Recent advances in protein-based nanoparticles and their applications in the delivery of bioactive compounds

ABSTRACT The target delivery of sensitive ingredients is affected by various factors including hostile processing, and environmental and storage conditions. These factors are significant challenges in the target delivery and stability of bioactive compounds. However, protein-based nanocarriers have the potential to address these challenges. Protein nanocomposites are of special interest in bioactive compound delivery due to their inexpensive synthesis, size, nontoxic nature, and quick removal from the human body. They can be prepared using several procedures including protein encapsulation, template-induced method, nano-spray drying, emulsion method, nanoprecipitation nano-spraying, crosslinking, adsorption, and desolation. A wide range of protein-based nanoparticles is being used in the delivery of bioactive components. The current review discusses the type of protein nanocarrier, methods for preparation, characterization, and potential applications.


Introduction
Drug delivery is known as the process of dispensing a pharmaceutical compound to achieve a therapeutic effect. [1]Whenever a drug is developed, its formulation is usually in the simplest possible dosage form that is effective in treating the intended condition. [2]The formulation of a new drug molecule is a timeconsuming process and requires a huge cost of money.The most widely used route of drug administration is the oral route.All the drugs before 1950 were made into capsule or pill formulations.Upon contact with water, these drugs release the drug immediately without any control of the drug release kinetics.Some drugs can have very poor permeability across the cells while others can have very poor water solubility.The process of making oral formulations is made difficult by these factors. [3]n 1952, a new technology, known as the Spansule technology was introduced by Smith Klein Beecham.With this technology, control of drug release kinetics was made possible, and sustained-release formulations were produced. [2]The methods of individualizing drug therapy, dose titration, and therapeutic drug monitoring have been used to improve the safety and efficacy ratio of old drugs.Other very attractive methods such as drug delivery at a controlled rate, slow, and targeted delivery have also been pursued strongly. [4]rug delivery systems (DDS) can be used to improve the pharmacological properties of conventional drugs.DDS uses nanoparticles and microparticles [5] and these systems are formed with the intent of altering or improving the pharmacological and therapeutic properties of the drugs. [6]They have particle carriers that enable them to function as a drug reservoir.We can obtain the controlled release of drugs at the desired sites through drug delivery systems as pharmacokinetics and  biodistribution of the associated drugs are altered through DDS. [6]Extensive use of nanoparticle-based drug delivery systems has been seen in the last few decades. [2]Accelerating nanotechnology has gained extensive research interest. [7]It has opened several new possibilities, especially in the medical sciences and has gained popularity in the field of drug delivery. [8]anoparticles are solid colloidal particles and can be produced in various shapes and sizes. [8]Their sizes range from about 10 nm to 100 nm. [9]Particle size, particle morphology, and surface properties of nanoparticles can be controlled.Nanoparticles can provide site-specific drug delivery.Many factors dictate the selection of the material of the nanoparticle.These include the size needed for the nanoparticle, the built-in properties of the drug, biocompatibility, and biodegradability, desired drug release profile, hydrophobicity and surface charge, and toxicity and antigenicity of the product. [8]Nanotechnology has been widely used in sensing devices, therapeutics, energy harvesting, diagnosis, coating, purification systems, and packaging among other applications.It has a special place in the sector of bio-medics as it has helped for producing abundant forms of imaging agents and delivery systems of drugs.The aim of using nanotechnology in the biopharmaceutical industry is to enhance the functioning of therapeutic drugs in vivo by improving parameters such as bioavailability. [10]everal nano-carrier systems for drug delivery have been developed since then, based on liposomes, metals, and synthetic polymers.However, each of them had its own set of flaws.Few nano-formulations approved by Food and Drug Administration have been presented in Table 2.For hydrophilic drugs, liposomes, for example, had reduced encapsulation quality of drugs, less stability, and increased release rates. [11]Nanoparticles developed from inorganic sources and metals have drawbacks such as low clearance rates, limited options for surface modification, and increased venomousness.Proteinaceous therapeutic molecules contained in the internal cores of nano-carriers formed by synthetic polymers such as poly lactic acid (PLA) and poly lactic-co-glycolic acid (PLGA) can assemble. [12]ince multiple biological media or organisms are involved, the engineered nanomaterials must have desirable biosafety and biocompatibility, which necessitates the selection of nanomaterial precursors or components. [13]Proteins are suitable candidates because they are an integral part of living organisms. [14]Proteins can also interact with both hydrophobic and hydrophilic moieties since they are amphiphilic.Proteins can be called "participants" that play various roles in nano materials.They imbue the protein-based nanomaterials and nano systems (PNNS) with a range of natural properties. [15]Protein nanocomposites containing protein nanoparticles and its conjugates have evolved as an advanced nano-carrier systems. [16]Protein nanoparticle synthesis is inexpensive and follow simple techniques that may be modified to get the desired size distribution. [8]Protein nanoparticles are nontoxic and smaller in size than typical nanoparticles made from metals or other types of inorganic sources, removed from the human body quickly [17] and have been shown to evoke a negligible immune response in humans. [18]urthermore, some modifications give these nanoparticles excellent stability during storage and invivo processes. [19]The nanoparticles of protein have the benefit of being functionalized along with moieties that assist with drug and other beneficial compound delivery to specific locations. [20]esearchers have successfully utilized protein nanoparticle conjugates in a range of applications in addition to protein nanoparticles. [21]ue to their special characteristics and vast range of uses, gels are polymeric materials that can incorporate significant volumes of water (hydrogels), air (aerogels), or oil (oleo-gels) into their three-dimensional networks. [22]From the early 20th century, either alginate-based gels for wound healing or hyaluronic acid-based injectable soft tissue fillers have been utilized as biomaterials. [23]Designing tough, stimuli-responsive and/or self-healing gels as well as cellcompatible gels for biomedical applications have been significant recent advances for gels. [24]icrogels that are hydrophilic and biocompatible can offer a novel method for delivering encapsulated drugs through the circulatory system. [25]Nano-carriers are much smaller than erythrocytes or lymphocytes, which are common blood cells.They are free to move in the bloodstream into the tiny capillaries and vessels after intravenous infusion, allowing for the site-or tissue-specific transport through physiological clearance mechanisms.After the nano-carriers have been taken up by the cells of the targeted tissue, the drug is then released. [26]The current research focuses on drug delivery by protein-based nanoparticles and nano-systems.

Template induced method
Works on soft and hard templates Effective in designing the nanoparticles and controlling the position of synthetic polymers.
Protein strain potential occurs [2]   Nano spray drying Nanoparticles processed in through carbon dioxide gas Step-by-step process Cost-effective Production of proteins nanoparticles on a small scale [3]   Emulsion/solvent extraction method

Single emulsion preparation and double emulsion preparations
Highly stable process An unstable process requiring stabilizers and surfactants [29]   Nanoprecipitation Miscible solvents used for protein and drug

Rapid technique
Helpful for small-sized particles The concentration and nature of surfactant affect the size of the particles [93]   Electro-spraying Protein and drugs dispersed in solvent to form protein nanoparticles High drug loading efficiency and self-dispersion Not suitable for complex proteins [4]   Crosslinking Chemical reagents can be used for the crosslinking process which incorporates reactivity.

Sustained drug delivery
Increased stability of proteins Time-consuming [5]   Adsorption/ Grafting Non-covalent interactions to attach proteins to surfaces

Low cost Efficient Mildest immobilization method
The protein can be desorbed. [6] Desolvation A desolvating agent is added and proteins are dehydrated and change their conformation from stretched to coil conformation.

Extensively used in the preparation of nanoparticles
Toxic crosslinkers are used. [4]

Protein based nano particles
A developing area of study called protein nanotechnology combines the various physicochemical characteristics of proteins with nanoscale technologies.As a result of this area's integration with medical formulations, a new category of nanoparticles known as protein (or protein-based) nanoparticles were formed (PNPs). [27Few methods to prepare protein based nanoparticles are presented in Table 1.
Nanoparticles are solid colloidal particles and can be produced in various shapes and sizes. [8]Their sizes range from about 10 nm to 100 nm. [9]Particle size, particle morphology, and surface properties of nanoparticles can be controlled.Nanoparticles can provide site-specific drug delivery.Many factors dictate the selection of the material of the nanoparticle.These include the size needed for the nanoparticle, the built-in properties of the drug, biocompatibility, and biodegradability, desired drug release profile, hydrophobicity and surface charge, and toxicity and antigenicity of the product. [8]Nanotechnology has been widely used in sensing devices, therapeutics, energy harvesting, diagnosis, coating, purification systems, and packaging among other applications.It has a special place in the sector of bio-medics as it has helped for producing abundant forms of imaging agents and delivery systems of drugs.The aim of using nanotechnology in the biopharmaceutical industry is to enhance the functioning of therapeutic drugs in vivo by improving parameters such as bioavailability. [10]rotein-based nanoparticles can be diffused into the cell through the process of endocytosis due to their small size. [28]Proteins have unique properties and functions in biological products.They can be used as a base for making nanoparticles.Protein nanoparticles have advantages over other types of nanoparticles due to their control over particle size, surface modifications, biodegradability, and stability.They are also less toxic (immunogenic). [29]Moreover, the activity, half-life, and stability of protein-based nanoparticles are mostly protected from renal clearance and degradation by enzymes.Due to these properties, protein-based nanoparticles can be used in various targeted drug deliveries like cancer therapy, tumor therapy, vaccines, and lung delivery as they are non-antigenic by nature. [30]

Role of protein-based nanoparticles in immunotherapy and drug delivery system
The human immune system plays a vital role in many important functions of the body such as the homeostatic function, defensive actions, tissue repair process, and clearance of dead cells. [31]Our immune system consists of cells that continuously move throughout the body and screen every single cell.These cells search for invading pathogens as well as cancerous or malfunctioning cells. [31]Once recognized, malfunctioning cells are eliminated immediately. [32]Immune cells can be classified as either of the two arms immune system's, innate or adaptive.Innate immune cells are assured to respond to pathogen invasion immediately. [31]They express receptors that can recognize the pathogen through molecular motifs.These cells then phagocytose the pathogens and produce chemicals that provide a rapid response to pathogens.Prominent cells under innate immunity are neutrophils and macrophages.The adaptive immune system comprises of B and T cells, CD4+ helper T cells, as well as CD8+ killer cells.Cytokines are released through CD4+ helper T cells which controls the functions of Natural Killer cells, B cells, and innate cells.CD8+ killer cells, on the other hand, act as killers of infected cells.T and B lymphocytes have a clonal nature upon encountering an antigen.These cells activate an immune response and back up the innate defense which clears the invading pathogens. [32]ancer is one of the leading cause of mortality throughout the world, and regardless of momentous struggles, it is nevertheless difficult to treat. [33]It is the unregulated proliferation of aberrant cells. [34]wing to inadequately targeted medication accretion, severe side effects, and resistance to drugs, old cancer cures like radiotherapy, surgery, and chemotherapy besides cancers are away from fitting, instigating cancer degeneration and later treatment failure. [35]mmunotherapy is one of the most suitable treatments for curing cancer.Immunotherapy is a type of cure that involves using a patient's immune system to combat a disease.Based on whether it induces or suppresses the immune response, immunotherapy can be classified as activation or suppression immunotherapy. [36]Immunotherapy uses monoclonal or adoptive cell therapy and is becoming a novel therapy for cancer. [37]The main motifs that are involved in cancer immunotherapy are the administration of cytokine, vaccines for cancer, and adoptive cell transfer therapy. [38]Role of proteinbased nanoparticles in caancer immunotherapy is shown in Fig. 1.Immunotherapies based on nanoparticles can be grouped into three main categories, each having its objectives.The first strategy is to use nanoparticle delivery systems.Nanoparticles can either act as nano-carriers or as nanovaccines.The objective of this strategy is to generate a specific cytotoxic response against the tumor by targeting the T lymphocytes.This can be done by reaching the lymph nodes.Moreover, another strategy named as adoptive cell transfer (ACT) therapy.In this therapy, the immune cells of the patient are isolated.These are then treated ex-vivo after which these cells are reinfused.The third strategy involves the infusion of therapeutic drugs into the tumor site. [32]everal diseases are treated with systematic drug delivery by nanoparticles; however, the major focus of nano drug delivery is cancer.Targeted delivery of drugs by nano-based systems, is greatly desired as chemotherapeutic agents for the treatment of cancer.The most effective pharmacological carriers for chemotherapeutic medicines are considered to be nanoparticles, which have been made for the diagnosis and treatment of cancer.Such targeted environments enable the elimination of malignant cells with the least amount of damage to healthy tissues.It is typically preferable to target tumors with nanocarriers to increase the killing power while reducing systemic toxicity. [39]owever, the first biomedical use of nanoparticles was not immunotherapy.Immunotherapy was known as the "Breakthrough of the Year" by researchers a few years ago.Following this, nanoparticles began to be used as immunotherapeutic agents after being used as chemotherapeutic agents.Furthermore, the FDA has accepted numerous anticancer medications based on nanoparticle formulations, which are now being tried in medical trials. [37]42][43] Particularly serum proteins have piqued interest which are being examined as naturally plagiaristic drug nano-carriers having biocompatible and biodegradable characteristics for a variety of biomedical uses, particularly cancer nano-medicine. [44]Due to their low noxiousness and immunogenicity, serum proteins, particularly proteins found endogenously, are commonly applied as carriers for imaging agents and small-molecule therapeutic preparations.They are also exceptional aspirants for pharmaceuticals and vaccine excipients. [45] range of protein-based nanoparticles is being used in cancer immunotherapy, each having its significance.48] Albumin-based nanocarrier systems are formed on the fact that albumin transports molecules across endothelial membranes and is biological transporter.It uses caveolae-mediated transcytosis.Many studies have demonstrated that albumin accumulates in solid tumors, making it an important anticancer medication carrier.Albumin nanoparticles can be taken by cancerous cells via the caveolae pathway. [49]Abraxane is a paclitaxel formulation.It is albumin-bound and is based on the concept that albumin may have the ability to target tumors intrinsically.Higher dosing can be achieved using Abraxane.The need for premedication with antihistamines and corticosteroids is precluded while using Abraxane. [50]Cationic bovine serum albumin (CBSA) is also being tested for treatment of the lung cancer.It is a novel siRNA delivery system that forms stable nanosized particles with siRNA.CBSA promotes the accumulation of siRNA in the lungs.A gene-silencing effect that induced cancer cell apoptosis was seen when Bcl-2 siRNA was introduced into the blood circulation using CBSA. [51]liadin nanoparticles are administered orally and are used for colon cancer-targeted drug therapy.This nano-system releases the drug gradually in 48 hours and causes apoptosis of cancer cells. [8]hese cancer nano-immunotherapies are of significant value in the treatment of cancer.But there are a few drawbacks to this technology of nano-immunotherapies. Nanoparticles show colloidal instability over physiological conditions, less blood circulation time, and harmful interactions with endothelial systems like macrophages. [52]However, the development of "Protein Corona" on the surface of the nanoparticles is a significant limitation of nano particle-based therapies. [53]Nanoformed materials having protein corona may have altered physicochemical characteristics and the result of therapy is affected. [54]In immunotherapies based on molecule recognition on the nanoparticle's surface, this problem is very important.

Gels
Gels are three-dimensional (3-D) cross-linked structures prepared up of two constituents, a solvent step and structuring materials.Organic or inorganic molecules can be used as structuring materials, and physical or chemical interactions can be used to crosslink them. [55]The gel matrix is formed as a result of connections between these structuring components.The use of protein biopolymers to make gels has gained popularity over the years.These biopolymers have desirable characteristics including hydrophilicity, biocompatibility, nontoxicity, and long-term stability, making them suitable for tissue engineering and drug delivery. [56]Natural sources, like plant and animal origins, are commonly used to produce these structuring entities.Proteins and peptides are economical and often selected against synthetic polymers since they are easily accessible.

Types of protein-based gels
The 3-D network of gels is designed in various lengths and scales ranging from macro to nanoscales.Fig. 2 presents few protein-based gels and their properties.Different kinds of protein-based gels are mentioned here.

Hydrogels
The word hydrogel comes from two words "hydro and gels" which means "water and jelly-like substance," respectively.They have the liquid phase in the form of an aqueous solvent.These are made up of hydrophilic polymers which are the building blocks of 3-D polymeric networks.Both chemical cross-linking and physical methods can be used for the formation of 3-D structures.Hydrogels are divided into conventional and stimuli-responsive. [57]Conventional hydrogels can absorb water.They exhibit constant equilibrium swelling.Changes in the pH, temperature, and magnetic and electric fields do not affect the swelling process of the hydrogel.Hydrogels use the mechanism of diffusion for drug release. [58]The stimuli-responsive hydrogels show changes in swelling properties because they are affected by the changes in their environment.These environmental changes include pH, temperature, chemical, and enzymatic changes.These gels provide drug delivery to localized areas.The stimuli-responsive gels have uses in both targeted drug delivery and tissue engineering. [59]

Microgels
Microgels are 3-D structures that are composed of cross-linked polymer molecules.They have micron sizes ranging from 0.5 to 5 micrometers.Microgels can undergo deformation during stress.Depending upon their composition, they give rise to surface activity and show responses to changes in temperature and pH.All these properties are making microgels an important option as colloidal units and stabilizing agents. [60,61]

Nanogels
These gels have nanoscale ranges, ranging in size from 1-100 nm are called nanogels.They can easily move through cellular membranes due to their small diameter and less surface-to-volume ratio. [62]ue to their smaller size and increased movement through cellular membranes, they increase the uptake of the drug by the cells that are used for targeted drug delivery.Nanogels can be classified based on cross-linking technology and their response to the environment. [63]eparation of microgels A common method to synthesize microgels is the emulsification method.A multiphase mixture is obtained by combining an organic phase and a polymeric aqueous phase.The multiphase mixture is then homogenized to form an emulsion.Spherical hydrogels are formed by the solidification of the internal droplets of the aqueous polymeric phase.Physical or chemical cross-linking methods are used for the solidification process.The hydrogels thus formed are called microgels if they are micron-sized. [64]he method of preparation of microgels determines the structure, electric charge, and physiochemical properties of microgels.Some factors that govern the properties of microgels are: (a) the nature of the crosslinking agent (b) shear stress (c) ionic strength (d) type of solvent (e) temperature (f) pH value, and (g) biopolymer concentration.The texture of microgels is tailored by the fabrication methods used for microgels including molecular association, self-association, and chemical methods which include extrusion method, atomization method, shearing method, emulsion-based process, and micro-molding methods. [65]

Drug delivery by protein-based microgels
Bio-macromolecular drugs, especially peptides and proteins, have become highly significant in drug development based on current innovations in the insight into human proteome, as well as advanced high throughput research methods. [66]For these drugs, drug delivery can offer several benefits, such as binding stability and biological evaluation retention, prevention from chemical and enzymatic depletion, drug release rate regulation, and reduced toxicity, immunity, and other biological adverse effects. [67]icrogels that are hydrophilic and biocompatible can offer a novel method for delivering encapsulated drugs through the circulatory system. [25]Nano-carriers are much smaller than erythrocytes or lymphocytes, which are common blood cells.They are free to move in the bloodstream into the tiny capillaries and vessels after intravenous infusion, allowing for site-or tissue-specific transport through physiological clearance mechanisms.After the nano-carriers have been taken up by the cells of the targeted tissue, the drug is then released. [26]nfortunately, the reticuloendothelial system (RES) easily removes the bulk of particulate DDSs after intravenous injection. [68]By phagocytosis, these elements gather in the macrophages of the liver and spleen.The adsorption of blood proteins to the particle surface causes nano-carrier uptake.Immunoglobulin and complement system components are examples of well-known opsonins that facilitate phagocytosis.Surface modifications of nano-carriers with Polyethylene oxide polymers have been used extensively to gain long circulation times in blood and localization in non-reticuloendothelial tissues. [69]

Triggering factors for microgels-based drug delivery
There are some triggering factors for drug delivery by microgels.These are as follows:

Temperature triggering
Forms of poly n-isopropyl acrylamide (PNIPAM), polyethylene oxide derivative, and cellulose ethers are among the polymers that display temperature-dependent swelling/deswelling transitions. [70]Solvency in such processes decreases as temperature rises, resulting in noticeable deswelling as temperature rises.Under certain situations, this causes temperature-induced "burying release," and in others, trapping causes the release of the drug to reduce.Temperature-dependent gel deswelling can be used for increased release of encapsulated drugs as well as to protect encapsulated compounds from enzymatic and hydrolytic degradation after administration in either case.Elashnikov et al., for instance, studied temperature-induced discharge of insulin from PNIPAM microgels and revealed that insulin discharge augmented as temperature increased.The microgels act like "sponges," releasing insulin when "squashed" as an outcome of their dramatic deswelling during the same temperature rise [71,72]

Triggering by electrostatics
Besides the temperature triggering of microgels, electrostatic triggering, such as pH, is gaining popularity.For example, when Eichenbaum et al., 1999.investigated the reaction of poly (methacrylic) microgels to ionic strength and pH, they discovered that these microgels expanded due to carboxyl group dissolution, an impact tested by an electrolyte. [73]an and Tam studied the effect of pH-dependent swelling on the release of procaine hydrochloride and the pH-dependent swelling of a methacrylic -ethyl acrylate microgel system, which showed that by increasing the pH, there was an increase in the microgel swelling due to an increase in network charge and the rate of drug release was also increased. [74]Ramesh Babu et al. also shared the same results for sodium alginate -acrylic acid microgels. [75]ternal triggering of microgels Microgels may also be affected by external fields such as magnetic fields, light, and ultrasound.To study the regulation of release of drugs in azo-dextran microgels by using cis-trans isomerization of an azobenzene moiety present in the cross-linking microgels, the release of aspirin and rhodamine was at a slow rate in the E-configuration and a high rate in Z-configuration. [76]kirtach et al., [77] Angelatos et al., [78] and Radt et al. [79] reported light triggering in polymer microcapsules, while triggering in those scenarios was achieved by incorporating metal nanoparticles, created heat by illumination of light, and triggering a shift in conductivity of temperature-sensitive polymer network.Concentrated drug release caused by light or ultrasound is of concern for example in the therapy of photodynamic and other localized therapies. [80]

Role of hydrophilic and hydrophobic microgels in drug delivery
Microgels can respond to different stimuli including pH, specific metabolites and ions, ionic strength, temperature, and external fields.They can also be used as injectables. [58]Polymer gels lack hydrophobic domains.For this reason, the use of polymer gels as carriers for poorly soluble drugs is limited.The aggregation of proteins and peptides and the conformational changes are encouraged at the hydrophobic surfaces.Microgels are hydrophilic and facilitate the maintenance of the biological effect of biomacromolecular drugs.Drugs of peptide and protein can be combined with minimum aggregation and conformational changes using microgels.There are a few papers that address the issues of microgel drug delivery. [81]The limited-production throughput of single-cell microgels becomes a major hurdle toward translating them into industrial and clinical applications. [82]teraction of microgels with proteins Electrostatic interactions are essential for the association of proteins and peptides with loaded microgels because of their hydrophilic and charge structure.A study looked into the absorption and discharge of lysozyme into and out of oxidized starch microgels with opposite charges. [83]The increase in the oxidation of starch causes an increase in the microgels' starch with a negative charge resulting in increased binding of strongly positively charged lysozyme.As ionic strength increases, the electrostatic affinity between lysozyme and microgel is filtered, leading to a lower adsorption driving force and a faster release rate.Both of these effects are similar to those seen with nonporous substrates. [84]Surprisingly, lysozyme adsorption affinity decreased as pH increased, implying that the charge on lysozyme is more significant in this regard than the starch's charge, a charge regulation impact. [85]he cross-linking density of microgels influences protein binding and transmission due to their porous structure.The problem of protein/peptide integration into opposite charge microgels regarding microgel particle sizes is a somewhat critical topic, despite the potential deswelling of loaded microgels on attachment to oppositely charged proteins/peptides, along with its high reliance on pH and ionic power. [86]For instance, based on both, the extent of microgel swelling and loading of lysozyme, lysozyme has been shown to spread non-uniformly inside poly (acrylic acid) microgels for a variety of conditions, developing a thick layer. [87]Since lysozyme oligomerizes in response to ionic strength, protein concentration, and pH. [88]This is also verified that cytochrome C, a smaller protein that is very similar to lysozyme other than the lack of self-assembly, may not form a shell under similar conditions. [89]

Conclusion
Nanotechnology holds great promise for improving immune therapy and as a potential anticancer agent.Proteins are novel applicants for cancer-based immunotherapy by nanoparticles.Proteins from a diversity of ways can be processed as nanoparticles through simple, cost-effective, and eco-friendly synthesis processes which use minimal hazardous materials.Each protein has a natural metabolization method that enables the nanoparticles to be easily degraded inside the recipient with nontoxic residues.They can be used as a natural alternative to the artificial and naturally occurring sources currently accessible for nanoparticle formation for targeted biomedical applications.However, more research is required to explain the factors that contribute to microgels and microcapsules' less-than-ideal performance as bio macromolecular drug delivery systems, such as inhomogeneous distribution inside microgels (shell formation), inadequate loading of drugs, and slow/incomplete bio macromolecular drug release, to fully exploit these chances.

Future prospects
Most developed and even emerging nations have recently expressed concern over the rising cost of healthcare.Governments must gain a greater understanding of the cost-effectiveness of nanopharmaceuticals in order to maximize cost efficiency. [90]It should be highlighted that the first stage in creating this market is to do a normal cost-effectiveness analysis that will show whether the extra health advantages that nano-pharmaceuticals have over conventional formulations might be justified by the higher price.By doing this, governments will have the freedom to lay down precise regulations and assess the financial benefits of expanding this market.The success of this industry completely depends on patient, public, and medical professional assurances regarding the efficacy and safety of nanomedicines.The future market will need to raise public awareness of the advantages, safety concerns associated with nanopharmaceuticals in order to overcome this obstacle. [91]Despite all of the aforementioned obstacles, the authors believe that over the next five years, the market for nanomedicines and nano-pharmaceuticals will grow, largely because of advancements in bio-nanotechnology and nanoengineering, the introduction of clear regulations on new nanotechnology-based drugs, increased funding from governmental institutes, increased agreement on environmental issues, and the formation of partnerships between nanomedicine startups and leading pharmaceutical companies. [92]

Figure 1 .
Figure 1.The role of protein-based nano-particles in cancer immunotherapy.

Figure 2 .
Figure 2. Different types of Protein-based gels and their properties.

Table 1 .
Methods to prepare protein nanoparticles.

Table 2 .
Food and Drug Administration (FDA) approved nano-formulations.