Electrospinning applications from diagnosis to treatment of diabetes

Abstract

Electrospinning is a facile, yet low cost and reproducible technique that can use both natural and synthetic polymers to address problems in diagnosis and treatment of diabetes. For the diagnosis of diabetes, effective continuous glucose monitoring of the blood glucose level can be achieved by using electrospun glucose biosensors. Electrospun nanofibers confer a high-surface area, micro-porosity, and potential to encapsulate drugs or biomolecules within nanofibers. Even though electrospinning has been used widely there is no review available till now with the applications of electrospinning specifically for the diagnosis and treatment of diabetes. In this critical review, recent advances of electrospinning to optimize the glucose sensing ability and a myriad of diabetic drug delivery techniques via electrospinning are discussed. Future perspectives of biodegradable nanofibers are also discussed in the last section, which highlights the current challenges, innovation and development of novel electrospun nanofibers for theranostics targeted to diabetics.

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M. V. Vellayappan

Mr Muthu Vignesh Vellayappan is currently pursuing a Masters in Biomedical Engineering with Research at Universiti Teknologi Malaysia. His research areas of interest are Bio-nanotechnology, Biomaterials, Nanofibers and Nanocomposites.

J. R. Venugopal

Dr Venugopal Jayarama Reddy is a senior research fellow at National University of Singapore. His research interest includes Tissue Engineering, Nanofibres fabrication and Nanomaterials.

S. Ray

Dr Sudip Ray is a senior research fellow at University of Auckland. His research interest includes Tissue Engineering, Nanofibres fabrication and Nanomaterials.

A. F. Ismail

Dr Ahmad Fauzi Ismail is the Deputy Vice-Chancellor (Research and Innovation) Universiti Teknologi Malaysia. His research interest includes Membrane Technology, Gas Separation, Reverse Osmosis, Ultrafiltration, and Nanofiltration.

S. Seal

Dr Sudipta Seal is the Director of Advanced Materials Processing and Analysis Center at University of Central Florida. His research interest includes Nano-bio assembly and system development, Nano Energetics and Nanomaterials.

S. K. Jaganathan

Dr Saravana Kumar Jaganathan is currently working as a senior lecturer at Universiti Teknologi Malaysia. His research interests are Cancer, Anticancer Compounds, Drug resistance, Biomechanics and Biomaterials.

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1. Introduction

Diabetes is one of the dreadful and major global health problems of the 21st century. According to a latest statistics, there are 415 million adults who are estimated to currently suffer from diabetes; meanwhile there are 318 million adults with impaired glucose tolerance, which puts them at huge risk of developing the disease in the future. By 2040 it is estimated that this count will rise to 642 million.¹ It is predicted that currently 1 in 11 adults have diabetes, 1 in 7 births is affected by gestational diabetes and the worst case is in every 6 seconds a person dies from diabetes. The estimated number of people with diabetes worldwide as well as per region in 2015 and 2040 (20–79 years) is represented in Fig. 1.¹

Fig. 1 Estimated number of people with diabetes worldwide as well as per region in 2015 and 2040 (20–79 years). The numbers shown are in millions.

The health risks associated with diabetes are numerous, like high risks of heart disease, kidney failure, or blindness. Such severe health complications can be minimized to a great extent via stringent personal control of blood glucose using blood glucose meter, glucose biosensor and etc. Millions of diabetic patients test their blood glucose levels on a regular basis daily, making glucose as the most commonly tested analyte annually. The glucose biosensors constitutes about 85% (nearly 5 billion US dollar) of the entire biosensor market.² Hence, due to the severe health complications and the extremely large financial burden triggered by diabetes, the reliable detection of glucose is evolving as a cornerstone in managing diabetes.

Continuous glucose monitoring (CGM) is competent in providing continuous “real-time” observation of glucose level of blood and permits realization of the concept of “artificial pancreas”.³ This would appreciably improve the diabetic management and minimize the complications induced by diabetes. Even though notable advancements has been made in this field recently, its widespread usage is prevented by the lack of performance of the glucose biosensors after implantation due to functionality loss and sensor failure.³ Owing to this reason, the conventional glucosemeters fall short to confer an overall pattern of blood glucose levels change in accordance to a patient's daily activity. Moreover, the diabetic patients count was found to increase drastically underscoring the need to develop a strategy immediately for increasing the glucose biosensors in vivo performance.

For treatment of diabetes, great advancements in drug delivery system (DDS) research has been made recently.⁴ DDS has been exhaustively researched in last two decades due to the high need for delivering biomolecules to different components our body. The conventional treatment of diabetes comprised of dosages which are given in the form of pills or injections. These techniques are found to possess setbacks such as poor solubility of drugs as well as bio-distribution, quick breakdown of the drugs in vivo, tissue damage on extravasation, drugs rapidly lose their activity, drugs exhibit cytotoxicity and ultimately lead to tissue damage and adverse side effects.⁵ The patient compliance is the prime bottleneck in current diabetes delivery devices, where there are diabetic patients irregularly follow or even discontinue their insulin therapy because of the pain or fear of injections.⁶ These bottlenecks can be circumvented by introducing an appropriate delivery system based on the medical situation.

The motivation behind DDS utilization is to maximize therapeutic efficiency, extend drug releasing time and stability, enhance the bioavailability of the drugs, minimize side effects, and increase patient compliance with the reduction of the dosage frequency. The DDS controls the rate of releasing drugs chemically or physically, and it is classified as controlled and responsive drug delivery. There are different drug loading techniques for the treatment of diabetes.^7–9 To circumvent complications associated with the conventional drug delivery methods, multitude of effort has been taken across the globe with nano-scale materials. Nano-scale DDS exploits the fact that they can demonstrate unique physical properties, electrical, mechanical and optical, properties that varies compared to conventional materials at macroscopic scale.¹⁰ Via advanced designing method, nano-scale DDS readily made integrate desirable modules, combining either biological and synthetic material, for different uses.¹¹ Amongst various nanomaterials, electrospun nanofibers possess a large specific surface area as well as porous structure.

The main aim of this review is to analyze the electrospinning process and summarize its role for diabetes diagnosis through glucose sensors and its treatment via DDS. It is envisaged that this review will equip the readers with knowledge on the holistic role of electrospinning in combating. A concise introduction on electrospinning process and its parameters were highlighted before exploring the role of electrospinning for diabetes applications.

2. Electrospinning

Electrospinning was identified as a simple method for polymer nanofibers preparation in the submicron range, which is intricate to be fabricated via standard mechanical fiber techniques. Electrospinning allows fabrication of continuous polymer fibers from polymer melt or solution possessing fiber diameter from nano- to micro-size.¹² The possibility to apply electrospinning on natural and synthetic polymers, polymer blends, composite with metal or ceramic particles, nanocomposites enable the electrospun product to serve as effective fibrous sensors, tissue engineering and drug delivery.^13–16 To mimic collagen nanofibers of extracellular matrix by biodegradable biopolymers or biocomposites as a replacement for injured tissues, electrospinning was singled out as the most promising fabrication method.^17–20

The electrospinning process is simple yet cost-effective compared to other nanofiber fabrication techniques. It consists of an apparatus that includes syringe (needle-tip) with syringe pump, flow rate, high voltage power supply (electric field) and grounded fiber collector which is generally a metal plate or rotating mandrel. Electrospinning is carried out at room temperature and atmospheric pressure, with either vertical or horizontal set-ups. It is essential to adjust electrospinning parameters to obtain uniform nanofibers.²¹ The parameters affecting the electrospinning process are represented in Fig. 2.

Fig. 2 Parameters affecting the electrospinning process.

The flow rate of polymer solution is associated with applied electric field, and its correlation is almost specific depending on the polymer–solvent system. The solution will be ejected from the tip of the capillary to elongate under the applied electric field and thereby form Taylor cone. Taylor's cone deformation results in beads formation and it occur due to greater feeding rate of polymer solution. By applying higher voltage, it could prevent beads formation, however it would lead to the dispersion of main fiber and result in non-uniform fiber diameter.²² Besides that, the higher voltage accelerates electrostatic forces of charged solution and prevents complete solvent evaporation process. Nevertheless, lower feed rate results in inability to eject polymer solution jet from the needle tip due to the drying of droplet. A minimum tip-to-collector distance is necessary for conferring the fibers sufficient time for drying before it reaches the collector.^23,24

The collector composition and geometry are significant parameters which affect morphology of electrospun fibers. Collectors like aluminium foil, conductive paper, conductive cloth, wire mesh, pin, parallel bars, rotating rod, rotating cylinder, liquid non solvents like methanol, ammonium hydroxide and others are utilized.²⁵ Uniform aligned fibrous scaffold are the most desired morphology in tissue engineering field, owing to imitation of collagen fibers of extracellular matrix. The role of geometry of the collector, specifically on fiber alignment, was been exhaustively studied.^26–29 Rotating collectors like mandrels or disks are easy ways to produce oriented fibers.³⁰ Generally, the use of rotating cylinder is also governed by the regulation of rotating speed to obtain desirable alignment.^31–33

There are different electrospinning techniques based on the syringe configuration for drug loading. They are co-electrospinning, side by side electrospinning, multi-jet electrospinning, co-axial electrospinning, emulsion electrospinning, and surface immobilization technique. Fig. 3 ascertains the different electrospinning techniques as well as different collectors along with the morphology of the resultant nanofibers.

Fig. 3 Major drug loading techniques and types of collectors used for electrospinning. (A) Co-electrospinning; (B) side by side electrospinning; (C) multi-jet electrospinning; (D) co-axial electrospinning; (E) emulsion electrospinning; (F) surface immobilization; (G) static plate; (H) rotating mandrel; (I) grid; (J) rotating disk.

3. Unraveling potentials of electrospinning – diagnosis to treatment of diabetes

A succinct insight on the various fundamentals, key processing parameters and different techniques of electrospinning which can be adopted to obtain a tailor made nanofiber patch were discussed in the previous section. These key concepts and fundamental principles of electrospinning for achieving an optimized electrospun material play a crucial role for fabricating electrospun materials with multifaceted applications in managing diabetes. Hence, the versatility of electrospinning to serve as a putative candidate ranging from diagnosis to treatment of diabetes is summarized in the upcoming sections.

3.1. Glucose biosensors for diabetic diagnosis

Glucose is the fundamental source of energy for most living organisms. Glucose is widely utilized for synthesis of molecules like citric acid, gluconic acid, vitamin C, and etc.³⁴ High usage of glucose demands for effective feedback control systems, where the glucose sensors role is obligatory. Key features of glucose biosensors such as the latest technologies, the pros and cons of CGM utilizing implantable sensing technologies, and role of electrospinning in improving the efficacy of implantable biosensors for long-term application are discussed in this section.

3.1.1 The importance of glucose biosensors. Blood glucose level detection and is mandatory for preventing devastating health problems. Appropriate glycemic control can be achieved only with proper, precise and timely glucose level monitoring.^35–37 Since glucose is comprised of sugar, techniques utilized for sensing of glucose often demands unique affinity molecules usage. A measurable signal is produced when the glucose binds with the affinity molecule, which can be exploited for the detection of glucose in the human body. The glucose sensors are divided into enzymatic and non-enzymatic sensors where the enzymatic glucose sensors are further classified as first, second and third generation glucose sensors. However, the stability of enzymes for prolonged period of storage or application have always been a limiting factor for enzymatic glucose sensors, made the non-enzymatic glucose sensor a cynosure for glucose sensing applications.³⁸

3.1.2 Electrospun electrochemical non-enzymatic glucose sensors. The non-enzymatic glucose sensor is comprised of non-enzymatic electrodes as glucose sensors and it is contemplated as the fourth generation sensors for glucose oxidation. Rather than satisfying the high requirement of delicate and complicated enzymes, non-enzymatic electrode works on the principle of directly oxidizing the glucose in the sample. Walther et al., studied the electrochemical oxidization of glucose with sulphuric acid at anode.³⁹ Following this breakthrough, a spectrum of techniques was investigated for glucose sensors. The process of glucose reduction in an electrolytic cell entail severe conditions like pH ≥ 11 or pH ≤ 1 and high voltages, otherwise will lead to electrode poisoning.³⁸ The various isomeric glucose forms become electrooxidized, irrespective of intermediates involvement, thereby forming gluconic acid as the ultimate stable product.³⁸ The end result which is the gluconic acid was demonstrated to possess half-life period of 10 minutes at pH 7.5.

The glucose electrooxidation is comprised of three potential ranges versus reversible hydrogen electrode (RHE) which are used to sense the glucose.³⁸ The initial potential ranging from 0.15 to 0.35 V is known as ‘hydrogen region’ confers good oxidation peak potential which is distinct for glucose. This is found to occur mostly in case of platinum electrode. Nevertheless, flat platinum electrodes will lose its sensitivity for sensing the glucose in a short span of time mainly due to the poisoning of the platinum electrodes by the chloride ions as well as organic and inorganic constituents in our blood like amino acids, acetaminophen, creatinine, epinephrine, urea and uric acid in the blood. They usually clump on the catalytic sites of platinum, thereby hindering oxidation of glucose. Potential difference ranging from 0.4 to 0.8 V versus RHE, is known as ‘double layer region’. It reduces the adsorption ability of the poisoning agents. However, other interfering agents will also be oxidized at the site of electrode. Ultimately, ‘platinum oxide region’, with potential difference ranging from 1.125 to 1.5 V form a layer of platinum oxide. Glucose will react with this platinum oxide layer meanwhile other lactone identical poisoning products are decomposed.³⁸

The glucose can be electro-oxidized at conventional electrodes but the oxidation rate of glucose is comparatively slower than the regular interfering compounds like uric acid and ascorbic acid in the human blood. Despite the concentration of blood glucose level was as high as 3 to 8 mM compared to the interfering substances which is just 0.1 mM, the faradic current produced by interfering agents oxidation is greater in comparison to glucose.³⁸ Efforts were taken for circumventing the interference by decreasing the over-potential for glucose oxidation via fabrication of electrodes made of wide range of alloys.^38,40,41 It was found that the glucose was oxidized at platinum (Pt)–lead (Pb) alloy surface at astonishingly lower potentials compared to pristine Pt where the electrode was almost not sensitive to common interfering substances.⁴²

The birth of nanotechnology has integrated novel nano-materials with contemporary electrodes for overcoming the interference problem caused by the common interfering substances. In addition, it also aided in decreasing or avoiding electrodes poisoning. Materials with carbon backbone in combination with electrospinning and nanoparticles (NPs) are being investigated exhaustively for glucose sensing application. Various types of carbon materials and NPs, using or not using polymer fillers are being studied extensively for coating on regular electrodes like platinum, gold and glassy carbon and for the non-enzymatic glucose sensors application.^43–48

Glucose sensing process of enzymeless sensors based on conventional semiconductor oxides commonly involves a surface redox reaction. Yet, most of conventional nanostructured electrodes are fabricated by printing the nano-object onto a conductive substrate like drop or spin coating following dispersion.⁴⁹ Here, the dense films will be formed and then lead to uneven thickness, poor reproducibility, and structural instability of the fabricated electrodes. As a result, the overall efficiency of the electrodes will be degraded. In order to overcome this, efforts have been taken for enhancing or modifying the traditional film into integrated porous electrospun network. This is performed to obtain a greater effective surface area, which may drastically improve the molecule diffusion and mass transport in sensor materials.^50,51

The requirement to achieve greater specific surface area and porosity has made researchers to improve the electrospinning technique. The ideal fiber diameter is expected to be below 20 nm for optimal glucose sensor performance, but an electrospun fiber attained from the traditional process typically has a diameter in the range 100–500 nm.⁵² Nanofibers with smaller size and nonwoven morphology is expected to have a better glucose biosensor performance. But we cannot generalize this as the sensor performance depends on the material type of also.⁵³ In addition to that, the aligned orientation of the nanofibers is found to possess improved detection limit and excellent sensitivity compared to other types nanofibers alignment.⁵⁴ Hence, the aforementioned features of electrospun nanofibers may modify and improve the sensor diagnostic properties like sensitivity, response time, and limit of detection.^55,56 In addition, it also possesses improved life time which is one of the cornerstone properties of glucose sensors for practical applications.⁵⁷

Long-term drug delivery can be achieved by using hydrophobic polymers. Yet, these materials are not appropriate for glucose biosensor coating, as the glucose analytes fail to diffuse through them.⁵⁸ Thus, selection of the materials for coating the glucose biosensors is quiet essential. The major setback associated with the electrospun glucose biosensors are novel polymeric materials that may be utilized for glucose sensor application have to developed and characterized. This process is time consuming and expensive owing to the need for a comprehensive understanding of the physicochemical properties as well as it may involve exhaustive toxicology studies.

Zheng et al., developed sensitive non-enzymatic glucose sensor with silver NPs modified cupricoxide nanofibers (Ag/CuO NFs).⁵⁹ The Ag/CuO NFs–indium tin oxide (ITO) electrode was produced via electrospinning of AgNO₃, Cu(NO₃)₂ and polyvinylpyrrolidone onto the surface of an ITO glass. This was followed with oxidization of Ag/CuO NFs by annealing in air. The Ag/CuO NFs–ITO electrode was utilized for glucose detection via cyclic voltammetry (CV) and chronoamperometry. The effects of electrospinning duration, AgNO₃ : Cu(NO₃)₂ mass ratio, calcination temperature and NaOH concentration on the sensitivity of glucose sensor were studied. It was found that compared with CuO NFs–ITO electrode, the AgNPs modified sample displayed a exceptional efficacy improvement in the electro-catalytic oxidation of glucose, demonstrating a high sensitivity of 1347 μA mM⁻¹ cm⁻² as well as low detection limit of 51.7 nM (S/N = 3). Furthermore, the electrode poisoning by electrode poisoning agent like chloride ion and the interferences from interferrants like L-ascorbic acid, uric acid, lactose, sucrose, fructose, and dopamine became insignificant. Hence, Ag/CuO NFs–ITO sensor can be exploited for the glucose detection in real samples. Ag/CuO NFs–ITO sensor is a plausible candidate for non-enzymatic glucose sensing attributing to its high sensitivity, quick response, exceptional stability and excellent reproducibility.

Similarly, in another work MCo (M = Cu, Fe, Ni, and Mn) NPs anchored and embedded in carbon nanofibers (CNF) were fabricated via electrospinning and then subjected to heat treatment process.⁶⁰ It was found that the 3D network films are assembled through bimetallic MCo alloys doped-extremely long CNF except for MnCo–CNF. Effectiveness of non-enzymatic glucose sensing was studied via cyclic voltammetry and chrono amperometry. Result shows that the catalytic abilities was found to follow order of CuCo–CNF > FeCo–CNF > NiCo–CNF > Co–CNF > MnCo–CNF. Structural advantages of 3D network films along with synergistic effect of Co(III)/Co(IV) and Cu(II)/Cu(III) redox couples, CuCo–CNFs exhibited exceptional glucose detection sensitivity and efficacy even in human blood samples. Moreover, it also shows good reproducibility, improved long-term stability, and outstanding anti-interference to electroactive molecules or chloride ions. Hence, improved catalytic activity, stability, selectivity as well as cost effective and easy fabrication make electrospun CuCo–CNFs a potential candidate for electro-chemical glucose sensor application.

Likewise, Pd(IV)-doped CuO composite nanofibers (PCNFs) was synthesized through electrospinning by Wang et al., and it was utilized to develop an amperometric non-enzymatic glucose sensor.⁶¹ The PCNFs exhibit significantly improved electrocatalytic activity for the glucose oxidation displaying notable lower overvoltage (0.32 V), ultrafast (1 second) and ultrasensitive current (1061.4 l μA mM⁻¹ cm⁻²) response with improved detection limit of up to 1.9 × 10⁻⁸ M (S/N = 3). In addition to that, it demonstrated exceptional selectivity, reproducibility and stability. Thus, PCNFs fabricated using electrospinning technique seems to be a potential candidate for developing the amperometric non-enzymatic glucose sensors.

Manesh et al., employed electrospinning for preparing nanofibrous membrane (NFM) made of poly(vinylidene fluoride) and poly(aminophenylboronic acid) (PVdF/PAPBA-NFM).⁶² PVdF/PAPBA-NFM demonstrated an exceptional linear response for detecting glucose at the concentration ranging from 1 to 15 mM with a quick response time of lesser than 6 seconds. The detection of glucose was demonstrated in presence of various common interferents like uric acid, ascorbic acid, acetaminophen and etc. using PVdF/PAPBA-NFM. The interferents were found did not instigate overlapping current signal at the time of determination of glucose concentration. In addition to that, PVdF/PAPBA-NFM was found to possess excellent reproducibility towards detection of glucose. Hence, the above studies ascertain the role of electrospinning in improving the efficiency of glucose biosensors which can be used for the diagnosis of diabetes. Comparison between conventional and electrospinning incorporated glucose biosensors is represented in Fig. 4. For the treatment of diabetes, different DDS can be adopted using electrospinning is discussed in the subsequent section. The various electrospun electrochemical non-enzymatic glucose sensors and its key findings are tabulated in Table 1.

Fig. 4 Comparison between conventional and electrospun glucose biosensors.

Table 1 Electrospun electrochemical non-enzymatic glucose sensors and its key findings

S. no.	Technique	Base material	Additive component	Further treatment	Form of electrospun material	Key findings	Ref.
1	Electrospinning	Silver (Ag) nanoparticles modified cupricoxide (CuO) nanofibers	Polyvinylpyrrolidone	Oxidization of Ag/CuO NFs by annealing in air	Combination of fibers and particles	Exceptional increase in the efficiency of the electro-catalytic oxidation of glucose	59
						Sensitivity of 1347 μA mM^−1 cm^−2 and a low detection limit of 51.7 nM (S/N = 3)
						Poisoning by chloride ion and the interferences from L-ascorbic acid, uric acid, lactose, sucrose, fructose, and dopamine were insignificant
2	Electrospinning	Carbon nanofibers (CNFs)	Series of bimetallic MCo (M = Cu, Fe, Ni, and Mn) nanoparticles	Thermal treatment process	Combination of fibers and particles	The catalytic abilities was highest for CuCo–CNFs compared to other metallic nanofibers	60
						CuCo–CNFs demonstrate the best detection efficiency even for the glucose detection in human serum samples
						Show good reproducibility, improved long-term stability, and outstanding anti-interference to electroactive molecules or Cl^−
3	Electrospinning	Polyurethane (PU)	Fibers were electrospun directly on the sensor surface	—	Fibers	Incrementing thickness as well as fibre diameters resulted in a statistically insignificant decrement in sensor sensitivity	34
						Sensors' linearity sustained for the glucose detection range of 2 to 30 mM
						Electrospun coatings also served as mass-transport limiting membrane
						Electrospun coatings have a controllable fibro-porous structure and thicknesses on miniature ellipsoid glucose biosensors
						This has minimal effect on pre-implantation sensitivity and also mass-transport limiting ability
4	Electrospinning	Copper oxide (C)	Pd(IV)-doped (P)		Fibers	PCNFs based glucose sensors exhibit significantly improved electrocatalytic activity for the oxidation of glucose	61
						Displayed appreciably lower overvoltage (0.32 V)
						Showed ultrafast (1 s) and ultrasensitive current (1061.4 l μA mM^−1 cm^−2) response
						Demonstrated lower detection limit of 1.9 × 10^−8 M (S/N = 3)
						Exhibited exceptional selectivity, reproducibility and stability
5	Electrospinning	Poly(vinylidene fluoride) (PVdF)	Poly(aminophenylboronic acid) (PAPBA)	—	Fibers	PVdF/PAPBA-NFM demonstrated an excellent linear response for the detection of glucose at the concentration range of 1 to 15 mM	62
						Exhibited a response time of less than 6 s
						The interferents did not confer notable overlapping current signal
						PVdF/PAPBA-NFM was found to possess better reproducibility towards glucose detection and storage stability

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3.1.3 Characterization of electrospun fibers on glucose sensors. From the Fig. 5a it can be seen that the glucose sensor was concealed with the electrospinning technique. Nanofibers covering the sensor observed to extend up to almost half of the overall length of the glucose sensor.³⁴ The electrospun fiber layer thickness was observed to be increased with increase in duration of electrospinning. However, it had ridges and groves forming spike-like structures with a hollow core Fig. 5a–c. The ridges may be formed because of the alteration in the electric field surrounding the sensor. In electrospinning when the fibers reach the collector the deposition pattern depends on the columbic interaction. Moreover, after deposition the residual charges on the surface further triggers deposition of the fibers on the surface of the glucose sensor. When the polymer jet reaches the surface of glucose sensor, it forms a loop where the entire polymer fiber does not contact electrode surface. This looping of fibers may be the reason for the hollow spike like structure formed. Hence, static collector system with the ellipsoid 3D space of the glucose sensor and coulombic interactions are responsible for the distinct electrospun coating of glucose biosensor.

Fig. 5 Optical microscopic images showing (a) morphology as a function of electrospinning time, (b) the cross-section on working electrode (c) the cross-section of the cut-off PU coating on the sensing element of the coil-type glucose biosensors, coated using static collector configuration-3. S – sensing element, AP – air pockets (c and d) and SEM (e–j) images showing the morphology of a coil-type biosensor without (d) and with (e–j) electrospun 8PU coating spun using a dynamic collector, showing the uniform covering of the miniature coil-type sensor including at its convex tip (g), while a closer look at its cross-section and surface revealed a uniform porosity (h and i) and random orientation of the electrospun fibres (j) respectively. Scale in d and e is in mm. Reprinted with permission from ref. 34. Copyright 2016, Elsevier.

In order to circumvent the limitation of the spike formation, the collector configuration and rotation speed was changed. Sensor was kept parallel to the auxiliary flat-plate collector which rotates at a speed of 660–690 rpm during the electrospinning process.³⁴ Spinneret tip as well as the collector was maintained at a distance of 22 cm. This has resulted in a fruitful outcome where uniform coating of electrospun fibers were obtained over the surface of the glucose sensor. The surface of coating was found to be even with no spikes as seen in Fig. 5e and f. The coating was uniform at the convex tip as shown in Fig. 5g of the sensing element. The coating can be controlled by altering the rotation speed of the collector. The Fig. 5h and i shows electrospun nanofibers on the glucose sensors. Thus, uniform coating by completely covering the ellipsoid shaped glucose sensor can be achieved through manipulation the fiber diameters and thickness using electrospinning technique. The uniform coating is necessary to make sure that the coated polyurethane nanofibers completely cover and fit snugly on the sensor surface. Moreover, this will also ensure that the coating sustain with the biosensor with sufficient adhesion to the sensor throughout the intended sensor lifetime.

3.1.4 Sensor sensitivity and linearity after covering glucose sensors by electrospinning and in vivo sensor functionality assessment. The glucose biosensor which was covered by electrospinning was tested for its efficiency at in vivo condition up to 84 days. It was found that different polyurethane membranes coated on both platinum–glucose oxidase–epoxy polyurethane and platinum–glucose oxidase base sensors did not deteriorate sensor response, sensitivity and linearity even up to eighty four days as showed in Fig. 6a–d.

Fig. 6 Long-term changes in sensor response sensitivity (a–c) and linearity (d) based on intermittent sensor function tests: all sensors were tested for sensitivity and linearity before and after applying the different coatings and data represented as % change of sensitivity or linearity for the individual sensors of 7th day testing after coating self-referenced to that on 7th day test before coating. Light microscopy images showing the implant sites for 6PU10Ge (e) H&E and (f) MT stained sections at 4 weeks after implantation and (g) MT stained section at 9 weeks after implantation. Reprinted with permission from ref. 34. Copyright 2016, Elsevier.

In addition to that, the polyurethane can be utilized as a mass transport limiting membrane substituting the conventional epoxy polyurethane outer layer resulting in extended linear glucose detection range up to 2–30 mm. Sensor sensitivity was also found to be stable until 84 days of testing. Efficacy of the electrospun covered glucose biosensors can also be studied using tissue staining techniques using hematoxylin and eosin (H&E) and Masson's trichrome (MT) staining.

The sensor membrane was found to be covered with connective tissue identical to the native tissue after 4 weeks which was observed using the H&E stain. Similarly, for the samples stained using MT, it was observed to have minimal collagen deposition in the scaffold in 4 weeks. However, for the nine weeks duration sample was found to possess a well organized collagen network combining the both electrospun network as well as host tissue as shown in Fig. 6e–g.⁶³ Hence, the electrospinning technique is a potential candidate for improving the glucose sensor sensitivity, linearity and good biocompatibility for the diagnosis of diabetes.

3.2. Nanofibers for diabetes

The electrospinning technique enables us to fabricate continuous fibers with diameters at a range of nanometers to micrometers.^64,65 The electrospun fibers ranging in nanometers size possess larger specific surface area and porous structure that can be exploited for DDS.^11,66,67 The DDS based on nanofibers have attracted tremendous attention of researchers in the pharmaceutical field. Controlled DDS are utilized for improving the therapeutic efficiency and maintain the safety of drugs which are delivered at a required rate.⁶⁸

The polymeric electrospun nanofibers can be used to load different drugs and bioactive molecules for DDS.⁶⁹ In DDS, drug release profile can be managed via precise control of the structure of the electrospun fibers.⁷⁰ Improved drug loading capacity, increased encapsulation efficacy, delivery of different treatment drugs at the same time, simple operation, and low in cost are some of the alluring properties of electrospinning in DDS.^71,72

There are many pros of utilizing the novel electrospun polymeric nanofibers as DDS.¹⁶ Presence of high surface area to volume ratio, of polymeric nanofibers provides a pathway for water insoluble drugs. Nowadays, count of poorly soluble drugs increased to a great extent and the formulation of poorly soluble compounds by oral delivery is one of the most common as well as challenging task.⁷³ The solid dispersion is contemplated as apt way to enhance the dissolution rates and bioavailability of poor water soluble drugs.⁷⁴ Nevertheless, the acceptance of solid dispersion systems are still restricted because of problems in contemporary preparation techniques, lack of reproducibility of physiochemical properties, formulation of dosages and low potential in scaling-up the manufacturing processes.⁷⁵

3.2.1 Water soluble and insoluble drugs for diabetes treatment using electrospinning. There are different drugs/proteins which can be used for the treatment of diabetes like insulin, lignaliptin, and etc. using electrospinning technique. The insulin is a water soluble peptide hormone produced by beta cells of pancreas. It regulates the metabolism of carbohydrate in the body. The chemical structure of insulin is given in Fig. 7a. When there is failure of insulin control in the body it leads to diabetes. In case of diabetes type 1, patients depend on external insulin whereas in type 2 the patients become insulin resistant leading to insulin deficiency. In recent times, biosynthetic human insulin of high purity has been manufactured by recombinant DNA technology. Insulin is generally administered via sub-cutaneous injections using syringes, insulin pumps, or insulin pen with disposable needle and nowadays even inhaled insulin is available in the market. However, insulin cannot be taken in oral route since the protein in the insulin will be reduced to fragments leading the failure of its activity in the digestive tract. Hence, novel ways of protecting insulin from the digestive tract are being explored by researchers and electrospinning is a captivating choice among them.

Fig. 7 Drugs for diabetes treatment using electrospinning. (a) Chemical structure of insulin (b) chemical structure of lignaliptin.

Linagliptin is white to yellowish in color slightly hygroscopic solid substance. It is very less soluble in water. The lignaliptin is used generally with other medications for lowering the blood glucose level for the treatment of type 2 diabetes. Lignaliptin will help to control high blood glucose level but does not cure diabetes. The chemical structure of lignaliptin is given in the Fig. 7b. Linagliptin usage was approved by the U.S. Food and Drug Administration (FDA) on 2 May 2011 for treatment of type 2 diabetes.⁷⁶ Currently, it is marketed by Boehringer Ingelheim and Lilly. Clinical trial result in 2010 demonstrates the lignaliptin can effectively reduce the blood glucose level.⁷³ However, the lignaliptin usage combined with insulin for treatment of diabetes is not yet performed.

Recently, the water soluble drugs encapsulated hydrogel nanofibrous scaffolds were used to accelerate angiogenesis and to improve wound healing. The sustained release profile of desferrioxamine (DFO), which prolongs up to 72 hours leads to appreciable enhancement in neovascularization.⁷⁷ Most developed scaffolds were found to degrade in 14 days, providing necessary space for cell proliferation and vessel formation. The in vitro study depicts that the scaffolds upregulate the expression of Hif-1α and vascular endothelial growth factor, thereby increasing the interaction between fibroblasts and endothelial cells. Meanwhile, the in vivo studies suggest a greater expression of angiogenesis related cytokines. Hence, it can be interpreted that the DFO released from hydrogel nanofibrous scaffolds of quick degradation can interact with the required prolyl-hydroxylases cofactors by acting as Fe²⁺ chelator and then upregulate the expression of Hif-1α, resulting in improved neovascularization.

Similarly, in another study DFO-loaded photo-crosslinked hydrogel (gelatin methacrylamide (Gelma)) was developed for speeding up the skin reconstruction in diabetic patients.⁷⁸ The controlled release of DFO peaking at 16 hours which was succeeded by a steady release after 48 hours via the swelling of the Gelma hydrogel led to a notable increase of neovascularization. Moreover, it was also found that DFO–Gelma was safe, reliable, and highly effective. Likewise, Wenguo Cui et al. also have recently explored on how this new therapy can be used to treat different diseases using injectable and bioresponsive hydrogels.⁷⁹ However, this treatment method may only attain a release profile within a certain therapeutic window and cannot self-regulate its release profile with the spatial and temporal variation along with the disease progression. Apart from the use of hydrogels, there are some strategies adopted by researchers to utilize the electrospinning technique for diabetes management. For instance, recently electrospun fibrous membranes were coated with basic fibroblast growth factor (bFGF) for repairing skin tissue among diabetic patients.⁸⁰

3.2.2 Different drug loading technique using electrospinning. The electrospinning technique offers electrospun nanofibers focusing on novel approach for improving the dissolution rate where dissolution of poorly soluble compounds may be increased compared to oral availability method. The polymeric nanofibers loaded with water-soluble drug itraconazole fabricated via electrostatic spinning, was investigated for its dissolution ability by varying the dissolution rate of the drug. Differential scanning calorimetry (DSC) result depicts that the melting endotherm for itraconazole was absent, hence it is anticipated that an amorphous dispersion or solution was formed.⁸¹

Drug release profile can also be fine tuned via modulation of the composition of the nanofibrous mats, process, altering the nanofibers morphology and their micro-structure. Core–shell nanofiber morphology is a promising structure for multifaceted biomedical engineering uses.^82,83 There are numerous fabrication techniques available till date for preparing ultrafine nanofibers. Xu et al., synthesized uniform core–shell through electrospinning water-in-oil emulsion.⁸⁴ After emulsion electrospinning, core–shell nanofibers were formed with two distinct polymers with one polymer acts core and the other functions as the shell having a sharp boundary between them.

Different emulsion composition as well as emulsification parameters resulted in variation in core and shell structure diameters. It was found that stretching and evaporation triggered de-emulsification and lead to the transformation of the emulsion forming core–sheath fibers. Similarly, concentric electrospinning is a potential approach to produce core–sheath fibers.⁸⁵

In case of coaxial drug delivery, the drug release with core polymer dissolution followed by slow drug release by sheath of the nanofiber. In layer by layer electrospun sheet, slow diffusion of the drug occurs as the sheath layer acts as barrier. For the surface chemically immobilized electrospun sheet, the cleavage of the cleavable linker occurs first followed by delivery of drug. On the contrary, simple diffusion of the drug occurs for the physical immobilized electrospun sheet. In the case of coelectrospun sheet, simple diffusion of the drug molecules occurs from the nanofibers whereas the nanofiber and nano-and micro sized devices, the drug is released from carriers and later the drug is released from the nanofibers. For the nanofiber and hydrogel type of drug delivery, the drug diffusion occurs at first from the nanofiber to the hydrogel followed by the drug release from hydrogels.⁸⁶ This different drug release process of spectrum of electrospun nanofiber sheet is represented in Fig. 8.

Fig. 8 Drug delivery system using electrospinning for diabetes treatment.

Recently, Yadav et al., developed drug for diabetes treatment by utilizing polymeric nanofibers where poly(vinyl alcohol) (PVA) nanofibers was loaded with linagliptin through electrospinning.⁸⁷ In vitro drug release study dictates the sustained lignaliptin release from the electrospun sheet. Studies performed on male Wistar rats elucidate the good pharmacodynamics properties of developed composite. Mucoadhesive strength study result ascertains that the drug loaded PVA nanofibers mat possess the highest mucoadhesion strength in comparison to the pure PVA film and blank PVA nanofibers, owing to its greater water retention ability and higher surface area. Besides that, the in vivo studies also depict that linagliptin was delivered properly with same active state and resulted in excellent outcome in compared to conventional treatment method. In addition to that, an encapsulation efficacy of 92% was achieved in this experimental formulation. Hence, all these data shows that lignaliptin can be delivered efficiently using electrospun nanofibers.

Conventional therapy for diabetes mellitus is subcutaneous administration of insulin and it is subjected to some serious setbacks like patient noncompliance and occasional hypoglycemia. Thus, the oral administration of insulin could be more convenient and may serve as a plausible route. Yet, oral administration of insulin is greatly restricted by the low bioavailability of insulin through the gastrointestinal tract. Hence, in a latest work, a semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber scaffolds (GIF) formulation was developed for transbuccal insulin delivery.⁸⁸ In this work the electrospinning of gelatin was performed and the electrospun nanofibers were converted into a sIPN following eosin Y-initiated polymerization of polyethylene glycol diacrylate (PEG-DA).

This resulting sIPN was later subjected to cytocompatibility, degradation rate and mechanical property tests using varying ratios of PEG-DA to eosin Y, for the determination of an appropriate formulation for transbuccal drug delivery. Insulin was co-electrospun with gelatin forming fibers and later converted into a sIPN-GIF utilizing this formulation. Insulin in vitro release kinetics was studied via Enzyme Linked Immuno Sorbent Assay (ELISA). Besides that, a bioactivity study of released insulin was performed in 3T3-L1 preadipocytes via western blotting and oil red-O staining.

Transbuccal permeability of released insulin was also found by in vitro porcine oral mucosa model. The result of the dictates that the sIPN-GF formulation of GF cross-linked by PEG-DA (1% w/v) with eosin Y (5% v/v) demonstrated no cytotoxic effect, a moderate degradation rate with degradation half-life of 49 min, and an appreciable increase in the mechanical properties. This formulation was implemented to fabricate sIPN-GIF. Insulin release was prolonged up to 4 h by sIPN-GIF. The released insulin fruitfully induced intracellular AKT phosphorylation as well as triggered adipocyte differentiation in 3T3-L1 preadipocytes. The transbuccal permeability of released insulin was found to be on the order of 10 cm s⁻¹. Hence, the insulin can be loaded within sIPN forming sIPN-GIF formulation after co-electrospinning and crosslinked thereby not losing its bioactivity.

Morphology of semi interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber scaffold (GIF) was examined using SEM as shown in Fig. 9.⁸⁸ It was found that the change in the morphology of the electrospun material was in corroboration with its degradation kinetics.

Fig. 9 Representative SEM images of GFs and sIPN-GFs. GF1 (a) and sIPN-GF1 (b) as well as sIPN-GF1 after 15 min of degradation (c) 30 min of degradation (d) 60 min of degradation (e) and 120 min of degradation (f) was observed by SEM. Reprinted with permission from ref. 88. Copyright 2016, Springer.

Hence, immediately after the incubation the sIPN-GIF starts to lose its fibrous structure and becomes more and more porous. At the first hour, the scaffold maintained its fiber structure to some extent. However, at the second hour the scaffold completely lost its three dimensional fibrous structure and broke into smaller pieces. Hence, this clearly shows that the drug is loaded in the nanofibers matrix in a random pattern and it is released when the nanofibers are degraded in course of time.

3.2.3 Physical characterization of electrospun nanofibers for drug delivery. The morphology of the polymeric nanofibers depicts an increase in surface roughness after insulin is loaded in the nanofibers. Surface roughness can be measured using AFM. Fig. 10 depicts the polyvinyl alcohol (PVA), PVA–sodium alginate (PSA) and insulin encapsulated in PVA–SA physical characterizations result.⁸⁹ The AFM results depicts that the average surface roughness decreases from 326 nm, 276 nm and 128 nm for PVA, PSA, and insulin encapsulated PSA respectively. It can be interpreted that the surface roughness nanofibrous patch decreases with the encapsulation of the insulin due to the change in the morphology of the individual fibers, thereby affecting the overall surface roughness.

Fig. 10 (a) AFM images of PVA, PVA–NaAlg and insulin loaded PVA–NaAlg nanofiber patch, (b) XRD spectra of PVA–NaAlg nanofiber, raw insulin and insulin loaded PVA–NaAlg nanofiber. (c) DSC and (d) TGA curves of raw insulin drug and drug loaded PVA–alginate nanofibers. Reproduced from ref. 89 with permission from the Royal Society of Chemistry.

The diameter of the nanofibers entrapped with insulin was found to be higher compared to the other samples nanofibers. The Fig. 10b shows the XRD spectra of PVA, PSA, and insulin encapsulated PSA. A characteristic peak was observed for PVA in case of PSA at 19.5° whereas raw insulin exhibits a broader peak at 2θ = 22.3. But, insulin encapsulated PSA exhibited a broader peak in comparison to the PSA showing that insulin losses it crystallinity. Hence, the insulin loaded in the nanofiber patch is present either in molecularly dispersed or metastable crystalline structure.⁹⁰

Stability of the insulin and the insulin loaded nanofiber patch can be studied using DSC and TGA and it is shown in Fig. 10c and d. It was reported that for the insulin drug, the exothermic peak owing to the heat loss occurs at the temperature of 300 °C (Fig. 10c). This is due to the interaction occurring in the insulin drug resulting in its degradation. On the other hand, the exothermic peak owing to heat loss in case of insulin loaded PSA was found to be higher (∼350 °C) than the insulin drug. This dictates that the stability of the drug is improved by encapsulating insulin between the polymeric chain molecules. Similarly, the TGA curve also shows that the stability of insulin loaded in the nanofiber patch is greater than the neat insulin as represented in Fig. 10d. It was found that the weight loss of neat insulin started comparatively at a lower temperature than the insulin encapsulated in PSA.⁸⁹

3.2.4 Electrospun nanofibers in vitro and in vivo drug release profile for diabetes treatment. The diabetic drug release profile can be studied using a simulated saliva solution at a pH of 6.5.⁸⁹ The drug release rate greatly depends on the loaded drug concentration as shown in Fig. 11a. Almost 99% cumulative insulin release was achieved in 10 hours in the study. It was found that the nanofibers release the insulin in a sustained manner. The drug release possess slight burst release initially but later becomes sustained up to a maximum of 10 hours where the insulin release profile will be ceased after that period. This suggests that there is drug present in the outer surface of the nanofiber patch leading to the small initial burst release. Further release becomes sustained and controlled since it depends on the degradation of the polymer to be released from the nanofiber patch. Hence, it can be summarized as the drug release is diffusion based for the first 2 to 3 hours and later the polymer erodes leading to a controlled release of insulin up to 10 hours. The Fig. 11b shows the in vivo release profile in male Wistar rats in five groups that are G1: nothing is placed, G2: a PSA nanofiber patch G3: 2.5 International Unit (I.U.) insulin encapsulated in a PSA nanofiber sheet, and G4: 2.5 I.U. insulin in solution form G5: 2.5 I.U. marketed formulation – Human Actrapid.

Fig. 11 (a) In vitro drug release profile with time from composite nanofiber patch (b) blood glucose concentration lowering effect in different groups of animals with time. Reproduced from ref. 89 with permission from the Royal Society of Chemistry.

All patches were placed under the tongue of the diabetic induced rats. It was found that the blood glucose concentration fluctuated throughout the sampling period for G1 which is common for streptozotocin (STZ) induced diabetic rats as STZ does not destroy the β cells. In case of G2 no impact on glucose reduction was observed and the rats were hyperglycemic with fluctuating glucose concentration. For G3, there was a decrease in glucose concentration. Blood glucose level was found to be between 113 ± 3.16 to 117 ± 4.24 mg dl⁻¹ and sustained for 10 hours then it increased to 159 ± 3.03 mg dl⁻¹ at 12 hours. It was observed that the drug release profile was sustained and controlled by the degradation of the polymer. The drug release commenced with a slight burst release but later slowed down and prolonged for 10 hours. Throughout the sampling period, no hypoglycemic case was observed in G3.⁸⁹ Hence, it can be concluded that the nanofiber patch fabricated by electrospinning technique can be used to attain sustained drug release profile for diabetes treatment. Electrospinning in diabetic drug delivery application and its key findings is tabulated in Table 2.

Table 2 Electrospinning in diabetic drug delivery application and its key findings

S. no.	Technique	Base material	Additive component	Form of electrospun material	Key findings	Ref.
1	Electrostatic spinning	Hydroxylpropylmethylcellulose	Water soluble drug, itraconazole	Fibers	DSC measurements result depicts that the melting endotherm for itraconazole was absent, thereby suggesting the formation of an amorphous solid dispersion	81
2	Emulsion electrospinning	Poly(ethylene oxide) (PEO) solution in water	Amphiphilic poly(ethylene glycol)-poly(L-lactic acid) (PEG-PLA) diblock copolymer	Core–sheath fibers	By varying the emulsion composition as well as emulsification parameters overall fiber size and the relative diameters of the core and the sheath can be changed	84
					Stretching and evaporation triggered de-emulsification and the transformation from the emulsion to the core–sheath fibers
3	Electrospinning	Poly(vinyl alcohol) (PVA)	Linagliptin	Fibers	Mucoadhesive strength results ascertained that the drug loaded PVA nanofiber patch had the highest mucoadhesion strength	87
					Have greater water holding capacity and surface area
					Controlled release array of the drug from the fabricated nanofiber patch was observed
					Linagliptin was delivered in its active state and exhibited visible results when compared to the commercial formulation
					Encapsulation efficacy of 92% of the experimental formulation was observed
4	Electrospinning	Gelatin	Insulin and eosin Y-initiated polymerization of polyethylene glycol diacrylate (PEG-DA)	Fibers	Resultant nanofiber demonstrated no cytotoxic effect, a moderate degradation rate with degradation half-life of 49 min	88
					Showed an appreciable increase in the mechanical properties
					Insulin release was prolonged up to 4 h by sIPN-GIF
					Insulin triggered induced AKT phosphorylation and triggered adipocyte differentiation in 3T3-L1 preadipocytes
					Transbuccal permeability of released insulin was found to be on the order of 10 cm
5	Electrospinning	PVA	Sodium alginate (SA) and insulin	Fibers	Mucoadhesive strength results established that the drug loaded PVA–SA nanofiber patch possessed the highest strength owing to its higher water retention capacity	89
					Sustained and controlled release pattern of the drug from the nanofiber patch
					Insulin was delivered in its active state and shown significant result when it is compared to the commercial formulation
					The insulin release profile was found to follow first order kinetics and it is followed by an initial burst release necessary to result in the desired therapeutic activity
					Encapsulation efficiency of 99% of the experimental formulation

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4. Current challenges and future prospects

There has been multifold growth in the fabrication of electrospun nanomaterials, confirmed by voluminous publications in the last two decades. Latest research has focused on many important aspects of electrospun nanomaterials like structure formation, functionalization, and potential implementation in devices with targeted applications. However, most of the studies performed using electrospinning is still in its infancy. Both experimental and theoretical modeling has to be conducted for achieving a precise control over the size and morphology of the electrospun fibers. Extensive research on nanofiber systems with ultrafine diameter less than 20 nm demonstrate several fascinating characteristics such as, increased specific surface area, high porosity and improved mechanical performance, which make them as favorites for the applications in ultrasensitive sensors for diabetes detection.

Sensing materials in form of nanostructures may appreciably enhance their performances in the existing devices or open the doors to new type of applications apart from glucose biosensors. For example, the research on engineering the secondary structures (porous and core–sheath or hollow) of electrospun nanomaterials will confer a novel platform for designing advanced electrode materials, catalyst supports, and sensing devices. Yet, integration of such nanomaterials into useful devices requires materials of precisely-controlled orientation, size, and other targeting characteristics as well as reproducibility locating them in specific positions and orientations.

In case of drug delivery for diabetes treatment, there are many novel DDS developed using electrospinning but still the optimum DDS for diabetes treatment is yet to be developed. For controlling the nanofibers for diabetes therapy, we have to understand quantitatively how the electrospinning technique transforms the electrospinning solution through a millimeter diameter syringe tip into solid nanofibers containing the anti-diabetic drugs. Apart from that, diabetic drug loading capacity of electro-spinning technique is still a setback. Hence more in depth studies on material selection, functionalization and process optimization are of dire necessity to facilitate the commercial success.

Currently several nanotechnology-based companies are producing electrospun nanofibers for diabetes applications. However, one of the main challenges of this processing technique is their low throughput, which restricts the commercialization scope. Hence a strong research focus should be given to scale up the process by modifying the structural aspect of the experimental set up and thereby reducing the cost of production to exploit commercial opportunities. Thus for example, more research on multiple nozzles has to be conducted, which can form a strong foundation for commercialization of electrospun products for diabetic treatment. The development of new electrospinning configurations such as solution blow spinning or use of twin-screw extruder can also provide novel nanofibers with greater potential for commercial scale-up. This twin-screw extruder based electrospinning process can offer better performance in compounding of various additives including nanoparticles. Moreover, human in vivo studies would be certainly required to support the commercial pathway for these pharmaceutical and medical based researches.

Acknowledgements

This work was supported partly by the Ministry of Higher Education Malaysia with the Grant Vot number: Q.J130000.2545.12H80. The authors would also like to thank the New Zealand Ministry of Business, Innovation & Employment (MBIE) for its support and funding for the NZ Product Accelerator (UOA X1309) and Biocide Tool Box (UOAX1410) research programs which supported this publication.

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