturity.

Frustratingly, colorectal cancer is the third deadliest cancer in the United States among both sexes and about 1 in 20 people are expected to be affected by colorectal cancer in their lifetime. Besides, the American cancer society estimates that a total of 93 090 new cases and 49 700 deaths will be reported due to of colon cancer by the end of this year. In colon cancer, the chance of developing metastasis is high, especially when it grows in the inner wall, where it has increased access to the lymph nodes or blood vessels through which it spreads to distant organs. Among various types, adenocarcinoma is reported in more than 95% of total colon cancer cases where the cancer starts in cells which give lubrication to mucus. Furthermore, carcinoid tumours, gastrointestinal stromal tumours, lymphomas and sarcomas are some of the rarely recorded colon cancer types.¹¹⁵ On the other hand the site-specific delivery of drugs to the colon is a complicated process since the preferred carrier system must tackle the attacks of gastrointestinal fluids, digestive enzymes, varying pH levels and long transit time. Hence, in the pharmaceutical field, it is referred as the “Black Box” of the gastrointestinal system.

5.2 Implantable, superhydrophobic fibrous mats

Like other cancers, chemotherapy, radiation therapy and surgery are the most commonly preferred clinical treatments for colon cancer. Among those, surgery is exclusively adopted for managing localized tumours, though it is severely hampered by the problem of post-operative rebound which is recorded in more than 40% of cases. To combat this scenario, chemotherapy and radiation are often pursued, but the problem of systemic side-effects, as discussed, offsets the benefits of this treatment.^116–118 This creates a huge demand for a potential system to deliver site-specific therapeutics by tackling the attack of gastrointestinal fluids. Recently, Stefan et al. developed a superhydrophobic, drug-loaded, implantable fibrous mat to avert this problem of locoregional tumour regeneration. They used a hydrophobic polymer dopant poly(glycerol monostearate-co-ε-caprolactone) (PGC-C18) to decrease the wettability of PCL and to enable controlled delivery of anti-cancer drugs (CPT-11 and SN-38).¹¹⁹ Among those, the drug SN-38 is an active metabolite of irinotecan, one of the most effective topoisomerase I inhibitors available on the market. The Topo-I enzyme plays a pivotal role in DNA replication and transcription processes, so disrupting the activity of Topo-I will cause DNA damage and transient S-phase arrest, which eventually leads to the formation of double strand (ds) DNA breakages. These defective DNA strands are reported to activate the various apoptotic cascades of cells.^120,121 The synthesized hydrophobic dopant and anti-cancer drugs were added to the PCL solution, then four different delivery systems were electrospun; namely pristine PCL, PCL doped with PGC-C18, PCL/PGC-C18/CPT-11 and PCL/PGC-C18/SN-38 using a rotating drum target. The contact angle results interpret the super-hydrophobic nature of doped PCL and the formation of microfibers was inferred from SEM images. Meanwhile, they also varied the electrospinning parameters to optimise the ranges, and the resultant large fiber morphology indicates the possibility of offering controlled local delivery for an extended period of time. The mechanical tests exposed the appropriate tensile strength of synthesized hydrophobic fibrous meshes required to avoid the problem of leakage or an instant burst of drug molecules. Furthermore, differential scanning calorimetry confirms complete dissolution of CPT-11 and SN-38 drugs in microfibers, and ultrasound studies proved the presence of entrapped air in hydrophobic meshes.

The utilization of super-hydrophobic surfaces will be a better choice to achieve a significant delay in the degradation of electrospun fibers. Interestingly, in this study, the authors designed 3D super-hydrophobic meshes entrapped with air, which depict to slowdown the degradation process. The PCL fibers doped with the higher concentration of PGC-C18 (10%) can successfully keep the sustained release of SN-38 for 70 days and CPT-11 for 50 days under in vitro conditions. They also proved the importance of entrapped air in influencing the release behaviour by showing that, in the absence of air, the 10% PGC-C18 doped PCL mat released a maximum dose of the SN-38 drug within 14 days and also had a massive initial burst. Furthermore, the distribution of drugs inside the fiber was varied based on its concentration; at high levels it was concealed in the centre of the fiber whereas at lower levels it was found mostly on the surface, which is clearly illustrated through confocal microscopy. The MTT assay performed using colorectal cancer cells (HT-29) exhibited the same trend of growth inhibition, and 1% SN-38 was enough to maintain the cytotoxicity for 90 days. In the interim, the CPT-11-loaded fibers showed a poor long-term effect in reducing the viability of colon cancer cells, and higher doses was required to provoke the same effect using 1% SN-38 drug loaded PCL. From the above inference, it can be concluded that the 3D superhydrophobic PCL has the potential to preserve the bioactivity of coupled substances, and the more active SN-38 drug can be used as a plausible replacement for the prodrug CPT-11 to elicit intense anti-cancer effects against colon cancer cells.¹¹⁹

5.3 Nanofibers-inspired drug-eluting stent for colon cancer treatment

Li and co-researchers fabricated a drug-eluting stent to rectify the problem of restenosis associated with colorectal cancer patients.¹²² Conventional stents available for clinical usage are hampered with the problem of tumour ingrowth and the granulation of tissue inside the lumen; moreover the treatment methods often practiced to restore this situation are associated with high mortality.^123–125 Hence, to address this clinical complication, the stent was coated with drug loaded nanofibers to repair lumen potency and avert intraluminal tumour growth. The drug 5-fluorouracil (5-FU) was used as an anti-cancer agent; it is one of the oldest and most eminent drugs, introduced in 1957 for clinical use. However, the problems of poor selectivity and undesired dispersion reduce its cytotoxic effects against colon cancer cells.

An electrospinning solution containing PLLA and a different concentration of 5-FU was fabricated into nanofibers using optimized parameters and deposited on a designed stent attached to the rotating drum target. At lower concentrations of 5-FU (1.6%), the fiber diameter of PLLA was decreased; in contrast fibers with an uneven morphology were obtained at maximum concentration, i.e. 12.8% of 5-FU. This indicates the uneven dispersion and increase in viscosity of the spinneret solution when adding high amounts of drug. Meanwhile, the establishment of chemical relations between the 5-FU and PLLA polymer was confirmed by FTIR illustration. As expected the total percentage of drug released was dependent on the loaded concentration and the synthesized nanofibers were able to maintain sustained release for 240 to 400 h. Accordingly the cell viability test conducted against HCT-116 cells shows that, after 96 h, normal growth of cancer cells was observed on pristine PLLA whereas on 12.8% 5-FU loaded nanofibers greater number of cells were lysed even better than the free 5-FU drug. Moreover, the MTT assay revealed that the proposed nanofiber system is able to maintain its cytotoxic effects for 120 h, and has a more potent IC₅₀ than the free drug as well. This confirms the controlled release of the drug which resulted in decreased drug resistance of the colon cancer cells employed.¹²²

5.4 Magnetic nanofibers for hyperthermic colon cancer treatment

Decades of cancer research have given rise to different types of therapeutic systems: hyperthermia is a newly explored cancer-curing treatment modality where elevated temperature is exploited to attack tumour tissue. In this technique, the temperature of oncogenic tissue will be typically raised to over 42 °C, simultaneously maintaining the normal tissue under harmless or tolerable temperature ranges. This works by utilizing high-temperature-sensitivity of cancer cells when compared with their counterparts. Various techniques like ultrasound, microwave and radio waves have chiefly been used as an energy source to destroy cancer cells by producing localized temperature elevation.To increase the effectiveness, it is often coupled with an available curative, such as chemotherapy or radiation therapy. However, the clinical application of this method is impeded by practical difficulties in providing spatiotemporal temperature distribution exclusively around the tumour tissues.^126–129 Recently, Lin and colleagues successfully demonstrated hyperthermic eradication of colon cancer cells by using magnetic nanoparticles or thermoseed-loaded chitosan nanofibers.¹³⁰ The thermoseeds of ferrite magnetite are low-toxicity nanoparticles which possess superparamagnetism in the nano-range. These attractive nanoparticles are approved by the FDA and have already found several applications in drug delivery, cancer tissue imaging, biomolecule separation, etc. The required heat is produced by applying a strong electromagnetic field using the coils placed outside, followed by intravenous administration of magnetic nanoparticles.^131,132

To overcome the hitches in using these nanoparticles, such as RES clearance, in vivo instability and mass diffusion, here the ferrite nanoparticles are entrapped inside chitosan/PEO nanofibers to produce a plausible outcome. Initially, the chitosan was functionalized with iminodiacetic acid (IDA) to fix a large number of magnetic nanoparticles; PEO/CS-IDA nanofibers were then synthesized using a conventional electrospinning setup. Later, the prepared Fe ions were sealed on the nanofiber surface by an immobilization method which consequently forms Fe₃O₄ nanoparticles by chemical co-precipitation and gives rise to magnetic nanofibrous composites. The electrospinning parameters were optimized to obtain smooth and beadless nanofibers; the magnetic nanoparticles were observed in large numbers on PEO/IDA-CS/Mag when compared to un-functionalized PEO/CS/Mag nanofibers. Meanwhile XRD patterns and TEM images confirmed the presence of desired crosslinking and successful assimilation of magnetic nanoparticles along the nanofibers’ surface. Finally, the LDH assay performed using mouse colon carcinoma (CT-26) revealed no significant changes in cytotoxicity of PEO/CS/Mag, PEO/IDA/Mag, PEO/CS-IDA/Mag nanofiber composite systems and free Fe₃O₄ nanoparticles. Under an applied magnetic field, a temperature of range 40–45 °C was created by the magnetic nanofibers and also showed better cell viability in the absence of magnetic nanoparticles; a maximum in cell deaths in all magnetic nanofibers under the applied field was reported. From the above observations, it can be concluded that these Fe₃O nanoparticles entrapped nanofibers can be chosen as a plausible treatment method without any systemic side-effects.¹³⁰

5.5 Nanofibers-assisted chemotherapy for colon cancer

As mentioned, the site-specific delivery of drugs for colon cancer is highly intricate, especially in the case of oral administration. The intended chemotherapeutic agent has to be intact during the prolonged transition time before it reaches the colon. It has to shield itself against the dynamism of the gastrointestinal tract, to prevent undesired dispersion, which often results in damage to normal cells. Establishment of an effective shield is a difficult task for conventional treatments available in the market, but the electrospun fibers will be very handy in a situation like this, as they not only retain bioactivity but also enable high loading. Motivated by this, Zhang et al. demonstrated the anti-cancer activity of multiple-drug-loaded electrospun nanofibers under in vitro and in vivo conditions. They used PLA as the carrier polymer to safely deliver the drugs 5-FU and oxaliplatin, whose cytotoxicity against colon tumours has been validated in several studies.¹³³ The drug oxaliplatin is the newest platinum based anticancer agent alongside cisplatin and carboplatin. Oxaliplatin exerts its cytotoxic effect by damaging the DNA of cells. Briefly, the apoptosis of cancer cells is reported to be caused by formation of DNA lesions, arrest of DNA synthesis, inhibition of RNA synthesis, and triggering of immunologic reactions. However, the underlying mechanisms of those effects have not yet been revealed completely.¹³⁴ A typical electrospinning setup was used to fabricate the anticipated nanofibers and the encapsulation efficiency of nanofibers was illustrated by various characterization studies. In the interim, the cell viability studies carried out on human colon cancer cells (CRC HCT8) showed the effective cytotoxicity of multiple-drug-loaded nanofibers which was later confirmed by MTT and flow-cytometry assays. Successively, they also reproduced the inferred results under in vivo condition using animal models. They implanted the synthesized nanofibers in CT26 tumour-bearing mice models then the anti-cancer activity was measured using histological and survival rate analysis. As in cell studies, the multiple-drug-loaded nanofibers exhibits superior tumour inhibition when compared with intravenous injection of free drugs and commendably increased the life expectancy of tumour-affected mice. This clearly proves the suitability of electrospinning-based combination therapy to battle the progression of colon cancer.¹³³

In another study, Liu et al. developed a novel drug delivery system to fight against colon cancer by utilizing the concept of difference in energy metabolism between tumour and normal cells. They produced PCL nanofibers loaded with sodium dichloroacetate (DCA) and diisopropylamine dichloroacetate (DADA). These were in-situ-administrated to C26 tumour-bearing mice separated into appropriate groups. Histological pathology performed after the 12th day of treatment revealed that in animals given PLA/DCA and PLA/DADA the suppression of tumour growth by 75% and 84% was accomplished, respectively. Approximately more than 95% of cancer reduction was achieved at the end of the 15th day. Therefore, DCA- or DADA-coupled PCL nanofibers can be employed to avert systemic side-effects by discriminating cancer and normal cells.¹³⁵

6. Conclusion and future prospects

The establishment of modern drug delivery systems is essential to control the current situation and future threats of cancer. In particular, nanotechnology-empowered delivery systems will play a pivotal role in fulfilling therapeutic goals such as increased drug absorption, distribution and minimum excretion. The current clinical approaches are badly hampered by the high occurrence of systemic side effects and recurrence of the disease. Accordingly the increasing death rates and economic loss, require timely implementation of effective alternatives. Meanwhile, modern drug delivery systems like hydrogels, nanoparticles, nanocomposites, micro/nanofibers, liposomes, micelles, nanotubes, dendrimers, quantum dots etc., have been reported to possibly avert these existing complications. The presence of a devoted vehicle arrangement to carry drugs, shielding materials and targeting molecules is the main advantage of modern delivery systems which enables them to offer site-specific medication. They reach precisely the cancerous site through passive or active targeting and can adjust the release of the desired biomolecules in response to the bodily environment, hence they are popularly termed as stimuli-responsive or intelligent drug-delivery vehicles. Among various alternatives, electrospinning-based micro/nanofibers have been reported to the best because of their high surface-to-area ratio, easy tailoring, cost-effectiveness and ability to retain the bioactivity of therapeutic agents. This helps researchers to achieve pharmacokinetics requirements such as high loading of drugs, targeted delivery, controlled and extended release, maintenance of the required drug concentration in the blood and avoidance of drug resistance. In this review, works reporting on the exploitation of electrospun micro/nanofibers to address lung, breast and colorectal cancers were discussed in detail. As mentioned, more than 40% of total cancer deaths are reported as due to the above types; moreover the chance of developing metastasis is also high in these types.

Various promising ways of delivering biomolecules ranging from synthetic drugs to DNA plasmids were illustrated using electrospun micro/nanofibers. In all cases, electrospinning-boosted drug delivery systems exhibited an excellent drug loading capacity, easy incorporation of desired agents, the required encapsulation efficiency and pre-determined/controlled release, thereby achieving advanced cytotoxicity as demonstrated in Fig. 7. Moreover, their pharmacokinetic properties can be modified productively based on the application: for instance, nanofibers can engineered to have an initial burst to maintain a high concentration of drugs in the blood and vice versa. This enables them to treat, correspondingly, developing tumours, advanced tumours and also post-operative tumours. Meanwhile, the importance of the electrospinning parameters and the loaded active substances in influencing the efficacy of the nanofibers was also indicated. The poor selection of parameter ranges and the addition of less compatible anti-cancer agents were always reported to affect the fiber diameter, surface area, degradation or drug-release behaviour and cytotoxicity (as listed in Table 1). However, these defects can be easily rectified by trying out different combinations of various parameters to achieve an ideal outcome. In addition, the use of statistical experimental-design methods to tailoring these essential factors, reducing the time consumed by practical approaches, has also been proposed. Most work reported only on the delivery of synthetic or natural anti-cancer agents and falls chiefly within the horizons of chemotherapy. To combat future demands, the applications of nanofibers need to be expanded to emerging alternatives like gene therapy and immunotherapy. The notion of gene therapy aims to attack oncogenic cells by suppressing or destroying their expressive genes so as to control their growth and induce apoptosis. This can be made possible by delivering DNA plasmids, miRNA, siRNA, RNAi and other genetic material. Clinical implementation was hindered by lack of site-specific delivery, an inability to maintain bioactivity and meagre bioavailability. However, in recent years, scientists have been able to cross these barriers with the help of nanotechnology. Meanwhile, the capabilities of electrospun nanofibers have attracted several researches on the delivery of genetic material through both viral and non-viral vectors. It has been reported to be used in the treatment of genetic disorders, infectious diseases and tissue engineering applications as well.⁹¹ In particular, nanofiber-based gene delivery or silencing has already proven to be a potential method for tissue engineering purposes such as bone regeneration, vascular repair or regeneration, skin regeneration and also for neural restoration.^136–140 Furthermore, the work done by Achille et al. meticulously showed the aptitude of electrospun fibers to achieve high bioactivity and also the significant eradication of breast cancer cells following the successful silencing of expressive oncogenic genes.⁹²

Fig. 7 Diagrammatic representation of cytotoxicity effects of various electrospun fibrous meshes.

Table 1 Effect of loaded drug/bioactive substances on the surface morphology of electrospun fibers

Carrier polymer	Drug/bioactive substances loaded	Key changes in fibre morphology	Reference
Polyethylene oxide (PEO), chitosan (CS) and graphene oxide (GO)	Doxorubicin (DOX)	Nanofibers with minimum diameter were obtained in lower concentration of GO until 0.5% due to increase in the conductivity of prepared solution	64
		At elevated GO concentration, nanofibers with beaded morphology were obtained due to increase in viscosity
		Accordingly, the drug loading capacity was also affected when increasing GO concentration because of π–π stacking between DOX and GO
Poly-L-lactic acid (PLLA)	Titanocene dichloride (TD)	Beaded nanofibers were observed while increasing the concentration of drug to more than 15%	71
		Furthermore, increasing drug concentration is also inferred to affect mechanical properties and release kinetics as well
PCL	Curcumin (CU), aloe vera (AV) and neem (NE).	Excessive addition of CU and NE reduced tensile strength, Young’s modulus and elasticity in contrast to AV	82
		Fibre diameter increased with increasing concentration of CU due to increase in viscosity
		However, the incorporation of CU was inferred to advance encapsulation and releasing behaviour of natural extracts
PCL/MWCNTs	Green tea polyphenols (GTP)	Increased levels of GTP increases fibre diameter due to increase in viscosity	83
		The plasticizing effects of GTP reduced Young’s modulus, maximum tensile strength and elongation-at-break-point of nanofibers
PCL	pKD-Cdk2-v5 plasmid (Cdk2i) and PCL/pKD-EGFP-v1 plasmid (EGFPi)	Tangled fibers with a grooved morphology were noted while adding both type of plasmids	92
		A drastic increase in fibre diameter from the sub-micron to micron level was noted after plasmid incorporation
Collagen (CG)/PNIPAA/chitosan (CS)	5-Fluorouracil (5-FU)	The nanofiber geometry was highly influenced by the concentration of chitosan but not by loaded drug	105
		At higher concentrations of chitosan, nanofibers with an uneven and beaded morphology were obtained mainly due to an increase in the viscosity of the electrospinning solution
		Because of this, a greater percentage of drug was deposited on the nanofiber surface in the form of aggregates and little was assimilated inside
Poly(D,L-lactide-co-glycolide) PLGA/PEO	Ferulic acid (FA)	Electrospraying was observed rather than nanofiber formation when increasing FA concentration to more than 6%	108
		Moreover, at higher concentrations of FA a serious crystallization problem arose, forming amorphous nanofibers
Poly L-lactide (PLLA), cellulose acetate (CA) and silk fibroin (SF)	Curcumin (CU)	Fibre diameter varies greatly depending on the concentration of CA, SF and CU, respectively	114
		All three electrospun fibers presented diversified distribution of pores based on the substances loaded
PLLA	5-FU	At lower concentrations of 5-FU (1.6%), the fibre diameter of PLLA was decreased	122
		Conversely, fibers with uneven morphology were obtained at maximum concentration, i.e. 12.8% of 5-FU
		This was mainly due to uneven dispersion and an increase in the viscosity of the spinneret solution when adding a high amount of drug

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Likewise, immune therapy is another exciting method which employs the patient’s defence system to scavenge cancer cells without the usage of any toxic chemicals. Here, man-made antibodies will be utilized to train our immune mechanism to outsmart disguised cancer cells. Its incorporation in nanofibers will help in both cancer prevention and treatment. In addition, the coupling of genetic materials or antibodies with chemotherapeutic agents may give rise to a new type of potential remedy, typically referred as combination therapy. Therefore, future researches on electrospun micro/nanofibers have to be broadened beyond chemotherapy and more works need to be carried out on advanced concepts to bring out the best products for clinical usage. At the same time, this process should not end with the laboratory, clinical trials must be encouraged to enable timely establishment of these potential alternatives.

Acknowledgements

This work was supported partly by the Ministry of Higher Education Malaysia with the Grant vot. no. R.J130000.7809.4F444 and Ref. no. PY/2014/03667.

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