# Polymeric Nanogels as Versatile Nanoplatforms for Biomedical Applications

Authors: Fakhara Sabir; Muhammad Imran Asad; Maimoona Qindeel; Iqra Afzal; Muhammad Junaid Dar; Kifayat Ullah Shah; Alam Zeb; Gul Majid Khan; Naveed Ahmed; Fakhar-ud Din
Journal: Journal of Nanomaterials
 (2019)

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## Abstract

Nanomaterials have found extensive biomedical applications in the past few years because of their small size, low molecular weight, larger surface area, enhanced biological, and chemical reactivity. Among these nanomaterials, nanogels (NGs) are promising drug delivery systems and are composed of cross-linked polymeric nanoparticles ranging from 100 to 200 nm. NGs represent an innovative zone of research with speedy developments taking place on a daily basis. An incredible amount of focus is placed on the fabrication of NGs with novel polymers to achieve better control over the drug release. This review article covers a number of aspects of NGs including their types, associated pros and cons, and methods of preparation along with technical and economical superiority and therapeutic efficacy over each other. The last part of review summarizes the applications of NGs in the drug delivery and treatment of various diseases including brain disease, cardiovascular diseases, oxidative stress, diabetes, cancer therapy, tissue engineering, gene therapy, inflammatory disorders, pain management, ophthalmic and autoimmune diseases, and their future challenges. NGs appear to be an outstanding nominee for drug delivery systems, and further study is required to explore their interactions at the cellular and molecular levels.


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## Body

## 1. Introduction

Appropriate drug delivery systems can be characterized by several factors including pharmacodynamic, pharmacokinetic, and physiochemical properties of the drug. Different carrier systems including hydrogels, nanogels (NGs), dendrimers, drug conjugates, and micelles have been used for several years for the effective delivery of drugs [1]. One of the most prominent and convenient systems among them is the hydrogel which could be attributed to its physiochemical and biological characteristics to achieve a site-specific delivery of incorporated drugs [2]. Previously, hydrogels with macro sizes were extensively used for various medical applications. However, with the advancement in nanotechnology, NGs were developed which are considered more suitable for optimum delivery at the target site due to their small size, ease of formulation, improved retention time, and swelling properties [3–6]. NGs are hydrogels with a submicron size range of 100-200 nm [7] or particles less than 200 nm composed of a cross-linked network of polymers via different functional groups such as carboxyl (COOH), hydroxyl (OH), amino (NH2), and sulphonic (HSO3) [8–10]. NGs are composed of physiochemical-bound natural and synthetic polymers [11], active ingredients, and solvents [12, 13]. NGs may consist of a charged or non–charged system of amphiphilic molecules. The drug loading of NGs requires a basic physiochemical interaction between functional groups of polymeric compounds and drug substance [14, 15].A nanosize regimen is designed to overcome some of the limitations of micron size particles, including surface area, site specificity, retention at targeting site, swelling behavior, drug loading, and release behavior. Ideally, NGs are considered as biocompatible, biodegradable, versatile, and safe from any kind of leakage [4, 16–18]. Size control is easy in NGs to induce active or passive delivery [19]. NGs are utilized in many medical situations including cancer and inflammatory conditions owing to their stimulus-responsive behavior. Under various diseased conditions (for example, cancer and inflammation), body functionality changes due to altering metabolic and/or physiological states. The conventional delivery systems are unable to respond to these minor physiological variations such as pH and temperature, thereby making it very challenging to display a proper drug release profile and therapeutic effects [17, 20, 21]. In such cases, NGs are very useful because their stimulus responsiveness increases several times leading to the required drug delivery at the targeted site for a desired therapeutic effect [22]. One of the many reasons for selecting NGs as a drug delivery system is their toxicity reduction through transdermal delivery of active pharmaceutical ingredients (API), an example of which includes aceclofenac-loaded NG [23]. This could also be attributed to the fact that NG mostly consists of polymers which are biodegradable and degraded into nontoxic metabolites. Furthermore, NG formulation for psoriatic skin is a new area where recently a number of trials are performed. For cancer treatment, a variety of polymeric NGs loaded with doxorubicin are reported earlier [24, 25].In this review, we discuss different aspects of NGs, their classification, their methods of preparation, and their advanced biomedical applications in various ailments including brain disorders, cardiac diseases, pain management, diabetes, tissue engineering, cancer treatment, gene therapy, and inflammatory disorders (Figure1). This review also highlights the advancement of nanotechnology in the field of NGs supported by the latest references from the literature.Figure 1
Advanced biomedical applications of NGs.


## 2. Types of NGs

NGs can be classified into different types on the basis of their structural properties including artificial chaperones, layer-by-layer NGs, functionalized NGs, core-shell NGs, and hollow NGs [26]. A comprehensive description of all the types is given below.


### 2.1. Artificial Chaperones

These are cross-linked, self-assembled particles with extensive applications in various fields of biomedicine [26]. They are used as a drug transporter and synthetic molecular chaperones. The representative diagram of artificial chaperones is given in Figure 2. Artificial chaperones are made up of cross-linked, bifunctional systems of polyion and anionic polymers for the transport of polynucleotides (cross-linked PEI-PEG or PEG-CL-PEI). One of the examples of artificial chaperones is the development of cholesterol-bearing pullulan (CHP) [27, 28] . They have multihydrophobic zones which can entrap hydrophobic drugs and proteins inside them. CHP has been majorly employed as a drug carrier, in particular for hydrophobic drugs [29–31]. Similarly, another class of artificial chaperones (polysaccharide-based hybrid NGs) was reported to offer extensive opportunities for diagnosis and therapy [32]. These NGs not only exhibited exceptional stability as a drug carrier for a model anticancer drug temozolomide but also offered a pH-triggered sustained release of drug molecules from the gel network [33]. Furthermore, in comparison to the liposome-loaded quantum dots (QDs), NG-bearing QDs have improved the capacity for imaging utilizing a lesser quantity [33, 34].Figure 2
Types of NG formulations.


### 2.2. Layer-by-Layer NGs

These are cross-linked, stimulus-responsive NGs and are also known as multilayer NGs (Figure2). These multilayers are formulated in different dimensions to expose their efficiency as a best carrier for stimulus responsiveness. Different templates are used such as rigid particles, microgels, and NGs. NG is the most successful approach, and unlike the others, it does not lead to deformability and deposition at the site of action or inside the body [35]. Initially, a single particle light scattering technique was used for formulating multilayer NGs. However, this technique is not suitable for thermosensitive microgels because of their soft, porous, and solvent-penetrable polymeric networks, characterized by the alleged volume phase transition from an engorged to a collapsed state during heating. Various scanning techniques are used for evaluating the thickness of layer-by-layer assembly such as confocal laser electron microscopy, dynamic light scattering (DLS), and fluorescence correlation spectroscopy [36–38].


### 2.3. Functionalized NGs

These types of NGs are a cross-linked water-soluble polymeric nanoparticle network that is formulated to overcome the stability issues associated with layer-by-layer NGs (Figure2). These are extensively used NGs; however, their formulation methods are very complicated and require high purification at every step [35], including the microemulsion or inverse microemulsion technique. For instance, water-soluble polymeric nanoparticles are incorporated in NGs using the inverse microemulsion method. In this regard, surface modification of layer-by-layer NGs is performed through cross-linking, covalent coupling, and physical, thermal, or chemical posttreatments [39]. This method of formation of functionalized NGs has advantages over other methods including its single-step process without the use of external cross-linkers, fast cross-linking reactions, no undesirable side products, and covalent grafting of active molecules (functional groups) to the surface. To formulate a stimulus-sensitive functional group, disulfide bonds are selected as these bonds are more sensitive to bioreductive agents such as glutathione (GSH) and thioredoxin. Additionally, thiol-containing functional groups, if added on polymers, result in higher reactivity which could be attributed to the functionalized property of pyridyl disulfide (PDS) bond of the thiol group as compared to several other disulfide functionalities. Therefore, nanoparticle-based NGs can easily be functionalized leading to their therapeutic application in control release formulations [4, 40].


### 2.4. Core-Shell NGs

These are cross-linked stimulus-sensitive NGs made up of polymers with different sensitivities and consisting of core and shell compartments. Core-shell NGs consist of two regions which are chemically coupled with one another (Figure2). This coupling or cross-linking also affects their stimulus-responsive property and makes them different from other branched polymers. Backfolding of cross-link chains is not possible in core-shell NGs. There are many stimuli to which core-shell NGs are sensitive including temperature, pressure, and pH [41, 42]. The formulation of core-shell NGs is a very critical process depending upon several parameters such as size, core-shell density, and inclusion of functional groups in core-shell compartments. Various methods are used for the preparation of core-shell NGs such as precipitation polymerization, batch polymerization, and seed polymerization. NGs coated with different nanoparticles such as gold nanoshells and gold nanorods are available in the market and are applied in different temperature-sensitive therapies [43, 44]. Amphoteric core-shell NGs provide important information relevant to specific properties of core-shell NG. An evaluation of the internal structure of NGs can be done through theoretical modeling [45].


### 2.5. Hairy NGs

Hairy NGs consists of a dual structure having a core and a shell. The shell is composed of linear polymeric chains with high dispersibility (Figure2). The core of hairy NGs consists of inorganic or polymeric material [46]. These nanomaterials respond to various stimuli including temperature, pH, light, and enzymes. Among all the other types of NGs, thermosensitive hairy NGs are of great importance because of their very small size and stimulus responsiveness [47].Different methods of preparation of hairy NGs are currently in use. One of them is grafting onto the process, but particles formed through this process have a high density. To address this issue, the controlled radical polymerization method is used, which provides various advantages on the formation of hairy NGs. Another method is the two-pot synthesis method which is generally and specifically used for hairy particles. The process runs over two parts: firstly synthesis, isolation, and purification of NG particles and secondly the synthesis of hairs or grafted straight chains over the particle surface [35]. The most recently developed method is the one-pot synthetic route in which NGs are synthesized by copolymerization of the monovinyl monomer and divinyl cross-linker. The advantage of one-pot synthesis is its convenience and the lack of the need of purification of the intermediates. The size of hairy NGs can be adjusted by changing the concentration of monomers. Thus, synthesis of hairy NG through the one-pot method is more advantageous among others [47, 48].


### 2.6. Hollow NGs

Hollow NGs are fabricated by temperature-sensitive polymers that are predominantly favorable constituents. The stimulus sensitivity, large size, composite covering, thickness and permeability, large storage capability, and release pattern describe their main features [39]. Therefore, the finding and regulation of all these features are of utmost significance for the preparation of hollow NGs. Hollow NGs with considerably cross-linked shells depict discrete temperature sensitivity but retain virtually no void (14% of the initial core volume) and therefore hardly become hollow. NGs with a rigid shell (smaller void as compared to the core size of the template) are certainly hollow but have low-temperature sensitivity [49].One of the many advantages of hollow NGs is their improved drug loading, which could be attributed to their greater storage volume (Figure2). Various methods used for their preparation include layer-by-layer assembly, self-assembly of lipids or block copolymers, template method, and ultrasonic fabrication [35]. However, loading capacities of these hollow nanoparticles may not significantly be enhanced as expected. To encounter this issue, hollow NGs can be synthesized with mesoporous channels penetrating from the shell to the hollow inner core. Hollow NGs prepared through this method have easy fabrication in the aqueous phase without any inclusion of the organic phase [50]. Hollow NGs can be formulated as a dual stimulus-responsive carrier from the continuous association of two graft copolymers into polymersomes [51].


## 2.1. Artificial Chaperones

These are cross-linked, self-assembled particles with extensive applications in various fields of biomedicine [26]. They are used as a drug transporter and synthetic molecular chaperones. The representative diagram of artificial chaperones is given in Figure 2. Artificial chaperones are made up of cross-linked, bifunctional systems of polyion and anionic polymers for the transport of polynucleotides (cross-linked PEI-PEG or PEG-CL-PEI). One of the examples of artificial chaperones is the development of cholesterol-bearing pullulan (CHP) [27, 28] . They have multihydrophobic zones which can entrap hydrophobic drugs and proteins inside them. CHP has been majorly employed as a drug carrier, in particular for hydrophobic drugs [29–31]. Similarly, another class of artificial chaperones (polysaccharide-based hybrid NGs) was reported to offer extensive opportunities for diagnosis and therapy [32]. These NGs not only exhibited exceptional stability as a drug carrier for a model anticancer drug temozolomide but also offered a pH-triggered sustained release of drug molecules from the gel network [33]. Furthermore, in comparison to the liposome-loaded quantum dots (QDs), NG-bearing QDs have improved the capacity for imaging utilizing a lesser quantity [33, 34].Figure 2
Types of NG formulations.


## 2.2. Layer-by-Layer NGs

These are cross-linked, stimulus-responsive NGs and are also known as multilayer NGs (Figure2). These multilayers are formulated in different dimensions to expose their efficiency as a best carrier for stimulus responsiveness. Different templates are used such as rigid particles, microgels, and NGs. NG is the most successful approach, and unlike the others, it does not lead to deformability and deposition at the site of action or inside the body [35]. Initially, a single particle light scattering technique was used for formulating multilayer NGs. However, this technique is not suitable for thermosensitive microgels because of their soft, porous, and solvent-penetrable polymeric networks, characterized by the alleged volume phase transition from an engorged to a collapsed state during heating. Various scanning techniques are used for evaluating the thickness of layer-by-layer assembly such as confocal laser electron microscopy, dynamic light scattering (DLS), and fluorescence correlation spectroscopy [36–38].


## 2.3. Functionalized NGs

These types of NGs are a cross-linked water-soluble polymeric nanoparticle network that is formulated to overcome the stability issues associated with layer-by-layer NGs (Figure2). These are extensively used NGs; however, their formulation methods are very complicated and require high purification at every step [35], including the microemulsion or inverse microemulsion technique. For instance, water-soluble polymeric nanoparticles are incorporated in NGs using the inverse microemulsion method. In this regard, surface modification of layer-by-layer NGs is performed through cross-linking, covalent coupling, and physical, thermal, or chemical posttreatments [39]. This method of formation of functionalized NGs has advantages over other methods including its single-step process without the use of external cross-linkers, fast cross-linking reactions, no undesirable side products, and covalent grafting of active molecules (functional groups) to the surface. To formulate a stimulus-sensitive functional group, disulfide bonds are selected as these bonds are more sensitive to bioreductive agents such as glutathione (GSH) and thioredoxin. Additionally, thiol-containing functional groups, if added on polymers, result in higher reactivity which could be attributed to the functionalized property of pyridyl disulfide (PDS) bond of the thiol group as compared to several other disulfide functionalities. Therefore, nanoparticle-based NGs can easily be functionalized leading to their therapeutic application in control release formulations [4, 40].


## 2.4. Core-Shell NGs

These are cross-linked stimulus-sensitive NGs made up of polymers with different sensitivities and consisting of core and shell compartments. Core-shell NGs consist of two regions which are chemically coupled with one another (Figure2). This coupling or cross-linking also affects their stimulus-responsive property and makes them different from other branched polymers. Backfolding of cross-link chains is not possible in core-shell NGs. There are many stimuli to which core-shell NGs are sensitive including temperature, pressure, and pH [41, 42]. The formulation of core-shell NGs is a very critical process depending upon several parameters such as size, core-shell density, and inclusion of functional groups in core-shell compartments. Various methods are used for the preparation of core-shell NGs such as precipitation polymerization, batch polymerization, and seed polymerization. NGs coated with different nanoparticles such as gold nanoshells and gold nanorods are available in the market and are applied in different temperature-sensitive therapies [43, 44]. Amphoteric core-shell NGs provide important information relevant to specific properties of core-shell NG. An evaluation of the internal structure of NGs can be done through theoretical modeling [45].


## 2.5. Hairy NGs

Hairy NGs consists of a dual structure having a core and a shell. The shell is composed of linear polymeric chains with high dispersibility (Figure2). The core of hairy NGs consists of inorganic or polymeric material [46]. These nanomaterials respond to various stimuli including temperature, pH, light, and enzymes. Among all the other types of NGs, thermosensitive hairy NGs are of great importance because of their very small size and stimulus responsiveness [47].Different methods of preparation of hairy NGs are currently in use. One of them is grafting onto the process, but particles formed through this process have a high density. To address this issue, the controlled radical polymerization method is used, which provides various advantages on the formation of hairy NGs. Another method is the two-pot synthesis method which is generally and specifically used for hairy particles. The process runs over two parts: firstly synthesis, isolation, and purification of NG particles and secondly the synthesis of hairs or grafted straight chains over the particle surface [35]. The most recently developed method is the one-pot synthetic route in which NGs are synthesized by copolymerization of the monovinyl monomer and divinyl cross-linker. The advantage of one-pot synthesis is its convenience and the lack of the need of purification of the intermediates. The size of hairy NGs can be adjusted by changing the concentration of monomers. Thus, synthesis of hairy NG through the one-pot method is more advantageous among others [47, 48].


## 2.6. Hollow NGs

Hollow NGs are fabricated by temperature-sensitive polymers that are predominantly favorable constituents. The stimulus sensitivity, large size, composite covering, thickness and permeability, large storage capability, and release pattern describe their main features [39]. Therefore, the finding and regulation of all these features are of utmost significance for the preparation of hollow NGs. Hollow NGs with considerably cross-linked shells depict discrete temperature sensitivity but retain virtually no void (14% of the initial core volume) and therefore hardly become hollow. NGs with a rigid shell (smaller void as compared to the core size of the template) are certainly hollow but have low-temperature sensitivity [49].One of the many advantages of hollow NGs is their improved drug loading, which could be attributed to their greater storage volume (Figure2). Various methods used for their preparation include layer-by-layer assembly, self-assembly of lipids or block copolymers, template method, and ultrasonic fabrication [35]. However, loading capacities of these hollow nanoparticles may not significantly be enhanced as expected. To encounter this issue, hollow NGs can be synthesized with mesoporous channels penetrating from the shell to the hollow inner core. Hollow NGs prepared through this method have easy fabrication in the aqueous phase without any inclusion of the organic phase [50]. Hollow NGs can be formulated as a dual stimulus-responsive carrier from the continuous association of two graft copolymers into polymersomes [51].


## 3. Synthesis of NGs

Various synthesis techniques are used for the development of NGs, depending upon their intended pharmacologic effect, desired characteristics, and quantity of the final dosage form. A descriptive detail of all the techniques is given below.


### 3.1. Polymerization of Monomers in a Homogeneous Phase or in a Microscale or Nanoscale Heterogeneous Environment

Uniform nucleation of the water-soluble monomer results in the formation of colloidal suspension of the polymer. This in turn is used to prepare stable NGs. This method is of great importance in cases where particle size control is of prime importance because particle size has a prominent role in the stability of colloidal formulations. This particle size control is accomplished by the use of the ionic surfactant, and there is an inverse relationship between particle size and surfactant concentration [52]. This method was utilized by Donini and coworkers for lipophilic and temperature-resistant drugs [53]. Similarly, Luisi and Straub reported copolymerization of monomers in reverse micelles for the entrapment of hydrophilic drug molecules [54].


### 3.2. Physical Self-Assembly of Interactive Polymers

In this technique, hydrogen bonding and van der Waals forces result in an interaction between drug moiety and solvent [55]. During the self-assembling process of NGs, micro- and macromolecules are captured inside them. This method is used to prepare protein-loaded NGs by the self-association of water-soluble polymers. Akiyoshi et al. developed insulin-loaded NGs using this technique. The particle size of NGs prepared with this technique had a particle size below 30 nm; however, it was dependent upon polymer concentration and various environmental factors including pH, temperature, and ionic strength. In a study, NGs with a particle size (120–150 nm) and enhanced stability were reported, using various ratios of two different polymers [30]. Furthermore, the reversible addition–fragmentation chain transfer (RAFT) technique [56] was used to make amphiphilic block copolymers which in turn were used to make NGs. The RAFT technique is a one-step production of PEGylated poly(N,N′-dimethylaminomethyl methacrylate) NG utilizing an amphiphilic trithiocarbonate which is a macro-RAFT agent along with hydrophobic (dodecyl) chain-assisting polymerization. Owing to the production of small particles, this technique is most suitable for the delivery of genes [57, 58]. The micellar behavior of amphiphilic block copolymers can be improved by alternating the temperature conditions and adding solvents [59, 60]. Similarly, a superficial behavior of NGs for site-specific targeting and their loading capacity can be enhanced.


### 3.3. Cross-Linking of Preformed Polymers

In this technique, oil in water emulsion followed by removal of solvent is used to prepare large-sized NGs [61]. Branched PEG (thiol-functionalized) and dimethyl sulfoxide containing DNA are mixed to obtain cross-linked NGs having a DNA by utilizing the oxidation process [62]. NGs obtained via this method are rod-shaped, spherical, and toroid-shaped. This method is suitable to controlling certain parameters like size and shape of the particles, as well as composition and surface properties of the NGs.


### 3.4. Novel Photochemical Approach

In the photochemical method, NGs are manufactured in an interlayer quartz flask (150 mL) furnished with a stirrer and a nitrogen gas inlet. A precise quantity of nanoparticles (usually 10 mg) is mixed with 60 mL deionized water containing 186 mg monomer. This mixture is stirred for 10 min followed by addition of 0.8 mL of 1 wt% cross-linker. Further, it is exposed to ultraviolet (UV) irradiation for 25 min. N2 is effervesced throughout the preparation procedure. The NGs are collected, washed many times with distilled water, and redispersed in distilled water for further use [63]. This method was used to prepare amino-functionalized magnetic NGs of coated ferric oxide nanoparticles using N-(2-aminoethyl)methylacrylamide and N,N′-methylene-bis-acrylamide for their application as an MRI contrast agent [64]. Likewise, DNA-loaded diacrylated Pluronic and glycidyl methyacrylated chito-oligosaccharide NGs were prepared by using UV light at a wavelength of 365 nm along with a photoinitiator [65]. These NGs were formulated to improve injectable deposition schemes for gene therapy which results in the enhanced indigenous transgene expression at injection sites.Photochemical internalization along with siRNA NGs is also used for the prolonged gene silencing. Basically, various nonviral siRNA carriers get attached to the endosomal layers resulting in limited gene silencing. However, photosensitizer (meso-tetraphenylporphine disulfonate) is used during formulation which is responsible for the rupture of the endosomal membrane leading to the release of genes into the cytoplasm, thereby improving the intracellular bioavailability of siRNA [66].


### 3.5. Novel Pullulan Chemistry Modification

In this method, chemical modification of pullulan is done. Cholesterol-based pullulan (CHP) NGs are prepared by using a combination of cholesterol in DMSO and pyridine. Modification is done by replacing 1.4 moieties of cholesterol per 100 glucoside units. Freeze-drying is a prerequisite for the formulations prepared via this technique [67]. The CHP-based technique behaves as an efficient carrier for protein NG formulations. Modification of the CHP method is also done by Michael addition reaction in which PEG replaces the acrylate and thiol groups [68]. Changes at the 1.1 unit of cholesteryl per 100 glucose units make it favorable to interact with the AB monomer as well as monomers responsible for the treatment of diseases like Alzheimer [65]. When modification is made by using 1.6 units of glucose, pullulan suitable for targeting folate receptors is developed. NGs are formed when pullulan and the photosensitizer are conjugated with carbodiimide followed by dialysis. Such types of NGs are effectively used in cancer treatment [69]. For example, acetylated chondroitin sulfate augments the discharge of doxorubicin in HeLa cells for three weeks, which is very helpful in cancer therapy [70]. Similarly, the release profile and absorption of methotrexate are changed by saturation of butyl acrylate (BA) and N-isopropylacrylamide with sodium carbonate, changing the absorption and release profile of methotrexate [71]. Additionally, pH-modified hydroxypropyl methylcellulose- (HPMC-) polyacrylic acid is made by eliminating cadmium ions and polyacrylic acid and is exercised for bioimaging by detecting the physicochemical surrounding [72].


## 3.1. Polymerization of Monomers in a Homogeneous Phase or in a Microscale or Nanoscale Heterogeneous Environment

Uniform nucleation of the water-soluble monomer results in the formation of colloidal suspension of the polymer. This in turn is used to prepare stable NGs. This method is of great importance in cases where particle size control is of prime importance because particle size has a prominent role in the stability of colloidal formulations. This particle size control is accomplished by the use of the ionic surfactant, and there is an inverse relationship between particle size and surfactant concentration [52]. This method was utilized by Donini and coworkers for lipophilic and temperature-resistant drugs [53]. Similarly, Luisi and Straub reported copolymerization of monomers in reverse micelles for the entrapment of hydrophilic drug molecules [54].


## 3.2. Physical Self-Assembly of Interactive Polymers

In this technique, hydrogen bonding and van der Waals forces result in an interaction between drug moiety and solvent [55]. During the self-assembling process of NGs, micro- and macromolecules are captured inside them. This method is used to prepare protein-loaded NGs by the self-association of water-soluble polymers. Akiyoshi et al. developed insulin-loaded NGs using this technique. The particle size of NGs prepared with this technique had a particle size below 30 nm; however, it was dependent upon polymer concentration and various environmental factors including pH, temperature, and ionic strength. In a study, NGs with a particle size (120–150 nm) and enhanced stability were reported, using various ratios of two different polymers [30]. Furthermore, the reversible addition–fragmentation chain transfer (RAFT) technique [56] was used to make amphiphilic block copolymers which in turn were used to make NGs. The RAFT technique is a one-step production of PEGylated poly(N,N′-dimethylaminomethyl methacrylate) NG utilizing an amphiphilic trithiocarbonate which is a macro-RAFT agent along with hydrophobic (dodecyl) chain-assisting polymerization. Owing to the production of small particles, this technique is most suitable for the delivery of genes [57, 58]. The micellar behavior of amphiphilic block copolymers can be improved by alternating the temperature conditions and adding solvents [59, 60]. Similarly, a superficial behavior of NGs for site-specific targeting and their loading capacity can be enhanced.


## 3.3. Cross-Linking of Preformed Polymers

In this technique, oil in water emulsion followed by removal of solvent is used to prepare large-sized NGs [61]. Branched PEG (thiol-functionalized) and dimethyl sulfoxide containing DNA are mixed to obtain cross-linked NGs having a DNA by utilizing the oxidation process [62]. NGs obtained via this method are rod-shaped, spherical, and toroid-shaped. This method is suitable to controlling certain parameters like size and shape of the particles, as well as composition and surface properties of the NGs.


## 3.4. Novel Photochemical Approach

In the photochemical method, NGs are manufactured in an interlayer quartz flask (150 mL) furnished with a stirrer and a nitrogen gas inlet. A precise quantity of nanoparticles (usually 10 mg) is mixed with 60 mL deionized water containing 186 mg monomer. This mixture is stirred for 10 min followed by addition of 0.8 mL of 1 wt% cross-linker. Further, it is exposed to ultraviolet (UV) irradiation for 25 min. N2 is effervesced throughout the preparation procedure. The NGs are collected, washed many times with distilled water, and redispersed in distilled water for further use [63]. This method was used to prepare amino-functionalized magnetic NGs of coated ferric oxide nanoparticles using N-(2-aminoethyl)methylacrylamide and N,N′-methylene-bis-acrylamide for their application as an MRI contrast agent [64]. Likewise, DNA-loaded diacrylated Pluronic and glycidyl methyacrylated chito-oligosaccharide NGs were prepared by using UV light at a wavelength of 365 nm along with a photoinitiator [65]. These NGs were formulated to improve injectable deposition schemes for gene therapy which results in the enhanced indigenous transgene expression at injection sites.Photochemical internalization along with siRNA NGs is also used for the prolonged gene silencing. Basically, various nonviral siRNA carriers get attached to the endosomal layers resulting in limited gene silencing. However, photosensitizer (meso-tetraphenylporphine disulfonate) is used during formulation which is responsible for the rupture of the endosomal membrane leading to the release of genes into the cytoplasm, thereby improving the intracellular bioavailability of siRNA [66].


## 3.5. Novel Pullulan Chemistry Modification

In this method, chemical modification of pullulan is done. Cholesterol-based pullulan (CHP) NGs are prepared by using a combination of cholesterol in DMSO and pyridine. Modification is done by replacing 1.4 moieties of cholesterol per 100 glucoside units. Freeze-drying is a prerequisite for the formulations prepared via this technique [67]. The CHP-based technique behaves as an efficient carrier for protein NG formulations. Modification of the CHP method is also done by Michael addition reaction in which PEG replaces the acrylate and thiol groups [68]. Changes at the 1.1 unit of cholesteryl per 100 glucose units make it favorable to interact with the AB monomer as well as monomers responsible for the treatment of diseases like Alzheimer [65]. When modification is made by using 1.6 units of glucose, pullulan suitable for targeting folate receptors is developed. NGs are formed when pullulan and the photosensitizer are conjugated with carbodiimide followed by dialysis. Such types of NGs are effectively used in cancer treatment [69]. For example, acetylated chondroitin sulfate augments the discharge of doxorubicin in HeLa cells for three weeks, which is very helpful in cancer therapy [70]. Similarly, the release profile and absorption of methotrexate are changed by saturation of butyl acrylate (BA) and N-isopropylacrylamide with sodium carbonate, changing the absorption and release profile of methotrexate [71]. Additionally, pH-modified hydroxypropyl methylcellulose- (HPMC-) polyacrylic acid is made by eliminating cadmium ions and polyacrylic acid and is exercised for bioimaging by detecting the physicochemical surrounding [72].


## 4. Biomedical Applications of NGs

### 4.1. Brain Diseases

Various nanoplatforms are recently utilized for the treatment of brain diseases including Alzheimer’s disease (AD), depression, migraine, and schizophrenia. NGs are one of those nanodecorated drug delivery systems. Their efficacy in brain diseases is because of their improved therapeutic effects, better mechanism of targeting, and biological efficiency. AD is the irretrievable neurodegenerative illness leading to progressive loss of memory and intellectual abilities [73]. Although various pathological conditions are believed as the possible reasons of AD, amyloid hypothesis is, however, widely accepted in this regard [74]. The anomalous buildup, accretion, and accumulation of amyloid β-protein (Aβ) lead to the cerebral extracellular amyloid blockade that causes neurotoxicity [75, 76]. Therefore, avoiding Aβ accumulation is believed as a favorable approach in the management of AD. For this purpose, NGs with a double inhibitor-modified hyaluronic acid function were fabricated with inhibiting capabilities of Aβ accumulation, resulting in the management of AD [77]. Likewise, Elnaggar et al. assimilated piperine, a phytopharmaceutical agent in NGs for its neuroprotective effect in AD [78].Similarly, NGs are reported to deliver olanzapine for the treatment of schizophrenia which is a brain disorder described by delusions and disordered behavior [79]. These NGs exhibited excellent entrapment and enhanced bioavailability. Moreover, Hu et al. prepared lidocaine hydrochloride-loaded NGs, for the management of migraine [80]. Lidocaine hydrochloride is a commonly used drug in the treatment of migraine; however, after incorporation in the NG, the drug showed better bioavailability with no toxicity. Furthermore, Dange et al. reported the development of venlafaxine-loaded NG for the treatment of depression [81]. This NG showed a quick onset of action with extended period of time as compared with the drug solution.


### 4.2. Cardiovascular Diseases

Cardiovascular diseases like myocardial infarction (MI) and heart failure are the main reason of human deaths globally [82]. Various strategies are adopted to treat these diseases including tissue engineering and stem cell transplantation [83, 84]. Among the different drug delivery systems, injectable NGs have been used to treat MI. These NGs have confirmed the improvement in cardiac condition via LaPlace’s Law, an act which is exhibited by increased wall thickness and reduced wall stress [85]. One such study reported the heart restoration using NG-encapsulated human cardiac stem cells in mice and pigs with MI [86]. This study concluded that synthetic porous NGs act as a promising cell carrier for allogeneic/xenogeneic cell rehabilitations. Most particularly, these NGs inhibit the entry of immune cells while promoting the regenerative capabilities of the heart.Another study demonstrated the development of thermoresponsive NGs to produce cell mass fragments for the treatment of ischemic diseases. Owing to their temperature-dependent behavior, the cell bodies are produced without proteolytic enzymes. The animal studies further exhibited the adherence of cell mass fragments with engraftment sites which in turn enhance the vascular density, hence treating the diseased condition of an infarcted heart [87].


### 4.3. Treatment of Oxidative Stress

Oxidative stress is a diseased condition in which the increased production of oxidants including hydroxyl radicals, singlet oxygen, and hydrogen peroxide lead to cellular disability. This increased level of oxidants may be produced by endogenous and exogenous sources which may result in development of many diseases including cardiovascular diseases, Parkinson’s disease, and acute renal failure [88, 89]. Various drug delivery systems are utilized to treat the oxidative stress; however, NGs are considered as reliable drug delivery vehicles in this regard [90–92]. For instance, quercetin-encapsulated poly(b-amino esters) NGs were developed for the treatment of cellular oxidative stress [93]. NGs with a size range at the nanoscale were developed with 25–38 drug wt% and constant drug release over a period of 45–48 h. These NGs demonstrated an antioxidant activity of the drug for a prolonged time period. Another study exhibited the development of ferulic acid-loaded NG with improved penetrability and augmented antioxidant activity in rats for the treatment of oxidative stress. This NG exhibited excellent stability and sustained release of the drug with outstanding antioxidant activity which could be attributed to the increase solubility of the drug and augmented permeability from the NG [94].


### 4.4. Diabetes Management

Diabetes, a very prevailing chronic disease around the globe, has grabbed the attention of scientists, and new ways of its management are reported. Recently, a new improved therapeutic regimen, noninvasive glucose checking techniques, and new methods of insulin administration have been reported [95, 96]. In this aspect, the preparation of glucose-sensitive NGs has addressed the major hurdles linked with diabetes management. Most particularly, these NGs exhibited sustained release of the insulin by glucose-dependent swelling and shrinking mechanisms [97, 98].


### 4.5. Cancer Therapy

Various anticancer drugs, e.g., doxorubicin, cisplatin, 5-fluorouracil, and temozolomide, can be incorporated in NGs for the treatment of cancer. Temperature- and pH-sensitive hydrogels of doxorubicin based on maleic acid poly-(N-isopropyl acrylamide) polymer were used in cancer therapy in which doxorubicin release was dependent on temperature and pH. Chitin-based NG of doxorubicin can be used for various types of cancers including lungs, breast, liver, and prostate cancer [99]. Similarly, photosensitizer agent chlorin e6 has been recently used for the photodynamic therapy of cancer using chitosan-based NGs [100]. A descriptive detail of the anticancer applications of NGs is given in Table 1.Table 1
Anticancer applications of NGs.
NG composition
Type of NG
Drug used
Method of preparation
Results and applications
References
PVA (polyvinyl alcohol)
Charge conversional and reduction-sensitive NG
Doxorubicin
Inverse nanoprecipitation
Better cell toxicity.Improved targeted intracellular drug release.
[114]
Dextrin with formaldehyde as a cross-linker
pH-sensitive NG
Doxorubicin
Emulsion cross-linking method
Efficacious antitumor activityIt is an important candidate for the treatment of colorectal cancer.
[104]
Poly(ethylene glycol)-b-poly(L-glutamic acid) (PEG-b-PGA)
Polypeptide-based NG
17-AAGDoxorubicin
Cross-linking method
Improved anticancer activityEffective cytotoxicity in a breast cancer cell panel
[33, 115]
P(N-isopropyl-acrylamide-co-butyl methacrylates)
Temperature-sensitive NG dispersion
Doxorubicin
Emulsion polymerization method
Improved efficacy for transarterial chemoembolization (TACE) of iohexol dispersion (IBi-D) was observed on rabbit VX2 liver tumors.
[116]
Poly (N-isopropylmethacrylamide) (PNiPMA), PDA-PEG, 4-methoxybenzoic acid (MBA)
pH, thermal, and redox potential triple-responsive expansile NG (TRN)
Pc 4
Targeted delivery of pc 4 to sigma 2 receptors in head and neck tumors.
[117]
Glycol chitosan (GC) conjugated with 2,3-dimethylmaleic acid (dma) and fullerene (C60) conjugate (GC-g-DMA-g-C60)
Acid pH-responsive NG
Photosensitizer drug
Two-step chemical grafting reaction
Beneficial to target endosomes and in vivo photodynamic therapy in different types of malignant tumors.
[118]
Dextrin with glyoxal as a cross-linker
pH-sensitive NG
Doxorubicin
Emulsion cross-linking method
Rapid release effective internalization of doxorubicin.Reduced side effects to cardiomyocytes and stem cells.
[119]
Chitin poly (L-lactic acid)
pH-responsive composite NG
Doxorubicin
Blood compatibility of the system was confirmed byin vitro coagulation assay and hemolytic assay.Effective for the treatment of liver cancer.
[120]
Chitin
pH-sensitive NG
5-Fluorouracil
Controlled regeneration chemistry method
Loosening of the epidermis after its interaction with negatively charged chitin with no inflammation.Important drug carriers for skin cancer therapy.
[121]
Folic acid conjugated poly(ethylene oxide)-b-poly(methacrylic acid)
Ligand-gated polyelectrolyte NG
Cisplatin
Cross-linking method
In vivo anticancer effect strengthens their use for the treatment of ovarian cancer.
[122]
Acetylated chondroitin sulfate (CS)
Self-organizing NG
Doxorubicin
Dialysis method
Drug was internalized into the cytoplasm through endocytosis.Effective drug carrier for anticancer therapy.
[70]
N-Isopropylacrylamide (NIPAM), poly(ethylene glycol) (PEG), poly(ethylene glycol) methyl ether methacrylate (mPEGMA)
pH-thermal dual-responsive NG
Cisplatin (CDDP)
Emulsion polymerization method
Extended circulation time.Reduced side effectsBetter antitumor activity for the treatment of breast cancer.
[123]
In situ immobilization of CdSe quantum dots in interior of hydroxypropyl cellulose poly(acrylic acid) (HPC-PAA)
pH and temperature-responsive NG
Temozolomide
Polymerization method
High drug loading, better stability, and pH-dependent sustained release.Used in cell imaging and optical pH sensing.
[32]


### 4.6. Tissue Engineering and Gene Therapy

NG-based formulations are widely used for tissue engineering and gene therapy. They are also used to deliver enzymes, genes, and proteins at a targeted site to achieve their intended effects. Artificial chaperons are usually utilized to modify polymers to carry enzymes and proteins. Similarly, pullulan is chemically modified by conjugating cholesterol moieties, and the functionalized molecules are self-assembled in water to develop NGs of up to 30 nm size. These NGs have an extraordinary biocompatibility which is utilized for bone regeneration [101, 102]. The properties usually depend on their size and density of NGs which alternatively depend upon the degree of substitution of the cholesterol fragments in NGs. Some of the NG formulations used in transport of enzymes, genes, and proteins are as follows (Table 2).Table 2
Applications of NG tissue engineering and gene therapy.
NG composition
Type of NG
Drug/agent used
Method of preparation
Results and applications
References
CHOPA-PEGSH
Hybrid NG
W9 peptide
Cross-linking
Bone repair, sustained release
[124]
Pullulan-collagen; 1,2,7,8-diepoxyoctane
PHD hybrid NG
1,2,7,8-Diepoxyoctane
Cross-linking
Tissue filler materials
[125]
Dendritic polyglycerol (dPG) and low-molecular-weight polyethylenimine
pH-sensitive NG
siRNA
Thiol-Michael nanoprecipitation method
In vitro gene silencing.Gene therapy
[126]
Chitosan–myristic acidNG (CMA)
Cross-linked NG
Aryldialkylphosphatase
“Self-assembly via chemical modification” method
Enhanced pH and thermal stabilityUsed for detoxification of paraoxon
[127]
Poly(N-isopropylacrylamide)-polyglycerol
Thermoresponsive NG
Biomacromolecules
Enhanced stability and release of proteinEffective for the topical delivery of biomacromolecules
[128]
Poly(N-vinyl pyrrolidone) (PVP)
Functionalized NG
Oligonucleotides (ODN)
Cross-linking and polymerization
Negligible cytotoxicityBypass cellular membranesEffective nanocarriers for gene delivery
[129]
Polyethyleneimine (PEI)
Microenvironment-responsive functional NG
Gene
Cross-linking and polymerization
Reduced cytotoxicityEnhanced transfection efficiencyPotential gene therapy
[106]
Poly(2-methacryloyloxyethyl phosphorylcholine), poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM)) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA))
Thermosensitive NG
Protein
Reversible addition−fragmentation chain transfer [56] and polymerization technique
Temperature-sensitive controlled release of proteins from biodegradable NG
[130]
Enzymatically synthesized glycogen (ESG) with cholesterol group
Artificial chaperon
Hydrophobic modification self-assembly method
Enhanced thermal stability of enzymeUsed for biomedical and protein engineering
[131]
Cholesteryl group-bearing pullulan (CHP) complexed with methyl-b-cyclodextrin (M-b-CD)
Artificial chaperon
Protein synthesis was not affected.Help in folding of active proteins.
[132]


### 4.7. Inflammatory Disorders

NGs are considered as important delivery systems for various anti-inflammatory agents. For instance, siRNA-loaded NGs were prepared by polymerization and chemical cross-linking. Structurally, it was polymethacrylic acid-co-N-vinyl-2-pyrrolidone (P[MAA-co-NVP]) cross-linked with a trypsin-degradable peptide linker. A maximum amount of drug was released in the intestinal environment due to its pH and enzyme sensitivity and hence proved to be a suitable candidate for the treatment of inflammatory bowel disease [103]. Similarly, two anti-inflammatory drugs spantide II and ketoprofen were loaded with (HPMC) and Carbopol-based NGs to achieve enhanced percutaneous delivery for the treatment of skin inflammation [104]. Additionally, anti-TNFα agent etanercept (ETR) was recently loaded with thermoresponsive NGs which not only resulted in effective delivery but showed enhanced anti-inflammatory responses [105].The NGs enhanced their ability to get deposited in skin’s epidermis and dermis for the therapy of topical inflammatory diseases. They are prepared by either solvent evaporation or emulsification method [106]. Photosensitizers tetraphenylporphyrin tetrasulfonate (TPPS4), tetra-phenyl-chlorin-tetra-carboxylate (TPCC4), and chlorin e6 (Ce6) have a hyaluronate ligand-gated chitosan with tripolyphosphate (TPP) as a cross-linker in their structure. Their potential for extending the retention time and reducing clearance from the inflamed joints enlists NGs as real contenders for the selective delivery of photosensitizers to macrophages. Ionic gelation is the method applied in their preparation [107]. Activated NGs of methotrexate have copolymerized N-isopropylacrylamide (NIPAM) and BA (poly(NIPAM-co-BA)) polymers in its composition. It is synthesized by the emulsion polymerization method. Their advantages include amplified release, elevated concentration gradient, building flux of methotrexate along with depressing PGE2 production, and hence effective anti-inflammatory effects [71, 108].


### 4.8. Pain Management

NGs have been successfully used for the local distribution of anesthetic medications for the pain management. They result in prolonged and sustained release of the incorporated drug [109]. Moreover, they resulted in lower cytotoxicity and enhanced drug uptake [110]. A detailed description of NGs as a local anesthetic drug delivery system is given in Table 3.Table 3
NGs for the management of pain.
NG composition
Type of NG
Drug/agent used
Method of preparation
Results and applications
References
NIPAAM, MAA
Magnetic NG
Bupivacaine
Free radical emulsion polymerization method
Rapid release at low temperature and pHEffective for the treatment of ankle block
[133]
Pluronic F127, hyaluronic acid (HA)
Thermogel
Bupivacaine
Easy to injectin situ gel for localized affect sustained release profileLess cytotoxic
[109]
Chitosan
Thermogel
Rupivacaine
Controlled releaseEfficacious delivery system for local anesthetic affect
[134]
Poly(N-isopropylacrylamide)(PNIPAM)
Temperature-sensitive NG
Bupivacaine
Polymerization
Less cytotoxic enhanced drug uptake
[135]
Alginate, chitosan
NG
Bupivacaine
Acceptable cytotoxicity and stabilitySlower drug release
[136]
Poly (e-caprolactone)–poly(ethylene glycol)–poly(e-caprolactone) (PCL–PEG–PCL)Pluronic F-127
Thermoresponsive NG
Lidocaine
Emulsion solvent evaporation method
Prolonged anesthetic affect with lesser toxicityEnhanced retention of local anesthetic
[137]
Methacrylic acid–ethyl acrylate cross-linked with diallyl phthalate
pH-sensitive NG
Bupivacaine
Emulsion polymerization
Enhanced pH-dependent anesthetic affect
[138]


### 4.9. Ophthalmic Diseases

NGs can be employed for ocular delivery with an advantage of enhanced residence time, controlled release of the loaded drug, increased corneal penetration, enhanced bioavailability, etc. These advantages offer improved patient compliance and also reduce dosing frequency. Some of the ophthalmic applications of NGs are given below (Table4).Table 4
NGs for ophthalmic delivery.
NG composition
Type of NG
Drug/agent used
Method of preparation
Results and applications
References
Nanodiamond, chitosan, poly(hydroxy ethyl methacrylate) matrix
Diamond NG
Timolol maleate
Spontaneous cluster formation
Lysozyme mediated sustained releaseEnhanced retention in the eyeLocalized delivery to treat glaucoma
[139]
Polyvinylpyrrolidone and acrylic acid (AAc)
NG
Pilocarpine
γ radiation-inducedPolymerization
Sustained drug release and improved bioavailability response
[140]
PLGA, chitosan
In situ NG
Levofloxacin
Sustained drug releaseEnhanced corneal retentionSlow drug clearance
[141]
Chitin
NG
Fluconazole
Controlled regeneration chemistry method
Good penetration to the corneaEffective for the treatment of corneal fungal infection
[142]
Cyclodextrin
NG
Dexamethasone
Emulsion-solventEvaporation
Controlled drug release by adhering to the ocular surface.Enhanced ocular bioavailability.Extended drug retention at eye surface
[143]
PLA, sodium alginate
In situ NG
5-Fluorouracil
Emulsion-solventEvaporation
Controlled drug releaseEnhanced retention of gelEffective ophthalmic delivery system for the treatment of conjunctival/corneal squamous cell carcinoma (CCSC)
[144]
N-Isopropyl acrylamide, 2-hydroxy-methacrylateLactide–dextran
Tacrolimus
Sustained drug release profileIncreased penetration to the cornea
[98]


### 4.10. Autoimmune Diseases

Autoimmune diseases can be effectively treated by using NG systems loaded with agents to be delivered to antigen-presenting cells to produce autoimmune responses. NGs containing KN93 and mycophenolic acid as therapeutic moieties are prepared by cross-linking and polymerization of the diacrylate-terminated co-block polymer of poly(lactic acid-co-ethylene glycol), CD [111]. The former specifically targeted CD4+T cells and reduced experimental autoimmune encephalomyelitis while later becoming effective for treating lupus by reducing cytokine production and enhancing immunosuppression [112, 113].


## 4.1. Brain Diseases

Various nanoplatforms are recently utilized for the treatment of brain diseases including Alzheimer’s disease (AD), depression, migraine, and schizophrenia. NGs are one of those nanodecorated drug delivery systems. Their efficacy in brain diseases is because of their improved therapeutic effects, better mechanism of targeting, and biological efficiency. AD is the irretrievable neurodegenerative illness leading to progressive loss of memory and intellectual abilities [73]. Although various pathological conditions are believed as the possible reasons of AD, amyloid hypothesis is, however, widely accepted in this regard [74]. The anomalous buildup, accretion, and accumulation of amyloid β-protein (Aβ) lead to the cerebral extracellular amyloid blockade that causes neurotoxicity [75, 76]. Therefore, avoiding Aβ accumulation is believed as a favorable approach in the management of AD. For this purpose, NGs with a double inhibitor-modified hyaluronic acid function were fabricated with inhibiting capabilities of Aβ accumulation, resulting in the management of AD [77]. Likewise, Elnaggar et al. assimilated piperine, a phytopharmaceutical agent in NGs for its neuroprotective effect in AD [78].Similarly, NGs are reported to deliver olanzapine for the treatment of schizophrenia which is a brain disorder described by delusions and disordered behavior [79]. These NGs exhibited excellent entrapment and enhanced bioavailability. Moreover, Hu et al. prepared lidocaine hydrochloride-loaded NGs, for the management of migraine [80]. Lidocaine hydrochloride is a commonly used drug in the treatment of migraine; however, after incorporation in the NG, the drug showed better bioavailability with no toxicity. Furthermore, Dange et al. reported the development of venlafaxine-loaded NG for the treatment of depression [81]. This NG showed a quick onset of action with extended period of time as compared with the drug solution.


## 4.2. Cardiovascular Diseases

Cardiovascular diseases like myocardial infarction (MI) and heart failure are the main reason of human deaths globally [82]. Various strategies are adopted to treat these diseases including tissue engineering and stem cell transplantation [83, 84]. Among the different drug delivery systems, injectable NGs have been used to treat MI. These NGs have confirmed the improvement in cardiac condition via LaPlace’s Law, an act which is exhibited by increased wall thickness and reduced wall stress [85]. One such study reported the heart restoration using NG-encapsulated human cardiac stem cells in mice and pigs with MI [86]. This study concluded that synthetic porous NGs act as a promising cell carrier for allogeneic/xenogeneic cell rehabilitations. Most particularly, these NGs inhibit the entry of immune cells while promoting the regenerative capabilities of the heart.Another study demonstrated the development of thermoresponsive NGs to produce cell mass fragments for the treatment of ischemic diseases. Owing to their temperature-dependent behavior, the cell bodies are produced without proteolytic enzymes. The animal studies further exhibited the adherence of cell mass fragments with engraftment sites which in turn enhance the vascular density, hence treating the diseased condition of an infarcted heart [87].


## 4.3. Treatment of Oxidative Stress

Oxidative stress is a diseased condition in which the increased production of oxidants including hydroxyl radicals, singlet oxygen, and hydrogen peroxide lead to cellular disability. This increased level of oxidants may be produced by endogenous and exogenous sources which may result in development of many diseases including cardiovascular diseases, Parkinson’s disease, and acute renal failure [88, 89]. Various drug delivery systems are utilized to treat the oxidative stress; however, NGs are considered as reliable drug delivery vehicles in this regard [90–92]. For instance, quercetin-encapsulated poly(b-amino esters) NGs were developed for the treatment of cellular oxidative stress [93]. NGs with a size range at the nanoscale were developed with 25–38 drug wt% and constant drug release over a period of 45–48 h. These NGs demonstrated an antioxidant activity of the drug for a prolonged time period. Another study exhibited the development of ferulic acid-loaded NG with improved penetrability and augmented antioxidant activity in rats for the treatment of oxidative stress. This NG exhibited excellent stability and sustained release of the drug with outstanding antioxidant activity which could be attributed to the increase solubility of the drug and augmented permeability from the NG [94].


## 4.4. Diabetes Management

Diabetes, a very prevailing chronic disease around the globe, has grabbed the attention of scientists, and new ways of its management are reported. Recently, a new improved therapeutic regimen, noninvasive glucose checking techniques, and new methods of insulin administration have been reported [95, 96]. In this aspect, the preparation of glucose-sensitive NGs has addressed the major hurdles linked with diabetes management. Most particularly, these NGs exhibited sustained release of the insulin by glucose-dependent swelling and shrinking mechanisms [97, 98].


## 4.5. Cancer Therapy

Various anticancer drugs, e.g., doxorubicin, cisplatin, 5-fluorouracil, and temozolomide, can be incorporated in NGs for the treatment of cancer. Temperature- and pH-sensitive hydrogels of doxorubicin based on maleic acid poly-(N-isopropyl acrylamide) polymer were used in cancer therapy in which doxorubicin release was dependent on temperature and pH. Chitin-based NG of doxorubicin can be used for various types of cancers including lungs, breast, liver, and prostate cancer [99]. Similarly, photosensitizer agent chlorin e6 has been recently used for the photodynamic therapy of cancer using chitosan-based NGs [100]. A descriptive detail of the anticancer applications of NGs is given in Table 1.Table 1
Anticancer applications of NGs.
NG composition
Type of NG
Drug used
Method of preparation
Results and applications
References
PVA (polyvinyl alcohol)
Charge conversional and reduction-sensitive NG
Doxorubicin
Inverse nanoprecipitation
Better cell toxicity.Improved targeted intracellular drug release.
[114]
Dextrin with formaldehyde as a cross-linker
pH-sensitive NG
Doxorubicin
Emulsion cross-linking method
Efficacious antitumor activityIt is an important candidate for the treatment of colorectal cancer.
[104]
Poly(ethylene glycol)-b-poly(L-glutamic acid) (PEG-b-PGA)
Polypeptide-based NG
17-AAGDoxorubicin
Cross-linking method
Improved anticancer activityEffective cytotoxicity in a breast cancer cell panel
[33, 115]
P(N-isopropyl-acrylamide-co-butyl methacrylates)
Temperature-sensitive NG dispersion
Doxorubicin
Emulsion polymerization method
Improved efficacy for transarterial chemoembolization (TACE) of iohexol dispersion (IBi-D) was observed on rabbit VX2 liver tumors.
[116]
Poly (N-isopropylmethacrylamide) (PNiPMA), PDA-PEG, 4-methoxybenzoic acid (MBA)
pH, thermal, and redox potential triple-responsive expansile NG (TRN)
Pc 4
Targeted delivery of pc 4 to sigma 2 receptors in head and neck tumors.
[117]
Glycol chitosan (GC) conjugated with 2,3-dimethylmaleic acid (dma) and fullerene (C60) conjugate (GC-g-DMA-g-C60)
Acid pH-responsive NG
Photosensitizer drug
Two-step chemical grafting reaction
Beneficial to target endosomes and in vivo photodynamic therapy in different types of malignant tumors.
[118]
Dextrin with glyoxal as a cross-linker
pH-sensitive NG
Doxorubicin
Emulsion cross-linking method
Rapid release effective internalization of doxorubicin.Reduced side effects to cardiomyocytes and stem cells.
[119]
Chitin poly (L-lactic acid)
pH-responsive composite NG
Doxorubicin
Blood compatibility of the system was confirmed byin vitro coagulation assay and hemolytic assay.Effective for the treatment of liver cancer.
[120]
Chitin
pH-sensitive NG
5-Fluorouracil
Controlled regeneration chemistry method
Loosening of the epidermis after its interaction with negatively charged chitin with no inflammation.Important drug carriers for skin cancer therapy.
[121]
Folic acid conjugated poly(ethylene oxide)-b-poly(methacrylic acid)
Ligand-gated polyelectrolyte NG
Cisplatin
Cross-linking method
In vivo anticancer effect strengthens their use for the treatment of ovarian cancer.
[122]
Acetylated chondroitin sulfate (CS)
Self-organizing NG
Doxorubicin
Dialysis method
Drug was internalized into the cytoplasm through endocytosis.Effective drug carrier for anticancer therapy.
[70]
N-Isopropylacrylamide (NIPAM), poly(ethylene glycol) (PEG), poly(ethylene glycol) methyl ether methacrylate (mPEGMA)
pH-thermal dual-responsive NG
Cisplatin (CDDP)
Emulsion polymerization method
Extended circulation time.Reduced side effectsBetter antitumor activity for the treatment of breast cancer.
[123]
In situ immobilization of CdSe quantum dots in interior of hydroxypropyl cellulose poly(acrylic acid) (HPC-PAA)
pH and temperature-responsive NG
Temozolomide
Polymerization method
High drug loading, better stability, and pH-dependent sustained release.Used in cell imaging and optical pH sensing.
[32]


## 4.6. Tissue Engineering and Gene Therapy

NG-based formulations are widely used for tissue engineering and gene therapy. They are also used to deliver enzymes, genes, and proteins at a targeted site to achieve their intended effects. Artificial chaperons are usually utilized to modify polymers to carry enzymes and proteins. Similarly, pullulan is chemically modified by conjugating cholesterol moieties, and the functionalized molecules are self-assembled in water to develop NGs of up to 30 nm size. These NGs have an extraordinary biocompatibility which is utilized for bone regeneration [101, 102]. The properties usually depend on their size and density of NGs which alternatively depend upon the degree of substitution of the cholesterol fragments in NGs. Some of the NG formulations used in transport of enzymes, genes, and proteins are as follows (Table 2).Table 2
Applications of NG tissue engineering and gene therapy.
NG composition
Type of NG
Drug/agent used
Method of preparation
Results and applications
References
CHOPA-PEGSH
Hybrid NG
W9 peptide
Cross-linking
Bone repair, sustained release
[124]
Pullulan-collagen; 1,2,7,8-diepoxyoctane
PHD hybrid NG
1,2,7,8-Diepoxyoctane
Cross-linking
Tissue filler materials
[125]
Dendritic polyglycerol (dPG) and low-molecular-weight polyethylenimine
pH-sensitive NG
siRNA
Thiol-Michael nanoprecipitation method
In vitro gene silencing.Gene therapy
[126]
Chitosan–myristic acidNG (CMA)
Cross-linked NG
Aryldialkylphosphatase
“Self-assembly via chemical modification” method
Enhanced pH and thermal stabilityUsed for detoxification of paraoxon
[127]
Poly(N-isopropylacrylamide)-polyglycerol
Thermoresponsive NG
Biomacromolecules
Enhanced stability and release of proteinEffective for the topical delivery of biomacromolecules
[128]
Poly(N-vinyl pyrrolidone) (PVP)
Functionalized NG
Oligonucleotides (ODN)
Cross-linking and polymerization
Negligible cytotoxicityBypass cellular membranesEffective nanocarriers for gene delivery
[129]
Polyethyleneimine (PEI)
Microenvironment-responsive functional NG
Gene
Cross-linking and polymerization
Reduced cytotoxicityEnhanced transfection efficiencyPotential gene therapy
[106]
Poly(2-methacryloyloxyethyl phosphorylcholine), poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM)) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA))
Thermosensitive NG
Protein
Reversible addition−fragmentation chain transfer [56] and polymerization technique
Temperature-sensitive controlled release of proteins from biodegradable NG
[130]
Enzymatically synthesized glycogen (ESG) with cholesterol group
Artificial chaperon
Hydrophobic modification self-assembly method
Enhanced thermal stability of enzymeUsed for biomedical and protein engineering
[131]
Cholesteryl group-bearing pullulan (CHP) complexed with methyl-b-cyclodextrin (M-b-CD)
Artificial chaperon
Protein synthesis was not affected.Help in folding of active proteins.
[132]


## 4.7. Inflammatory Disorders

NGs are considered as important delivery systems for various anti-inflammatory agents. For instance, siRNA-loaded NGs were prepared by polymerization and chemical cross-linking. Structurally, it was polymethacrylic acid-co-N-vinyl-2-pyrrolidone (P[MAA-co-NVP]) cross-linked with a trypsin-degradable peptide linker. A maximum amount of drug was released in the intestinal environment due to its pH and enzyme sensitivity and hence proved to be a suitable candidate for the treatment of inflammatory bowel disease [103]. Similarly, two anti-inflammatory drugs spantide II and ketoprofen were loaded with (HPMC) and Carbopol-based NGs to achieve enhanced percutaneous delivery for the treatment of skin inflammation [104]. Additionally, anti-TNFα agent etanercept (ETR) was recently loaded with thermoresponsive NGs which not only resulted in effective delivery but showed enhanced anti-inflammatory responses [105].The NGs enhanced their ability to get deposited in skin’s epidermis and dermis for the therapy of topical inflammatory diseases. They are prepared by either solvent evaporation or emulsification method [106]. Photosensitizers tetraphenylporphyrin tetrasulfonate (TPPS4), tetra-phenyl-chlorin-tetra-carboxylate (TPCC4), and chlorin e6 (Ce6) have a hyaluronate ligand-gated chitosan with tripolyphosphate (TPP) as a cross-linker in their structure. Their potential for extending the retention time and reducing clearance from the inflamed joints enlists NGs as real contenders for the selective delivery of photosensitizers to macrophages. Ionic gelation is the method applied in their preparation [107]. Activated NGs of methotrexate have copolymerized N-isopropylacrylamide (NIPAM) and BA (poly(NIPAM-co-BA)) polymers in its composition. It is synthesized by the emulsion polymerization method. Their advantages include amplified release, elevated concentration gradient, building flux of methotrexate along with depressing PGE2 production, and hence effective anti-inflammatory effects [71, 108].


## 4.8. Pain Management

NGs have been successfully used for the local distribution of anesthetic medications for the pain management. They result in prolonged and sustained release of the incorporated drug [109]. Moreover, they resulted in lower cytotoxicity and enhanced drug uptake [110]. A detailed description of NGs as a local anesthetic drug delivery system is given in Table 3.Table 3
NGs for the management of pain.
NG composition
Type of NG
Drug/agent used
Method of preparation
Results and applications
References
NIPAAM, MAA
Magnetic NG
Bupivacaine
Free radical emulsion polymerization method
Rapid release at low temperature and pHEffective for the treatment of ankle block
[133]
Pluronic F127, hyaluronic acid (HA)
Thermogel
Bupivacaine
Easy to injectin situ gel for localized affect sustained release profileLess cytotoxic
[109]
Chitosan
Thermogel
Rupivacaine
Controlled releaseEfficacious delivery system for local anesthetic affect
[134]
Poly(N-isopropylacrylamide)(PNIPAM)
Temperature-sensitive NG
Bupivacaine
Polymerization
Less cytotoxic enhanced drug uptake
[135]
Alginate, chitosan
NG
Bupivacaine
Acceptable cytotoxicity and stabilitySlower drug release
[136]
Poly (e-caprolactone)–poly(ethylene glycol)–poly(e-caprolactone) (PCL–PEG–PCL)Pluronic F-127
Thermoresponsive NG
Lidocaine
Emulsion solvent evaporation method
Prolonged anesthetic affect with lesser toxicityEnhanced retention of local anesthetic
[137]
Methacrylic acid–ethyl acrylate cross-linked with diallyl phthalate
pH-sensitive NG
Bupivacaine
Emulsion polymerization
Enhanced pH-dependent anesthetic affect
[138]


## 4.9. Ophthalmic Diseases

NGs can be employed for ocular delivery with an advantage of enhanced residence time, controlled release of the loaded drug, increased corneal penetration, enhanced bioavailability, etc. These advantages offer improved patient compliance and also reduce dosing frequency. Some of the ophthalmic applications of NGs are given below (Table4).Table 4
NGs for ophthalmic delivery.
NG composition
Type of NG
Drug/agent used
Method of preparation
Results and applications
References
Nanodiamond, chitosan, poly(hydroxy ethyl methacrylate) matrix
Diamond NG
Timolol maleate
Spontaneous cluster formation
Lysozyme mediated sustained releaseEnhanced retention in the eyeLocalized delivery to treat glaucoma
[139]
Polyvinylpyrrolidone and acrylic acid (AAc)
NG
Pilocarpine
γ radiation-inducedPolymerization
Sustained drug release and improved bioavailability response
[140]
PLGA, chitosan
In situ NG
Levofloxacin
Sustained drug releaseEnhanced corneal retentionSlow drug clearance
[141]
Chitin
NG
Fluconazole
Controlled regeneration chemistry method
Good penetration to the corneaEffective for the treatment of corneal fungal infection
[142]
Cyclodextrin
NG
Dexamethasone
Emulsion-solventEvaporation
Controlled drug release by adhering to the ocular surface.Enhanced ocular bioavailability.Extended drug retention at eye surface
[143]
PLA, sodium alginate
In situ NG
5-Fluorouracil
Emulsion-solventEvaporation
Controlled drug releaseEnhanced retention of gelEffective ophthalmic delivery system for the treatment of conjunctival/corneal squamous cell carcinoma (CCSC)
[144]
N-Isopropyl acrylamide, 2-hydroxy-methacrylateLactide–dextran
Tacrolimus
Sustained drug release profileIncreased penetration to the cornea
[98]


## 4.10. Autoimmune Diseases

Autoimmune diseases can be effectively treated by using NG systems loaded with agents to be delivered to antigen-presenting cells to produce autoimmune responses. NGs containing KN93 and mycophenolic acid as therapeutic moieties are prepared by cross-linking and polymerization of the diacrylate-terminated co-block polymer of poly(lactic acid-co-ethylene glycol), CD [111]. The former specifically targeted CD4+T cells and reduced experimental autoimmune encephalomyelitis while later becoming effective for treating lupus by reducing cytokine production and enhancing immunosuppression [112, 113].


## 5. Conclusion

The vehicle for drug delivery may have numerous components that need to be effectual, productive, and finely tuned. NGs are versatile and attractive delivery systems having combined attributes of both nanoparticles and hydrogel. Ease in synthesis and purification of this delivery system provides exceptional drug encapsulation efficiency, response to numerous environmental stimuli, higher level of stability, and biologic consistency as compared to other delivery systems, also allowing for convenient functionalization to target cells. The size control for several applications in the delivery of drugs can be tailor-made for lesser cytotoxic with unique and versatile fabrication of NGs by designing a nontoxic delivery vehicle which become metabolized into harmless components in the body. NGs are proficiently internalized by the target cells, avoid accumulation in nontarget tissues, and thereby lower the therapeutic dosage and minimize harmful side effects. The effectiveness and compatibility are enhanced multifolds by the NG delivery system with safety mostly for hydrophilic, hydrophobic, and small drug molecules due to their chemical conformation and formulations that are unsuitable for other preparations. These minute transporters can also hold an amalgamation of purpose depending on two or more agents for diagnosis, imaging, controlled release, and site-specific targeting. These practicalities of NGs have unlocked the opportunities for more development in the field of biomedical applications and drug delivery.


### 5.1. Future Perspectives

Nanomaterials have gained increased clinical interest in recent times on account of a drastic need for improvements in conventional drug delivery and diagnostic tools. Drug delivery scientists over the past three decades have extensively investigated various nanomaterials for drug delivery applications. Owing to their extremely small size with large surface area, these nanomaterials have produced delivery systems with altered basic properties and bioactivity of drug cargos, improved pharmacokinetics, reduced toxicity, controlled drug release, and targeted delivery of therapeutics. In this context, NGs offer versatile platforms with combined properties of cross-linking gelling materials and nanotechnology. Hydrogel properties improve the physicochemical characteristics of NGs, while nanometric size facilitates their transport and biodistribution in different sites of the body. NG technology has earned a wide use in biomedicine ranging from drug delivery to tissue engineering, from imaging to diagnosis and biosensing. Surface functionalization and stimulus responsiveness have added a lot to the advantages and applications of NGs.A widespread application and versatility of NGs hold them with a great potential for future innovative research to cover the yet unmet needs. A tremendous amount of research is currently in progress to design and fabricate NGs with novel polymers to have more control over the release of their payloads. Likewise, a multitude of preparation techniques have been explored in the past few years to synthesize NGs with the desired set of attributes for various applications. Targeted delivery of NGs by surface functionalization is an area that still has a lot of potential for research in the days to come. However, antibody-conjugated NGs have newly been developed for the targeted delivery of anticancer drugs. However, targeting only a single cancer antigen is improbable because of the heterogeneous expression of cancer antigens in tumor sites. Development of multitargeted NG systems will result in superior cancer diagnostics and therapeutics. Furthermore, a design of NGs in terms of high uptake in selected cancer cells needs to be improved through the collaboration of polymer chemists and biologists. They can elucidate the specific interactions of biomolecules and receptors, which are then prudently attached to NG systems for a more precise targeted delivery. Investigation is required to determine the mechanisms of uptake of NGs at the neuron and/or glial cell level within the central nervous system. It will confirm that NGs prefer a cytosolic destination over an endosomal target. This sort of studies is essential if NGs are ever to be projected as specific drug delivery systems for targeting at the subcellular level.Whereas NGs have provided a substantial advancement in the current drug delivery and therapeutic and diagnostic tools, a number of shortcomings need urgent attention. Development of cost-effective methods and resolution of technological issues are required for a large-scale production of NGs. A number of questions pertaining to pharmacokinetics and pharmacodynamics need to be answered. Provided these shortcomings are satisfied, NGs can translate into efficient next-generation pharmaceuticals with enhanced clinical care in the near future.


## 5.1. Future Perspectives

Nanomaterials have gained increased clinical interest in recent times on account of a drastic need for improvements in conventional drug delivery and diagnostic tools. Drug delivery scientists over the past three decades have extensively investigated various nanomaterials for drug delivery applications. Owing to their extremely small size with large surface area, these nanomaterials have produced delivery systems with altered basic properties and bioactivity of drug cargos, improved pharmacokinetics, reduced toxicity, controlled drug release, and targeted delivery of therapeutics. In this context, NGs offer versatile platforms with combined properties of cross-linking gelling materials and nanotechnology. Hydrogel properties improve the physicochemical characteristics of NGs, while nanometric size facilitates their transport and biodistribution in different sites of the body. NG technology has earned a wide use in biomedicine ranging from drug delivery to tissue engineering, from imaging to diagnosis and biosensing. Surface functionalization and stimulus responsiveness have added a lot to the advantages and applications of NGs.A widespread application and versatility of NGs hold them with a great potential for future innovative research to cover the yet unmet needs. A tremendous amount of research is currently in progress to design and fabricate NGs with novel polymers to have more control over the release of their payloads. Likewise, a multitude of preparation techniques have been explored in the past few years to synthesize NGs with the desired set of attributes for various applications. Targeted delivery of NGs by surface functionalization is an area that still has a lot of potential for research in the days to come. However, antibody-conjugated NGs have newly been developed for the targeted delivery of anticancer drugs. However, targeting only a single cancer antigen is improbable because of the heterogeneous expression of cancer antigens in tumor sites. Development of multitargeted NG systems will result in superior cancer diagnostics and therapeutics. Furthermore, a design of NGs in terms of high uptake in selected cancer cells needs to be improved through the collaboration of polymer chemists and biologists. They can elucidate the specific interactions of biomolecules and receptors, which are then prudently attached to NG systems for a more precise targeted delivery. Investigation is required to determine the mechanisms of uptake of NGs at the neuron and/or glial cell level within the central nervous system. It will confirm that NGs prefer a cytosolic destination over an endosomal target. This sort of studies is essential if NGs are ever to be projected as specific drug delivery systems for targeting at the subcellular level.Whereas NGs have provided a substantial advancement in the current drug delivery and therapeutic and diagnostic tools, a number of shortcomings need urgent attention. Development of cost-effective methods and resolution of technological issues are required for a large-scale production of NGs. A number of questions pertaining to pharmacokinetics and pharmacodynamics need to be answered. Provided these shortcomings are satisfied, NGs can translate into efficient next-generation pharmaceuticals with enhanced clinical care in the near future.



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*Source: 1526186-2019-05-16.xml*