An emerging era in manufacturing of drug delivery systems: Nanofabrication techniques

Abstract

Nanotechnology has the capability of making significant contributions to healthcare. Nanofabrication of multifunctional nano- or micro-character systems is becoming incredibly influential in various sectors like electronics, photonics, energy, and biomedical gadgets worldwide. The invention of such items led to the merger of moderate cost and excellent quality nano or micro-characters into 3D structures. Nanofabrication techniques have many benefits as the primary technology for manipulating cellular surroundings to research signaling processes. The inherent nanoscale mechanisms of cyto-reactions include the existence and death of cells, stem cell segmentation, multiplication, cellular relocation, etc. Nanofabrication is essential in developing various nano-formulations like solid lipid nanoparticles, nanostructured lipid carriers, liposomes, niosomes, nanoemulsions, microemulsions etc. Despite the initial development cost in designing the nanofabrication-based products, it has also reduced the total cost of the healthcare system by considering the added benefits compared to the other standard formulations. Thus, the current review mainly focuses on nanofabrication techniques, advantages, disadvantages, applications in developing various nanocarrier systems, challenges and future perspectives.

Graphical abstract

1. Introduction

“Nanotechnology” is assembling and organizing materials at the nanoscale, binding the distinct features exhibited by the nanoscale. Nanotechnology offers various innovative skills for medical, paramedical, transport, commercial, business, and technology fields. Nanotechnology has the capability of making significant contributions to healthcare. Nanobots can be employed to unblock congestion in a patient's artery. Operative procedures are becoming efficient and precise. It could ultimately aid in curing hereditary diseases by repairing defective genes. Nanotechnology can even improve drug e development by customizing compounds at the molecular level to enhance efficacy and reduce adverse effects. Using single-wall carbon nano-tubes, experts designed an intelligent patch to identify and manage an infestation in lesions. By measuring hydrogen peroxide levels, the nano-tubes help to predict illnesses. Thus, whenever an infestation is discovered, the patch is digitally controlled by a small portable sensor and transferred to the mobile, which alerts the person or a medical practitioner [1].

The term “nanofabrication” is referred to a toolkit that allows the modification of materials in the nanometer range. It is a technique for manufacturing objects with unique designs with 1–100 nm size. Hence it is a suite of processes for designing, expanding, reshaping, and extracting substances with control, accuracy, and reproducibility at the nano-level [2,3]. Nanofabrication of multifunctional nano or micro characters is becoming incredibly influential in electronics, photonics, energy, and biomedical gadgets worldwide. The invention of such items having particular properties stems from the merger of moderate cost and high-quality nano or micro-characters to 3D structures [4].

1.1. Advantages of nanofabrication techniques

Nanofabrication techniques provide the following advantages,

• i)It aids in the development of transportable, compact implant gadgets.
• ii)Few samples or reagents are required during testing and processing, which spares efforts and makes the process economical.
• iii)Nanotechnology delivers a way to reduce the long diffusing period, which serves as a barrier to developing products and technologies.
• iv)For instance, point-of-care diagnosis at the person's bedside allows doctors to detect illness faster than traditional laboratory-scale screening techniques. The evolution of such point-of-care testing devices has been aided by progress in nanoelectronics and biological sensor technologies.
• v)It minimizes the diagnosis period of the disease. The clinician will be ready to adopt superior treatment choices, resulting in better patient care and lower healthcare cost.
• vi)Nanofabrication allows us to study biochemical or physiological activities at molecular or cellular scales and design artificial gadgets that can link to physiological organisms.
• vii)The potential of regulating attributes to the nanoscale level for large-scale manufacturing of materials or electronics in a repeatable manner [5].
• viii)The drug substances' penetration, bio-availability, and durability are improved using nanofabrication techniques such as microfluidic, emulsification solvent evaporation, and complexation [Example: Chitosan-PLGA-PEG complexes] [6].
• ix)Nanomaterials effectively increased availability and lowered bio-active toxic effects. Due to their convenience of production, capacity to effectively absorb bio-actives into the matrix, and subsequent regulated delivery via diffusion or erosion pathways, artificial polymer-based nanoparticles are now considered the most accessible transportation units [7].

Example: Degradable PLGA is widely employed:

• x)Nanofabrication maintains the proportionality of the dosage and allows the development of small dosage forms, thereby reducing toxicity.
• xi)The diversity between fasting and fed state is reduced [8].

In addition, nanofabrication techniques are becoming the most potential technique in science, particularly cell physiology. Nanofabrication techniques have many benefits as the primary technology for manipulating cellular surroundings to understand signaling processes. First, manufacturing approaches generate a localized cell environment resembling biological and pathophysiological conditions. Second, the environment could be created accurately and repeatable to examine living cells' responses in various environments. Third, controlling the specific biochemical and cellular environment is simple, i. e. modulating the structure and abundance of extracellular matrix (ECM) proteins. Lastly, the nano techniques aid in identifying and creating 3D frameworks, which could be used to build tissues and medicine's reconstruction. It is achieved by cultivating or co-cultivating cells in a habitat analogous to the body's circumstances. As a result, nanofabrication methods are becoming essential in current biomedical research [9].

1.1.1. Disadvantages of nanofabrication techniques

Nanofabrication techniques suffer from the following disadvantages,

• i)The processes stimulated by biological stimuli, devices communicating with the human body, smart drug transport, nano-chip for nanomaterial discharge, and transporters for high-tech polymers in the administration of some peptides and proteins pose numerous practical hurdles [8].
• ii)It is challenging to describe nanotechnology at the present stage accurately. For example, thin-film techniques are hard to classify [10].
• iii)Nanofabrication can develop newer poisons and contaminants; the ecosystem might have detrimental consequences [11].

Example: Nemmar et al. observed cardiac oxidative stress and damage to DNA post administration of iron oxide nanoparticles in mice via intravenous route [12]. Magaye et al. noted a cardiac toxicity-arrhythmia and harmful impact on liver, lung and spleen after administration of nickel nanoparticles intravenously in rats [13]. Arfian et al. observed liver damage post administration of zirconia oxide nanoparticles (particle size 100 nm. Similarly, iron oxide nanoparticles showed liver toxicity in mice [14].

iv) Nanofabrication opens the door to minuscule wearable sensors that are practically invisible but misused. It could be easy to construct nuclear bombs or other hazardous weapons. The so-called "smart bullets" are computerized bullets that might be commanded and targeted precisely [1]. The different nano-drug delivery systems used in the biomedical sciences are shown in Fig. 1.

Fig. 1.

Different nano-drug delivery systems used in the biomedical sciences.

This review is an exhaustive account of nanofabrication, nanofabrication techniques, and recent advances in nanofabrication techniques. It also describes the applications of nanofabrication techniques in the preparation of nanocarriers. It also covers the regulatory aspects and challenges associated with nanofabrication techniques.

2. Nanofabrication techniques

2.1. Extrusion

2.1.1. Hot-melt extrusion (HME)

The method which has evolved in the previous decade and is used by many industries is Hot-melt extrusion (HME). It is the technique of driving a substance via an orifice or mold within regulated circumstances to generate a fresh substance (the extrudate). Extruders, particularly twin-screw type, have traditionally been used in the culinary preparation and polymer-producing industries. Multi-screw extruder-dependant technology has advanced across developed nations, notably the most significant initial breakthroughs in Germany, UK, and the US [15]. HME was used earlier to generate orally administered solid dosage formulations for several commercialized drugs, including Norvir (ritonavir), Kaletra (ritonavir or lopinavir), Onmel (itraconazole), etc. [16].

2.1.1.1. Mechanism of HME

The extruder consists of a stationary cylindrical barrel and one or more rotating screws. The barrel is frequently produced in separate pieces bolted or clamped together. The shape of the extruded product is controlled by an end-plate die attached to the barrel's end. The heat needed to melt or fuse the material is provided by the friction created as it is sheared between revolving screws and the barrel wall, along with electric or liquid heaters positioned on the barrels. Starting material is supplied into the feed section directly from a hopper. The material is brought there as a solid plug to get to the transition zone, where it is combined, crushed, melted, and plasticized. Compression is created by reducing the thread pitch while keeping the flight depth constant or by reducing the flight depth while keeping the thread pitch constant. The typical distance between the screw diameter and barrel width is between 0.1 and 0.2 mm. The material enters the metering zone as a homogeneous plastic melt appropriate for extrusion. Material is transferred from one screw to the other as the screws revolve due to the flight of one screw element wiping the flank of the neighboring screw. The material is moved along the extruder barrel [17].

2.1.1.2. HME for solid drug manufacturing

Extruders of the pharma sector are being developed as uninterrupted operating machines for mixing pharmaceuticals and manufacturing solid drug formulations, topical films, and wet granules. The transporter is liquefied, combined with a therapeutic component, de-vaporized, and subsequently forced across a mold of melt extrusion. On the other hand, wet granulation is mixing powder ingredients with solutions to confer structural and additional specific properties. Extruders produce higher-performance and cost-effective materials since they permit constant working if the equipment is operated under favorable circumstances. Simultaneously, it is predicted that device manufacturing companies may progress to refine device construction layouts and establish innovative items which maintain pace with the production demands of the dosage forms [15,18].

2.1.2. Extruder design and applications

HME is also used to fabricate solid lipid nanoparticles (SLN) -containing active ingredients [19]. This was accomplished by combining the oil and water phases with the emulsifying agent in a hot melt extruder's cylinder at a higher temperature than the lipid's melting point. A large-pressure homogenizer was then linked with the hot melt extruder to reduce the particle size to <200 nm. The active ingredient's physical and chemical attributes, the lipid's physio-chemical features, and the stabilizer selection are important excipient parameters. The crucial procedure variables for the HME technique are screw structure, screw velocity, thermal environments, and restraints for the temperatures at every region [20].

Using the hot-melt extrusion (HME) technique, Bhupendra Raj Gir and colleagues improved the solubility, bioavailability, and stability of Telmisartan (TEL) s olid dispersion. A variety of formulations were prepared using varying amounts of the drug (10–60% w/w), the hydrophilic polymer Soluplus® (30–90% w/w), and the pH-modifier sodium carbonate (0–10% w/w). The prepared solid dispersion tablets demonstrated a 30 times better-dissolving profile, superior stability and high drug content. When in vivo experiments are carried out on rats, TEL solid dispersion exhibits superior serum concentration, C_max and area under the curve (AUC_0–∞). This research improved the solubility and bioavailability of hydrophobic medications drugs without affecting the drugs' physical stability by adding a pH modifier, a hydrophilic polymer, and amorphizing crystalline pharmaceuticals in solid dispersion made using HME [20].

Another study used the hot-melt extruding method to create amorphous solid dispersion of the BCS class II drug Rivaroxaban. The method utilized a 1:1 and 1:4 of polyvinylpyrrolidone and vinyl Acetate 64, respectively, keeping a barrel temperature of 200–240 °C, and a screw speed of 15 rpm. This study assessed bioavailability, solubility, and dissolution behavior. A in vitro dissolution investigation revealed an 80% cumulative drug release post 120 min, pharmacokinetic research in rats showed increased absorption compared to plain drugs in terms of rate and volume [21].

2.2. High-pressure homogenization (HPH)

In pharma and industries, the HPH decreases the size, combines, and stabilizes mixtures such as micro or macro-emulsions or suspension. It causes intense localized tensions, which result in a significant drop in particulate size. The system is appropriate for scale-up because it is used in uninterrupted manufacturing at a large scale [22,23]. Fluid is forced via a tiny aperture at intense pressure in HPH. The fluid put into the homogenizer is known as premix (micro-suspensions, coarser emulsion, or dispersion). Based on the category of device used, the working force in HPH may range between 50 and 500 MPa [24]. The devices capable of working at pressures of ≥200 MPa are classified as ultra-high-pressure homogenizers (UHPH) [25]. The space between the valve seat and the forcer is generated, causing particle break-up due to a quick mixture flow across an opening at elevated pressure [26,27].

2.2.1. Mechanism of HPH

For HPH, a pump is used to push fluid through a valve, where it is then pushed through a small aperture. Since, during homogenization, a rise in temperature (about 2.5 °C per 10 MPa) corresponding to the fluid used is seen in the fluid downstream of the valve considered in the HPH. According to the literature, this is typically attributed to the viscous strains brought on by the high fluid velocity that impinge on the homogenizer's ceramic valve and cause a considerable portion of the mechanical energy to be lost as heat in the fluid [28].

2.2.2. Hot homogenization

Hot homogenization occurs above the lipid's melting point. High shear mixing device creates pre-emulsion of drug-loaded lipid melt and aqueous emulsifier phase (same temperature) [29].

Bhalekar made darunavir solid lipid nanoparticles (SLN) using hot homogenization. SEM, DSC, and PXRD examination of freeze-dried SLN showed complete drug entrapment and amorphous nature. Studies in 0.1 N HCl and 6.8 pH buffer showed 84 and 80% release after 12 h. The apparent permeability of SLN through rat gut at 37 °C after 30 min was 24 × 10−6, but at 4 °C, it was 5.6 × 10−6, suggesting endocytic mechanisms in SLN uptake. Accelerated stability test results indicated stable formulation [30].

In another article, hot homogenization was used to make alendronate-loaded SLNs. Drug determination using o-phthalaldehyde derivatization demonstrated high encapsulation efficiency (70–85%). The particle size and drug leakage of drug-loaded SLNs in aqueous dispersions were evaluated over two weeks. SEM images indicated nanometer-sized spherical particles, validating particle size analyzer findings. Flow cytometry and MTT assays verified the minimal toxicity of alendronate-loaded SLNs [31].

Güney used heat homogenization to make ascorbic acid-loaded SLNs. Nano zeta sizer ZS and HPLC analyzed the resulting SLN formulations. Ascorbic acid SLNs showed excellent encapsulation efficiency and sustained release behavior. Ascorbic acid-loaded -SLNs showed higher cytotoxicity than free ascorbic acid against H-Ras 5RP7 cells without harming NIH/3T3 control cells [32].

2.2.3. Cold homogenization

Like hot HPH, the Cold HPH technique also involves the dissolution of the medication in melting lipids. The heated drug lipid blend is cooled and solidified with liquid nitrogen or dry ice, then pulverized to microparticles in a ball mill or mortar. Microparticles are distributed in a cold aqueous surfactant solution and homogenized below room temperature to create LNPs. This cold HPH methodology is suitable for hydrophilic or thermolabile medicines because it avoids temperature-induced drug breakdown and distribution into the aqueous phase [33]. Larger particles and a wider size dispersion are disadvantages of cold homogenized samples. Solubilization of the drug in melting lipids and temperature generation during homogenization may not completely prevent temperature-induced drug degradation [34].

Efavirenz, a BCS class II medication, inhibits the non-nucleoside reverse transcriptase enzyme. The preparation and assessment of Efavirenz nanosuspension for the treatment of neuro-AIDS was done in one study. Efavirenz is a substrate for drug-resistant proteins at the Blood Brain Barrier (BBB) that are susceptible to efflux and cannot effectively enter the brain. Drug delivery mechanisms must currently be developed to target viral reservoirs in the brain effectively. To meet this need, researchers have created an Efavirenz nanosuspension for medication delivery from the nose to the brain that avoids the blood-brain barrier. The mean particle size of the nanosuspension created by high-pressure homogenization was 223 nm, the PDI was 0.2, and the zeta potential was −21.2 mV. Histopathology analysis of goat nasal mucosa revealed that the formulation had no adverse effects on the nasal tissues. Gamma scintigraphy investigations and in-vivo testing using Wistar rats revealed drug transport to the central nervous system (CNS) following nasal administration. Finally, the study concluded that efavirenz nanoparticle transfer directly from the nose to the brain is suggested by pharmacokinetic characteristics with a drug-targeting potential of about 99.46% [35].

2.3. Electrospinning (ES)

Electrospinning is among the most straightforward process for preparing nanostructures. This method provided versatility, convenience, scalability, economic benefits, and the ability to produce a vast array of polymeric materials [36]. The background of ES shows that it was first used in 1902. Ever since the science of ES has progressed, and after almost 3 decades, electrospun fibers were created for the very first time. In the later 6 decades, high strong volt power was applied to create fibers from a polymer mixture with a size spectrum of <5 m. The name "electrospinning" originated with the phrase "electrostatic fiber spin." Ultra-thin fibers of many polymers made by solvent mixture increased focus when the size declined to the nanometer range [37]. Fig. 2 depicts a diagrammatic illustration explaining the concept of electrospinning of polymeric nanoparticles.

Fig. 2.

Depicts a diagrammatic illustration explaining the concept of electrospinning of polymeric nanoparticles.

2.3.1. Mechanism of ES

A metallic needle known as a spinneret, a high-voltage power generator, and a gathering plate make up an electrospinning apparatus's three major parts. The initial step in the procedure is to place the reaction mixture in a syringe. For accuracy, it is made sure that the solvent in the syringe is air bubble-free. A syringe is attached to a metal needle. A syringe pump controls the solution's flow rate when it delivers via the metallic needle. A "Taylor cone" is produced by applying a high-voltage power source (often between 1 and 30 kV) to a liquid droplet of the suitable solvent at the orifice of the needle. The droplet is stretched and electrified, and the generated particles are evenly dispersed across the surface. The two main electrostatic forces that droplet encounters are electrostatic repulsion and coulomb forces. The electrostatic repulsion counteracts surface tension, whereas an external electric field generates coulomb forces. To deposit continuous and uniform nanofibres on the counter-electrode, the electrified jet will undergo a process of stretching and thrashing. These homogeneous nanofibres with nanometer-scale widths are gathered at a distance on a collection plate. From the needle reaches the collector plate, the solvent is lost from the polymer solution. The collector can be used to gather a nanofibre deposition [38].

2.3.2. Factors affecting optimization of ES

The ES may be tweaked to produce nanoparticles with specific shapes, sizes, and other properties. As a result, nanoparticles might have a smoother texture, rougher texture, or core-shell structure. These structures or diameters are influenced by various factors such as induced current, stream velocity, the composition of the mixture, rheology, mixture's capacitance, length among the receiver and syringe, solvent dissolving active moiety or additives, etc [[39], [40], [41]].

2.3.3. Types of ES [40,42]

The different types of electrospinning are shown in Fig. 3.

Fig. 3.

Types of electrospinning.

2.3.4. Advantages and applications of electrospinning in the pharma sector

It can provide distinct characteristics such as the potential to rotate a broad spectrum of artificial and natural polymeric materials, enhanced drug entrapment efficacy, large surface-to-volume fraction, customizable surface characteristics, and lower burst discharge. It also aids in efficient surface functionalization by choosing the appropriate solvent-polymer-drug framework [42]. By optimizing processing factors such as induced current, liquid volume, stream velocity, and thickness, the shape of nanofibers could be transformed into nano-shaped ribbons or beads.

Drug-embedded electrospun nanomaterials have many functions like anti-tumor, anti-hypertensive, anti-viral, and anti-microbial activities. It is also demonstrated that both aqueous and lipidic drugs could be electrospun successfully [40,42]. By employing electrospinning fiber for localized administration of drugs, the amount of the medicine could be reduced, causing undesired adverse events. The drug release rate is also influenced by fiber type and diameter. The method could be applied to adjust drug formulations to reduce drug dependence, eliminate adverse reactions, improve practical durability during storage, and increase the therapeutic availability of the drug [43].

Fibers with a wide range of molecular constituents, micro or macro-structures, and topologies are employed in various pharmaceutical uses. The nanofiber nature of electrospun substances resembles the ECM of biological tissues to some degree. It offers simple integration of aqueous and lipoidal compounds using a blend or coaxial ES techniques. Nanomaterials made utilizing this approach are incredibly permeable and pliable, with a large surface area, excellent drug encapsulation, performance, and customized drug discharge qualities. As a result, ES is considered a dynamic strategy used in the formulation of different compounds. It helps speed up the ES strategy's advancement, allowing electrospun substances to be used in additional drug administration [43].

Conventional topical dosage may have low intraocular bioavailability. Therefore, researchers developed chitosan/polyvinyl alcohol/polyvinyl pyrrolidone (CS/PVA-PVP) nanofibers with azithromycin (AZM)-loaded fibers that have extended antibacterial action via electrospinning. Using a Micrococcus luteus microbiological assay, the drug's release from nanofibers was assessed both in vitro and in vivo. The homogeneous thickness, weight, and medication content of all the formulations were found in the range to make them stable. The diameter of nanofibers ranged from 119 nm to 171 nm. According to the data, the crosslinked AZM nanofibers released the drug into the tear fluid over 184 h, which was slower and more controlled than the non-crosslinked ones. Based on these findings, the produced nanofibers may be considered suitable and non-invasive inserts for the prolonged ophthalmic delivery of AZM [44].

To examine the synergistic effects to speed wound healing, zinc oxide (ZnO) nanoparticles and glucosamine (GA)-loaded poly (methyl methacrylate) (PMMA)/polyethylene glycol (PEG) nanofibrous scaffolds were successfully developed. Using L929 fibroblasts and the MTT assay, the in vitro biocompatibility testing for the nanofibrous grafts was carried out. All nanofibrous grafts have been demonstrated to accelerate in vivo healing investigations using male albino rats to measure burn healing activity. Compared to the other three nanofibrous grafts, the ZnO nanoparticles and the GA-modified group induce fibroblasts to create immature collagen, which speeds up re-epithelialization [45].

2.4. Microfluidizer

Micro-fluidization is the patented blending process that employs a microfluidizer. The high pressure drives the medicinal substances via an interacting compartment. The fluid in this compartment is later split into separate flows that are subsequently reunited at extreme speeds [46]. A high-throughput technology is manufacturing consistent nanoemulsion using large-pressure microfluidics. Fast streaming flows of pre-mixed dispersion are driven into solid stainless-steel microchannels created by lithography or micromachining. In the past, various sectors adopted the microfluidizer® technique to design attributes that can only be achieved using nano components such as nanoparticles, nanoemulsion, nanoencapsulation, etc. The technique's benefits range from improved durability to product optimization and improved administration of lipoidal compounds to their desired sites. This technique is often used to make time-based release items, including pharmaceuticals, aesthetics, nutritional supplements, and insecticides. Unsurprisingly, industrial processing engineers as diverse as medicines, immunizations, specialty chemicals, biotech, cosmetology, food production, and energies all realized that microfluidics is an excellent technique for preparing nanomaterials [47].

Various industries formerly used the microfluidizer® approach to produce characteristics that could only be produced by utilizing nanoscale components, including nanoparticles, nanoemulsion, and nanoencapsulation. The benefits of the approach range from increased durability to product modification and enhanced delivery of lipoidal chemicals to target areas. Microfluidizer® devices improve traditional homogenizers regarding reproducibility, smooth scaling, smaller particle size dispersion, and particulate size reduction. The microfluidizer converts hydraulic tension into a considerable friction force more effectively and consistently by retaining consistent pressure and avoiding temperature impact by changing the chemical and physical properties of any fragile substance than alternative techniques like high-pressure valve homogenization technique in which significantly less operational power is converted to heat [47]. Micro-fluidization is a comparatively recent high-pressure homogenization approach incapable of producing tiny multilamellar vesicles by reducing the surface area of the vesicle. This liposomal mixture is circulated in the interacting compartment at great force. The mixture is split into separate flows in the interacting compartment and later merged rapidly, forming tiny and evenly formed liposomal vesicles [48].

2.4.1. Mechanism of Microfluidizer®

Despite being a high-pressure processing technology, high-pressure homogenization differs significantly from microfluidization in terms of how it operates. In high-pressure homogenization, the rapid limitation of flow that causes pressure fluctuations causes the shear forces. The treatment produces wider size distributions due to the shift in pressure. On the other hand, in micro-fluidization, the instruments fixed geometry results in the delivery of consistent pressure pro-files with compression and suction strokes. It generates particles with smaller sizes and narrower size distribution due to the continuous cycles of constant and zero pressure [49].

A full-factorial experimental approach was used to microfluidize a milk cream emulsion loaded with curcumin at various pressures (50–200 MPa) and passes (1–4). The average particle size (358.2 nm) was significantly reduced after microfluidization. After processing, it was discovered that the microfluidized emulsion's encapsulation effectiveness was much higher (97.88%) than the control (91.21%). For microfluidized emulsions compared to controls and found that there was a two-fold (100%) increase in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and a 25% increase in ferric-reducing antioxidant power (FRAP). After microfluidization, bioaccessibility noticeably increased by 30% [50].

3. Nanocarriers developed by nanofabrication methods

Several nanocarrier systems can be developed using all the nanofabrication methods discussed above. Such nanocarrier systems include,

3.1. Nanoparticulate systems

There are two types of nanoparticulate systems as follows,

3.1.1. Solid lipid nanoparticles (SLN)

SLN is a colloid transporter established in the past years, replacing regular transporters (liposome, emulsion, or polymer nanoparticle). These are a novel class of submicron-level lipid microemulsions in which a solid lipid form replaces lipids' fluid state [51].

3.1.1.1. Preparation methods of solid lipid nanoparticles

• i)High shear mixing or ultrasonic method
• ii)High-pressure homogenization (HPH): (Hot homogenization and cold homogenization)
• iii)Solvent evaporation method
• iv)Micro-emulsion dependant method
• v)Double-emulsion technique
• vi)Spray-drying technique

Compared to a high shear blending or ultrasonic, HPH is a superior strategy for producing SLN. The median particulate dimension of typical SLN suspension generated by the HPH method is < 500 nm, with a minimal microparticle concentration. Solids, lipids, and emulsifiers mixed with solvent or water make up SLNs. Triglyceride (tri-stearin), partial glycerides (Imwitor), fatty acid (stearic/palmitic acid), steroid (cholesterol), and wax (cetyl palmitate) are some of the lipids employed for the preparation of SLN. Several emulsifying agents and their combination (such as Pluronic F127 and F68) stabilize the fatty solution. Using multiple emulsifying agents may help minimize the aggregation of nanoparticles [51].

One study examined the potential for drug-loaded SLNs to enhance clarithromycin's therapeutic potential in topical ocular treatment and boost ocular penetration. High-speed stirring and the ultrasonication technique were used to fabricate SLNs. 3² full factorial and fractional factorial designs were used to screen and optimize clarithromycin-loaded SLNs. Optimized SLNs were tested in rabbits for ocular morphology, penetration, irritation, stability, and pharmacokinetics. The Weibull model kinetics was followed by the release profile of SLNs, which indicated an 80% drug release in 8 h. As shown by a 150% increase in C_max (1066 ng/mL) and a 2.8-fold improvement in AUC (5736 ng/mL) as compared to the control solution (C_max: 655 ng/mL and AUC: 2067 ng h/mL). Thus, the study concluded that clarithromycin bioavailability was significantly improved when formulated as SLNs [51].

Another study developed solvent-free solid lipid nanoparticles (SLNs) loaded with buspirone (BUS) for nose-to-brain transport and optimized them using the Box-Behnken design with three factors and three levels. Particle size, PDI, zeta potential, entrapment efficiency and in vitro drug release of the optimized batch were 218.60 ± 9.18 nm, 0.305 ± 0.012, 26.47 ± 2.36 mV, 70.13 ± 4.21%, and 93.36 ± 8.63% respectively. BUS-SLNs administered intranasally had an AUC_0-∞ in the brain 2.18 times higher than BUS-solution and 2.66 times higher than BUS-SLNs administered intravenously. When comparing BUS-SLNs via the nasal route to BUS-Solution intranasal, the values for drug targeting efficiency (DTE) (882.59%) and nose-to-direct brain transport (DTP) percentage (88.67%) were greater [52]. Hence, the study suggested that SLNs act as a potential drug delivery system for transporting BUS into the brain via the nasal route.

3.1.1.2. Polymeric nanoparticles

The substance is dissolved, encapsulated, entrapped or linked to a nanoparticle matrix in polymeric nanoparticles (PNPs). It comprises biocompatible and biodegradable polymers ranging from 10 to 1000 nm. Effective medication, protein, and DNA delivery to target cells and organs are made possible using polymer-based nanoparticles. Their nanoscale size encourages stabilization in circulation and efficient diffusion across cell membranes. The production of endless and diverse molecular patterns that may be incorporated into distinctive nanoparticle constructions with a wide range of potential medicinal uses [53].

3.1.1.3. Preparation methods of the polymeric nanoparticles (PNs)

Various techniques have been used to create polymeric nanoparticles depending on their intended use and the physicochemical properties of the compounds. The various preparation techniques have been developed, and these can be categorized into two groups: those that use formulated polymers and are based on the polymerization of monomers, such as microemulsion and interfacial polymerization, and those taking advantage of preformed polymers that include nanoprecipitation, solvent evaporation, dialysis, and supercritical fluid extraction. These techniques may be further divided into two groups: one-step procedures where nanoparticle production does not need emulsification and two-step procedures involving developing an emulsification system followed by nanoparticle formation in the second stage of the process [54].

One clinical study showed that the oral bioavailability of curcumin-loaded polymeric NPs was 5.6 times greater oral bioavailability of curcumin-than-loaded polymeric NPs compared to pure curcumin. Like curcumin, silymarin from an oral polymeric nanoemulsion is four times more effective than standard silymarin solution in vivo studies. Natural polymers with biodegradable, biocompatible, and mucoadhesive qualities, including chitosan, dextran, heparin, or hyaluronan, have been employed extensively in drug delivery research. Moreover, biomimetics has been introduced in material design to synthesize more sophisticated and highly sought nanocarriers. In this scenario, the surface of the carbon nanotubes is modified with the appropriate ligands, or chitosan nanoparticles are created [55].

In vitro colorimetric tests, such as Alamar blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), are frequently used to test the cytotoxic profile of nanoparticles for the viability of specific cell lines. Cell viability above 70% is often regarded as evidence of the evaluated nanoparticle formulation's minimal cytotoxicity. HepG2 (a kind of human hepatoma cell line), Caco-2 (a type of human epithelial colorectal adenocarcinoma cell line), and Y-79 have all been used as test subjects to determine the cytotoxicity of PLGA nanoparticles loading triterpenoids with potential anticancer activity (Human retinoblastoma cell line) [56].

3.1.2. Inorganic nanoparticles

In both preclinical and clinical studies, inorganic nanoparticles such as gold, iron oxide, silver, or silica are being researched to treat, diagnose, and detect numerous diseases. In addition, many inorganic substances used to create nanoparticles have a long history of use in medical settings for various therapeutic purposes. The use of platinum, cisplatin, carboplatin, oxaliplatin, etc., for treating cancer and silver ions as antibacterial agents are two prominent examples of inorganic compounds used for therapeutic applications. Additionally, specific inorganic nanoparticles can respond to particular outside stimuli, such as magnetic fields or near-infrared (NIR) light, to improve magnetic imaging or promote the release of drugs on demand, respectively. Additionally, various inorganic nanoparticles may be created and adjusted to make it easy for ligands or polymers to be incorporated into them, enhancing their biological activity [57].

Polyethylene glycol (PEG)-coated gold nanoparticles demonstrate longer circulation durations in vivo following systemic injection under inductively coupled plasma mass spectrometry (ICP-MS). Around 54% of the PEG-modified gold nanoparticles administered intravenously to mice were discovered in the blood post 0.5 h [56,57].

3.1.3. Current literature for fabrication of nanoparticles with proteins or nucleic acids

Using polyanion tripolyphosphate (TPP) as the coacervation crosslink agent Bovine Serum Albumin (BSA) was employed as a model protein for the development of nanoparticles. It was encapsulated using either the incorporation approach or the incubation method to create chitosan-BSA-TPP nanoparticles. Particle size, morphology, zeta potential, BSA encapsulation effectiveness, and subsequent release kinetics of the BSA-loaded chitosan-TPP nanoparticles were characterized. It was discovered that these properties primarily depended on chitosan molecular weight, chitosan concentration, BSA loading concentration, and chitosan/TPP mass ratio. The 200–580 nm size range and highly positive zeta potential of the BSA-loaded nanoparticles were produced under various circumstances. A swelling and particle breakdown process was seen in the morphological alteration of the BSA-loaded particles using detailed sequential time-frame TEM imaging. This study showed that the polyionic coacervation process for fabricating protein-loaded chitosan nanoparticles offers specific preparation conditions and a separate processing window for the manipulation of the physiochemical properties of the nanoparticles. The initial burst release was detected due to surface protein desorption and diffusion from sublayers, which was eminently apparent only after 6 h [58].

In work by Tohid Piri-Gharaghie et al., pcDNA3.1 (+) plasmid (pDNA) and Chitosan Nanoparticle (CSNP) complexes were created and studied. The properties of the pDNA/CSNP combination were examined using SEM, XRD, DLS, TGA, and FTIR. By tagging free plasmids with the fluorescent intercalating dye OliGreen, the ability of CSNP to form complexes with pDNA was examined. In the presence of chitosanase, the stability of pDNA/CSNP was assessed. Real-time PCR was used to measure the absorbance rate in BALB/c mice, and Surface-Enhanced Raman Spectroscopy (SERS) was used to locate the pDNA. The ideal pDNA/CSNP ratio for plasmid complex formation was determined to be 1:2 (w.w) using spectroscopy. SERS demonstrated that a portion of the pDNA was present on the complex outer surface at these optimal complex formation ratios, further supported by spectroscopic and gel digest studies. The rate of gene uptake was substantially higher at a dosage of 1:2 (w.w) of pDNA/CSNP than in other groups, according to real-time PCR results of plasmid absorption in mouse thigh tissue (P < 0.001). This study's findings showed how precisely pDNA fits into polymer nanostructured delivery systems, enabling formulation adjustments for targeted distribution [59].

3.1.4. Nanostructured lipid carrier (NLC)

NLCs are lipid-based nanocarriers developed from SLN, which combine solid and liquid lipids. This system was designed to overcome the limitations of SLNs; therefore, NLCs have a higher drug-loading capacity and can avoid drug expulsion during storage by preventing lipid crystallization. NLCs are a mixture of solid and liquid lipids like glyceryl tricaprylate, ethyl oleate, isopropyl myristate, and glyceryl dioleate. These nanoparticles can be loaded with hydrophilic and hydrophobic drugs, have site-specific targeting, controlled drug release, and low in vivo toxicity [[60], [61], [62]].

3.1.4.1. Preparation methods of the nanostructured lipid carrier

NLCs comprise an unorganized solid lipid network consisting of a combination of liquid plus solid lipid and an aqueous layer holding one or more surfactants. The solid form of lipids is often combined with the liquid form of lipids in a 70:30 to 99.9:0.1 proportion, including a surfactant component ranging from 1.5 to 5% w/v. Despite various techniques for preparing NLC (such as micro-emulsification and solvent displacement) being available, the HPH approach is favored over the others. The heated surfactant mixture is introduced into the premixed and molten lipid carrying the drug (at a temperature of about 10 °C higher than the melting point of the lipid). A hot nanoemulsion is obtained after homogenizing this microemulsion at intense pressure. Before the homogenization procedure, a pre-emulsification phase is commonly conducted. The nanoemulsion is cooled, resulting in the formation of the NLC [42].

Shah et al. used nanostructured lipid carriers' (NLCs) capability to increase raloxifene hydrochloride's oral bioavailability (RLX). The solvent diffusion approach created RLX-loaded NLCs using glyceryl monostearate and Capmul MCM C8 as the respective solid and liquid lipids. When the liquid lipid concentration in the formulation increased from 5% w/w to 15% w/w, the statistical analysis revealed a noticeable improvement in entrapment efficiency. The results of an in vitro release research demonstrated an initial 8 h burst release, followed by a 36 h continuous release. A TEM investigation confirmed a smooth surface and discrete spherical nanoparticles. In vivo pharmacokinetic analysis that compared the bioavailability of improved NLCs formulation plain drug solution revealed 3.75-fold improvements. These findings suggested that NLCs have the potential to significantly increase the oral bioavailability of poorly soluble RLX [63].

A possible topical administration method for clobetasol propionate (CP) was created using a nanostructured lipid carrier (NLC)-based gel. The NLC formulation has a particle size of 137.9 nm, a zeta potential of 20.5 mV, a polydispersity index of 0.224 and in vitro release of 85.42% after 24 h. The permeation analysis showed the steady-state flux, permeability coefficient, and enhancement ratio for the NLC-based gel formulation compared to commercial clobetasol propionate [64].

3.2. Vesicular drug delivery systems

3.2.1. Liposomes

Liposomes are microscopic phospholipid-based vesicles composed of one or more phospholipid bilayers in a hydrophilic center. When amphoteric liposomes are scattered in fluid, the lipophilic sections of the subunits try to assemble, whereas the lipophobic regions of the subunits disclose to the fluid. As a result, they can form bilayer-structured spherical vesicles [65].

With continuous drug release for up to 96 h, imatinib mesylate-loaded liposomes were developed with particle sizes under 150 nm. On N2a cells, the cytotoxicity of the liposomal formulation was compared with a simple drug solution, and it did not exhibit any toxicity at doses up to 25 g/mL. The nanocarrier formulation was then compared to a drug solution for brain deposition following nose-to-brain injection. Thus, liposomes substantially enhanced the brain deposition of drug from formulation compared to pure drug solution [66].

It was considered that liposomes had considerable potential for treating brain disorders. As a result, one study aimed to create a geniposide liposome (GE-LP) as a brain delivery method for treating cerebral ischemia-reperfusion injury (CIRI). The formulation was developed into the GE-LP using a reverse-phase evaporation technique. GE solution and GE-LP activity were studied in mice plasma. The outcomes showed that, compared to the GE solution, GE-LP revealed a greater bioavailability and brain targeting threefold longer distribution half-life. The middle cerebral artery occlusion rat model was used to assess the in vivo neuroprotective effects, and GE-LP showed a better tendency to prevent the injury of CIRI [67].

3.2.1.1. Preparation techniques of liposomes

The various techniques in the preparation of liposomes are mentioned below (Table 1),

Table 1.

Preparation techniques of liposomes; Reproduced/Adapted from The Journal of Supercritical Fluids 2020, 165 (1), 104984 with permission from Elsevier Ltd. Amsterdam, Copyright 2020 [68].

Technique	Lipids used	Types of vesicles	Advantages	Disadvantages	Reference
Solvent Injection	Egg phosphatidylcholine (EPC) Distearoylphosphatidylcholine (DSPC) Dioleoyl phosphatidylcholine (DOPC) Dierucoyl phosphatidylcholine (DEPC) Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) Dipalmitoyl phosphatidylcholine (DPPC) Dimyristoylphosphatidylcholine (DMPC) Dipalmitoylphosphatidylglycerol (DPPG) Distearoylphosphatidylglycerol (DSPG) Dioleoyl phosphatidylserine (DOPS) Dioleoylphosphatidylserine (DOPS) N-(carbonyl-Methoxypolyethlyeneglycol-2000)-distearolyphosphatidylethanolamine (MPEG-2000-DSPE) Cholesterol (Chol) Sphingomyelin (SM)	SUVs, SMVs	Easy technique	Removal of organic is difficult, More time require, Nozzle blockage possibility, Needs sterilization	[[69], [70], [71]]
Thin-film hydration (Bangham method)		MLVs	Easy process Straightforward strategy	Limited to small scale Time-consuming process Sterilization is required Difficulty in removing the organic solvent Leading to large vesicles without control on particle size Resulted in low encapsulation efficiency (Especially for water-soluble compounds)	[72,73]
Reverse phase evaporation		LUVs, MLVs	Simple method Provide sufficient entrapment efficiency	Time-consuming method Sterilization required Utilizes more amount of organic solvent (Not suited for peptide encapsulation)	[[74], [75], [76], [77]]
Detergent removal		LUVs, MLVs	Uniform product Good control over particle size Simple method	Leads to detergent residue in the product Require more time Organic solvent removal is difficult Entrapment efficiency is low Sterilization is required	[[78], [79], [80]]
Heating technique		SUVs, MLVs	A rapid and easy technique No sterilization is required Absence of contamination (Example: Organic solvent)	Involves the use of high temperature	[[81], [82], [83], [84]]
Membrane extrusion technique		MLVs	Absence of contamination (Example: Organic solvent) Rapid and easy process Reasonable control over particle size	Limitations in large-scale production May lead to clogging of pores	[[85], [86], [87]]
Microfluidic technique 1. Microfluidic droplets (MD) 2. Pulsed jet flow microfluidics (PJM) 3.Micro hydrodynamic focusing		GUVs (Using MD) GUVs (Using PJM) SUVs, LUVs (Using MHF)	Reasonable control over particle size	Not suitable for large-scale production Organic solvent removal is difficult	[[88], [89], [90], [91], [92]]

(SUVs- Small unilamellar vesicles, LUV's-Large unilamellar vesicles, MLVs- Multilamaller vesicles, GUVs- Giant unilamellar vesicles)

3.2.2. Niosomes

Niosomes are a vesicular technology that could be employed for long-term, regulated, and specific drug administration. Niosomes could also have cholesterol, analogs, and non-ionic surfactants. Cholesterol gives structural stiffness, whereas the charged particle maintains the mixture's integrity [93,94]. The preparation methods of niosomes are mentioned in Table 2.

Table 2.

Preparation methods of niosomes; Reproduced/Adapted from European Journal of Pharmaceutics and Biopharmaceutics 2019, 144; 18–39. With permission from Elsevier Ltd. Amsterdam, Copyright 2020 [97].

Method	Advantages	Disadvantages	References
Ether injection method	A simple method for lab-scale research	Not suitable for heat-sensitive drugs	[98,99]
Reverse phase evaporation method	High encapsulation efficiency is achieved	Needs organic solvents	[100]
Thin film hydration (hand shaking) method	A simple method for lab-scale research	Needs organic solvents	[99,101,102]
Emulsion method	A simple method for lab-scale research	Needs organic solvents	[103]
Lipid injection method	No requirement of organic solvents	Not suitable for heat-sensitive drugs	[103]
Bubble method	No requirement of organic solvents	Not suitable for heat-sensitive drugs	[104]
Supercritical reverse phase evaporation method	No requirement of organic solvents	Need special equipment for this method	[105]
Micro fluidization	No requirement of organic solvents	Not suitable for heat-sensitive drugs	[103]

One research was interested in increasing the oral bioavailability of rosuvastatin using specially formulated niosomes that utilized the film hydration process and sonication to increase their aqueous solubility. The rosuvastatin permeation utilizing niosomes was significantly improved in ex vivo intestinal permeability compared to the rosuvastatin solution (95.5% and 40.1% post 2 h). Due to the high surface area of niosomes and their lymphatic uptake via the transcellular route, the in vivo pharmacokinetic parameters in the rat model confirmed the improvement in the oral bioavailability with optimized rosuvastatin-loaded niosomes (relative bioavailability = 2.01) than rosuvastatin suspension [95].

A biological macromolecule called hyaluronic acid was used to load drugs into niosomes. Cholesterol was employed as the lipid and Span 80 as the non-ionic surfactant to prepare the niosomes using the ethanol injection technique. The particle size of HA-loaded niosomes was 177.6 nm, and entrapment efficiency was greater than 95%. The drug release from HA-loaded niosomes demonstrated a regulated release for up to 4 days. HA had a higher plasma concentration when administered through aerosol than free HA solution. According to in vivo pharmacokinetic study, organ biodistribution investigations also revealed that HA was more locally concentrated in the lungs than in other organs [96].

3.3. Emulsions

There are two types of emulsion systems as follows,

3.3.1. Nanoemulsion

Nanoemulsions are O/W, or W/O emulsions of two immiscible solutions stabilized with a surfactant. The average particle size is typically <500 nm. They have a transparent or cloudy appearance instead of the milk-white color of coarse emulsions. Nanoemulsion, submicron emulsion, and mini emulsion are occasionally referenced equally, yet they must never be mistaken for microemulsion. Although they possess identical particle dimensions to microemulsions, they significantly differ in terms of morphological characteristics and long thermodynamic durability [106,107]. Nanoemulsions are prepared using various methods, as shown in Table 3.

Table 3.

Different nanofabrication techniques used in the preparation of nanoemulsions [107].

Route	Active ingredient	Dispersed phase	Surfactant	Technique	Role	Size (nm)
Oral	Paclitaxel	Pine nut oil	Egg lecithin	Ultrasonication	Cancer (Enhance oral BA)	90–120
	Primaquine	Miglylol 812	Poloxamer 188	Homogenization + HPH	Malaria (Decreasing dosage)	10–200
Topical	Ceramide	Sphingolipid	Lipoid	Ultra turrax + HPH	Change dermal penetration	210 ± 18
	Blank	Snake oil	Soybean lecithin	Ultra turrax + HPH	Study of dermal transport capacity	75–300
Parenteral	Insulin	Self-assembling protein	intricate Polyvinyl alcohol	Spontaneous emulsification	Prevention of enzymatic deterioration	200–500
	Thalidomide	Soybean/castor/olive oil	Tween-80	Spontaneous emulsification	To manage the lesser dissolution	200
Intranasal	Quetiapine	Capmul MCM	Tween-80	Vortex + Ultrasonication	Nose-to-brain drug delivery with enhanced potential	144 ± 0.5
	Selegiline	Grape seed oil	Solutol®, Labrasol®	High energy emulsification	Nose to brain delivery with a better BA	60

The disease with the most significant risk to life is cancer. A popular novel drug delivery system for encapsulating water-insoluble medications and delivering them into malignant tissue after intravenous injection is polyethylene glycol nanoemulsion (PEG-NE). It was formulated and loaded with brucin (anti-carcinogenic) drug by Elsewedy and co-workers (BRU-NE). For BRU-NE, homogenous nanoemulsions were produced with particle sizes less than 140 nm and viscosities under 3.3 cp. The total serum protein adsorbed was less than 17.33 ± 0.76 g/mol total lipid. The in vitro drug release was less than 65% for PEG-NE 24 h. Additionally, PEG-NEs might significantly reduce the viability of cancer cells [108].

It has been established that the natural flavonoid isoliquiritigenin (ISL) can also cure, but due to its low aqueous solubility, a nanoemulsion of ISL was prepared by researchers (ISL-NE). The in vitro drug release and ex vivo corneal permeation testing showed that ISL-NE released more drugs and penetrated more than ISL suspension (ISL-Susp). After a single dosage of ISL-NE, the bioavailability of ISL-NE was 5.76 times greater in tears, 7.80 times higher in the cornea, and 2.13 times higher in the aqueous humour than ISL-Sups [109].

3.3.2. Microemulsion

Microemulsions are monophasic blends of two immiscible solutions stabilized with a thermodynamically steady, isotropic, and clear surfactant. These emulsions depend on surfactant self-assemblies and exhibit characteristics like traditional surfactants, such as micellar mixtures and lyotropic liquids crystals [[110], [111], [112]]. A diagrammatic representation of various nanofabrication techniques is shown in Fig. 4.

Fig. 4.

A diagrammatic representation of various nanofabrication techniques.

In one research work, an oil-in-water lignin microemulsion was formulated and characterized to increase the bioavailability of valproic acid (VPA). Microemulsion demonstrated long-lasting release and excellent encapsulation efficiency. Microemulsions were evaluated for their cytotoxic activities using the MTT colorimetric assay and the LDH leakage assay on malignant (MCF7 and HeLa) and healthy (HUVEC) cell lines. Microemulsions showed high encapsulation efficiency and sustained drug release. Compared to control rats treated with a control sample, in vivo treatment of free VPA at a dosage of 20 mg/kg dramatically elevated blood biochemical markers and liver lipid peroxidation in rats. In addition, liver lipid peroxidation was markedly accelerated by the 20 mg/kg free VPA. Thus, surfactant-based microemulsions were promising for effectively delivering VPA to target progressive cancer cells and inhibiting unwanted side effects leading to tissue injuries [113].

A calcium channel blocker, elodipine, has low oral bioavailability (15%) because it dissolves poorly in water and has a high rate of first-pass metabolism. Akshay and co-workers studied dynamic surface tension and properties of microemulsions to increase the oral bioavailability of felodipine by increasing the drug's permeability in the gut. The higher oral bioavailability of the microemulsion (relative bioavailability = 21.9) compared to the felodipine suspension was demonstrated by the in vivo pharmacokinetic parameters in the rat model. This improvement was attributed due to the high surface area of the oil droplets and lymphatic uptake via the transcellular route [114].

4. Comparative study of nanofabrication techniques

A comparative study of various nanofabrication techniques is shown in Table 4.

Table 4.

Comparison of various nanofabrication methods.

Formulation Technique	Nanocarrier system	API	Material	Therapeutic Outcome	Reference
Emulsion-solvent evaporation method	Lf-TMC NPs	Huperzine A	Lactoferrin and N-trimethylated chitosan	Good sustained release effect, adhesion and targeting ability and broad application as an intranasal drug delivery carrier.	[115]
Ionic-gelation-method	CS NPs	Bromocriptine	Chitosan	Nanocarrier has increased brain uptake of bromocriptine, also enhanced its antioxidant activity and made an effective system for the treatment of Parkinson's disease	[116]
Ionic-gelation method	CG NPs	Rasagiline	Chitosan glutamate	These could be a promising delivery system for the treatment of Parkinson's disease	[117]
Double emulsion-solvent evaporation method	PCL NPs	Carboplatin	Poly (ε-caprolactone)	Better nasal absorption and sustained release profile can be used in the intranasal administration of carboplatin for improved brain delivery.	[118]
Melt emulsification ultra-sonification method	NLC	Teriflunomide	Compritol 888 ATO (Solid-Lipid) and Maisine 35-1 (Liquid-Lipid)	Enhanced the nasal residence time	[119]
High-energy emulsification method	NE	Selegiline	Grape seed oil	Enhanced the uptake of selegiline to the brain, improved brain bioavailability and restored normal dopamine levels.	[120]
Emulsion-solvent-diffusion method Emulsion-solvent evaporation method	PNPs SLNs	Tarenflurbil	Poly (lactic-co-glycolic) acid	Desirable brain biodistribution profiles effectively deliver. Tarenflurbil to the brain for the treatment of Alzheimer's disease.	[121]
Thin-film hydration and rehydration method	NLs	Lamotrigine	Phospholipid 90G and Cholesterol	High entrapment in the lipid bilayer, high release rate and better penetration than suspension.	[122]
Double emulsion-solvent evaporation method	PLGA NPs	Venlafaxine	Poly (lactic-co-glycolic acid)	Plain nanoparticles showed a fast and highest ability to reach the brain after intranasal administration via nose-to-brain delivery compared to functionalized nanoparticles.	[123]
Modified double-emulsion (W/O/W0) method	CS PCL NPs	Eugenol	Chitosan and Poly (ε-caprolactone)	Enhancement of drug bioavailability can help in treating cerebral ischemia effectively.	[124]
Ionic-gelation-method	CNs	Pramipexole Dihydrochloride	Chitosan	Showed significant brain targeting potential compared to other formulations so that it can be utilized for effective brain targeting via the intranasal route for Parkinson's disease treatment.	[125]
Solvent displacement method	PLGA-PEG NPs	Pioglitazone	Poly (lactic-co-glycolic) acid-Poly ethylene glycol copolymer	Nasal mucosa showed enhanced drug permeation in the tissues in the treatment of Alzheimer's disease, and it could be a promising delivery route in the treatment of Alzheimer's disease.	[126]
Microemulsion method	NLC	Pioglitazone	Liquid lipid Capmul MCM and Solid lipid tripalmitin	The formulation improves the permeability of Pioglitazone across nasal mucosa and enhances the concentration of drugs reaching the brain.	[126]
Double emulsion-solvent evaporation method	Lf-BNPs	Dopamine	Borneol and lactoferrin	NPs increased Dopamine absorption into the brain, demonstrating that co-modification significantly enhanced the transport of drugs toward the brain.	[127]
High-pressure homogenization technique	MNE	Kaempferol	Chitosan	MNE decreases the viability of glioma cells by enhancing apoptosis and seems to be an essential carrier for cancer treatment.	[126]

(Lf-TMC NPs-Lactoferrin and N-trimethylated chitosan Nanoparticles, CS NPs- Chitosan Nanoparticles, CG NPs- Chitosan glutamate Nanoparticles, PCL NPs- Poly(ε-caprolactone) Nanoparticles, NLC- Nanostructured lipid carrier, PNPs-Polymeric Nanoparticles, SLN's- Solid lipid Nanoparticles, PLGA NP's- Poly (lactic-co-glycolic acid Nanoparticles, PEG NPs- Poly ethylene glycol nanoparticles, NLC- Nanostructured lipid carrier, Lf-BNPs- Borneol and lactoferrin nanoparticles, NL's- Nanolipids, NE's- Nanoemulsions).

5. Regulatory aspects of nanofabrication techniques

Nanotechnology is an emerging science used in many diverse fields. It has wide applications in developing products, including foods, textiles, electronics, and pharmaceuticals. They exhibit physical, chemical, and biological properties essential in designing and developing various products. The choice of materials and methods significantly impacts product design. Pharmaceutical product development is not an exception due to end-use by human beings related to patients' lives. Hence much care is taken while selecting materials and processes. Nanotechnology is used to develop pharmaceuticals like drugs, biological products, medical devices and various drug delivery systems. The food and Drug Administration (FDA) has regulated the design and development of pharmaceutical products by controlling the materials, ingredients, processes, process parameters and other substances. FDA has published many guidelines describing views and recommendations in selecting materials and techniques for developing pharmaceuticals. FDA considers planning for safety assessment while selecting appropriate materials and procedural requirements. This includes legal frameworks and characteristics of specific products. FDA significantly focuses on safety, effectiveness, and public health by considering two points while using nanotechnology: particle dimension and particle dimension-dependent properties [29,[128], [129], [130]].

FDA advises industries to consult with the FDA in the early development of processes and fulfill the regulatory requirements. These points are considered an initial screening tool that can be applied to all the FDA regulatory requirements. FDA will ask for the following criteria based on the above two points:

• •Whether a material or end product is engineered to have at least one external dimension or an internal or surface structure in the nanoscale range of approximately 1 nm to 100 nm, it checks over the change in the properties that directly impact safety, efficacy and public health issues.
• •Whether a material or end product is engineered to exhibit properties or phenomena, including physical or chemical properties or biological effects, that are attributable, even if these dimensions fall outside the nanoscale range [[128], [129], [130], [131]]. The marketed nanofabricated products are shown in Table 5.

Table 5.

Marketed nanofabricated products.

Product	Technology	Manufacturer	Application	References
Emend®	Elan Drug Delivery Nanocrystals®	Merck & Co., Inc., NJ, USA	Used in the prevention of nausea and vomiting during cancer chemotherapy.	[133,134]
Ostim®	Nano crystallization Technology	Osartis GmbH & Co. KG, Dieburg, Germany)	A bone grafting material, especially for dental and orthopedic surgeries.	[135,136]
Rapamune®	Nano crystallization technology involving bead/pearl formation	Wyeth Pharmaceuticals Inc., the USA	Used to prevent kidney transplant rejection	[137]
Doxil®	Liposomal Technology	Alza, Pakistan, is also known as Caelyx®; FDA approved it in 1995	Used in the treatment of various cancers.	[[138], [139], [140]]
DaunoXome®	Liposomal Technology	Galen, Craigavon, UK, approved by FDA in 1996	Used in treating cancers like leukemia.	[141]
Cimazia®	PEGylation Technology	UCB, Brussels, Belgium	Used to treat rheumatoid arthritis, ankylosing spondylitis and Crohn's disease etc.	[142]
Adagen®	PEGylation Technology	Enzon, Inc., NJ, USA	Used to treat immunodeficiency disorders.	[141]
Estrasorb®	Micellar-emulsion encapsulation	Novavax, Inc., MD, USA	Used in the treatment of vasomotor symptoms due to menopause.	[143]
Ontak®	Protein conjugation	Eisai, Japan	Used to treat leukemia and lymphoma.	[144]
Feraheme®	Metallic Nanosuspension	AMANG Pharmaceuticals, MA, USA	Used to treat the anemia	[145]
Argus II®	Implantable retinal electrode system	Second sight	Used to treat the severe profound retinitis pigmentosa.	[146]
Durasert®	Ocular miniatured, sustained-release drug delivery	pSivida (EyePoint Pharmaceuticals, Inc.)	Used to treat cytomegalovirus retinitis.	[146]
Albuminus Focus Nano active®	Albuminus Nanocarrier stents and nanocoating technology	Envision Scientific	Used in the treatment of diabetes mellitus and myocardial infarction.	[147]

The General Medicinal Product Regulation for nanomedicines is implemented by the European Medical Agency (EMA). The EMA established the European Nanomedicines Expert Group in 2009 to address the growing need for nanomedicine assessment. A proposal for FDA advice on drug products, including biological products using nanomaterials, was published in the USA in 2017. Additionally, the FDA assesses each nanotechnology case-to-case basis rather than attempting to label it as safe or dangerous. Manufacturers are encouraged to communicate with FDA while manufacturing nanotechnological products so that both parties can agree on regulatory concerns. FDA would continue postmarket surveillance even after approval to safeguard consumers. Other organizations have also helped to regulate nanomedicines, like the National Cancer Institute's Nanotechnology Characterization Laboratory (NCL-NCI), which has done so for more than ten years. The UK's Medicines and Healthcare products Regulatory Authority (MHRA) oversees the regulation of medications there.

Regarding FDA clearance of nanomedicine, there is no clear advice available; instead, it seems that each application is handled individually. Similar to the US, organizations throughout the UK and EU, such as the European Nanomedicine Characterisation Laboratory (EU-NCL), supply and continuously improve expertise on preclinical characterization tests of nanomedicine. Canada relies on the current legal frameworks for the approval of nanotechnology products. Health Canada urges producers to communicate with the respective regulatory authority early in product development to identify and evaluate the product's risks and qualities. Medical portfolio representatives from regulatory organizations, including Health Canada and the Canadian Institutes of Health Research (CIHR), make up the nanotechnology working group created in Canada to gather and discuss concerns relating to nanotechnology [132].

6. Challenges of nanofabrication techniques

There has been rapid development in the field of nanotechnology and nanomedicine. Many products have been approved since 1980, and revolutionary development has been observed. Many of these novel products are based on the fact that they belong to different categories, either therapeutic agents, medical devices, or carriers for the drug. As mentioned in Table 2, many nanostructured forms are available in the market, like nanocrystals, nanoparticles, nanocomposites, liposomal formulations, PEGylated polymeric nanoparticles, metallic nanoparticles and other forms of nanostructures. Nanotechnology has attracted scientists more significantly to develop pharmaceutical and medicinal products. The main reason for this is the many drawbacks associated with conventional products and the limitations in delivering these products [[148], [149], [150]].

Although nanofabricated products are influencing the current need, many issues have been addressed in their development. These challenges include cost, complexity in the design and development process, commercial scalability, commercial viability, and, more importantly, ethics in the development process [151,152].

Few companies are well versed with better equipment to develop nanofabricated products from laboratory to industry; hence, nanostructured products hold a minimal share in the pharmaceutical market. Very few start-up companies and little funding with inadequate resources are major hurdles in the development process of nanofabricated projects. More tie-ups between academics and industries may help develop these products at their initial stage of product development [151,153].

Regulatory frameworks have more concerned about nanofabricated products' safety and toxicity. During the development stage, intermediates and sometimes finished products may harm the patients. So, it is vital to calculate the risk/benefit ratio while developing these products. Pre-clinical and human clinical trials are concerned with assessing nanofabricated outcomes [[154], [155], [156]].

7. Future prospective of nanofabrication techniques

In recent years, nanofabrication techniques have been vital for the fabrication of nano-formulations. Nanofabrication techniques manufacture different nanoformulations, including solid lipid nanoparticles, polymeric nanoparticles, nanostructured lipid carriers, microemulsions and liposomes. Nanofabrication techniques help to manufacture nano-products at a larger scale with better product characteristics and aid in improving the different properties of compounds. These nano-formulations are used to manage diseases like cancer, HIV, Alzheimer's disease, Parkinson's disease, psoriasis, rheumatoid arthritis etc. Future research may lead to the creation of multifunctional nanocarriers using various nanofabrication techniques that may treat multiple diseases simultaneously. The main objective of nanotechnology-based drug delivery systems is to enhance appropriate therapy, diagnostics, and monitoring while also lowering costs and improving the quality of life of patients, effectiveness and safety. In the future, nanofabrication techniques will manufacture different formulations beyond small molecules like vaccines, monoclonal antibodies or personalized therapies. Nanofabrication techniques will be adopted to improve compounds' loading, encapsulation, yield and stability compared to other methods. We anticipate many more nanofabrication techniques will emerge for developing novel drug delivery systems.

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.