Recent advances in lipid-protein conjugate-based delivery systems in nutraceutical, drug, and gene delivery

Food Hydrocolloids for Health 2 (2022) 100054 Contents lists available at ScienceDirect Food Hydrocolloids for Health journal homepage: www.elsevier.com/locate/fhfh

Recent advances in lipid-protein conjugate-based delivery systems in nutraceutical, drug, and gene delivery Thilini Dissanayake a,b, Xiaohong Sun c, Lord Abbey d, Nandika Bandara a,b,d,∗ a

Department of Food and Human Nutritional Sciences, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg R3T 2N2, Canada Richardson Centre for Food Technology and Research (RCFTR), 196, Innovation Drive, Winnipeg, Manitoba R3T 6C5 Canada c College of Food and Biological Engineering, Qiqihar University, Qiqihar, Heilongjiang 161006, China d Department of Plant, Food & Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada b

a r t i c l e i n f o

Keywords: Lipid-protein conjugation Drug delivery Gene delivery Nutraceutical delivery Bioavailability

a b s t r a c t Lipid and protein-based delivery systems have long been used to deliver active compounds such as drugs, genes, and nutraceuticals. These delivery systems are fabricated to overcome issues of pure active compounds, which include rapid release and metabolism, poor solubility, low stability, poor bioavailability, poor bioaccessibility, and toxicity. However, there are limitations of lipids and proteins that restrict their efficient use in the delivery systems. Lipid-protein conjugation is an emerging technique for fabricating novel delivery systems that provide the advantages of having both proteins and lipids in one delivery system. In addition, these conjugates have a much better synergistic effect and desirable properties inside the body than single carriers. Among them, colloidal and biological stability, enhanced mechanical strength, controlled release, higher circulation time, targeted delivery, less cytotoxicity, higher loading capacity, co-encapsulation, and enhanced bioavailability are key outcomes. Despite recent technological advancement, there are still drawbacks to lipid-protein conjugate-based delivery systems that should be addressed in future research studies. This review is focused on critically evaluating the importance of lipid and protein as delivery systems, benefits of lipid-protein conjugation, conjugation methods, various applications of lipid-protein conjugation in drugs, genes, and nutraceutical delivery, and identifying research challenges and future research directions.

1. Introduction Consumer awareness of human health and nutrition is growing continuously due to the increasing number of chronic diseases. Drugs, nutraceuticals, and transfected genes are effective prophylactic or therapeutic agents for managing chronic diseases such as cancers, cardiovascular diseases, and diabetes (Chaudhari et al., 2021; Pooja et al., 2016; Zhu et al., 2019; Zuvin et al., 2019). However, their therapeutic efficacy and in vivo potential physiological benefits are limited due to poor bioavailability, low aqueous solubility, chemical instability, interactions with excipients or food components, poor absorption, transformation in the gastrointestinal fluids, fast metabolism and circulation, and low targeted delivery (Chen et al., 2016; Dima et al., 2020; Hong et al., 2020; McClements et al., 2015; Shishir et al., 2018). Therefore, encapsulation of bioactives and drugs is explored as a promising technique to address these challenges where lipid or protein carriers are one of the most extensively explored vehicles in encapsulation (Bandara et al., 2018; Devi et al., 2017; Simões et al., 2017).

Lipids have many desirable characteristics, such as being generally recognized as safe (GRAS), biodegradability, biocompatibility, industrial production and scale-up capabilities, and excellent functionality in the emulsification (Simões et al., 2017). Liposomes, emulsions, microemulsions, nanoemulsions, multiple emulsions, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (Assadpour & Mahdi Jafari, 2018; McClements, 2015), and hollow solid lipid nanoparticles (Yang & Ciftci, 2016) are some of the most common lipid-based delivery systems. On the other hand, food protein is another biopolymer widely used in food formulations due to its biodegradability, biocompatibility, stability, and numerous functional properties (Dima et al., 2020; Hong et al., 2020). Furthermore, Fathi et al. (2018) have mentioned that the properties of proteins like surface activity, structure formation, and antioxidant activity increase the value of proteins as nanocarriers. Despite the numerous advantages of lipid and protein-based nanocarriers, many limitations impede the effective use of lipids and proteins as carriers. For example, due to interactions between lipids and serum proteins, lipid-based delivery systems result in in-vivo colloidal and

Corresponding author at: Richardson Centre for Functional Foods and Nutraceuticals, 196, Innovation Drive, Winnipeg, Manitoba R3T 2N2. Canada. E-mail address: Nandika.Bandara@umanitoba.ca (N. Bandara).

https://doi.org/10.1016/j.fhfh.2022.100054 Received 4 October 2021; Received in revised form 15 December 2021; Accepted 20 January 2022 2667-0259/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

T. Dissanayake, X. Sun, L. Abbey et al. Food Hydrocolloids for Health 2 (2022) 100054 biological instability. In addition, several nonspecific binding to erythrocytes, lymphocytes, and endothelial cells also contribute to the instability of lipid-based systems (Simões et al., 2000). Moreover, reticuloendothelial system (RES) clearance of lipids due to opsonization by complement proteins, fibronectin, and immunoglobulins and rapid drug release from liquid-state lipid carriers limit the efficacy of lipid-based delivery systems (Kandadi et al., 2012; Yingchoncharoen et al., 2016). Conjugation of proteins and lipids can result in delivery systems with desirable characteristics of both proteins and lipids. Further, conjugation overcomes most limitations related to existing protein- or lipidbased nano delivery systems. For example, lipid-protein conjugated drug delivery systems revealed excellent properties, including increased targeted delivery, controlled release, reduced cytotoxicity, and enhanced therapeutic efficacy (ElMasry et al., 2018; Jain et al., 2012; Pooja et al., 2016; Tang et al., 2015). Apart from the targeted delivery, enhancing drug stability by protein or lipid coating is another benefit of conjugation (Tao et al., 2019). Studies showed that lipid-protein nanoparticles enhanced the controlled release of active compounds when encapsulated in lipid-protein conjugated delivery systems. Controlled release causes higher circulation time and increased bioavailability of active compounds (Chen et al., 2020; Hall et al., 2021; Li et al., 2021; Ruttala et al., 2017; Tezgel et al., 2020). Furthermore, Lipid-protein conjugates can enhance the therapeutic efficacy of drugs by their high solubility while being directly delivered to target sites to reduce the toxicity to regular cells (He et al., 2015). The existence of several constraints that limit the application of lipid-protein conjugated delivery systems is well acknowledged. In some cases, the toxicity of the conjugates themselves has been recorded (Elzoghby et al., 2012; Gaber et al., 2017). Furthermore, the objective of conjugation is to give novel properties to conjugated delivery systems that are not shown by individual protein or lipid-based delivery systems. However, it cannot be achieved easily due to the complex nature of this process and the inability to predict how these systems perform inside the body. However, the different techniques and applications of lipidprotein conjugated delivery systems can be found in other literature, making it difficult for potential users of such information to get easy access. Therefore, the objective of this review is to critically evaluate the benefits of lipid-protein conjugates over single carriers, techniques for manufacturing lipid-protein conjugates, characterization methods for conjugates, various applications of lipid-protein conjugation in drugs, genes, and nutraceuticals delivery, and the future research directions.

gelatin has high potential as a protein-based nanoparticle to deliver bioactive compounds (Hathout & Omran, 2016). Albumin also plays a significant role in encapsulation and delivery systems. As the most abundant protein in human plasma (Kratz & Elsadek, 2012), albumin contributes to many physiological processes, including maintaining colloidal osmotic pressure, transporting hormones and fatty acids, delivering nutrients to cells, and balancing plasma pH (Neumann et al., 2010). Furthermore, albumin maintains its stability under different processing conditions. Albumin can be heated at 60 °C for 10 h without any deleterious effect, and it is stable in the pH range of 4 to 9 (Neumann et al., 2010). Moreover, the availability of albumin is high as it is the most abundant protein in plasma, and this can encourage commercial-level production of albumin-based delivery systems. In terms of half-life, it has a 19 days blood circulation period (Kratz, 2008). In targeted drug delivery, albumin is widely used due to its ability to reach and accumulate in targeted sites in the body (Elsadek & Kratz, 2012; Kratz, 2008). Due to the presence of a considerable amount of charged amino acids and functional groups, nanoparticles made with albumin are easy to conjugate with target ligands (Ren et al., 2013). As Bandara et al. (2018) discussed, canola protein can be used as a potential source for delivery systems by further developing by conjugation with other polymers and producing emulsion-based systems. Based on the dry weight, 36–40% of protein presents in the canola meal after defatting. However, other non-protein components such as fiber, polymeric phenolics, phytates, and sinapine present in the cellular components and coat of canola seed limit the suitability of canola meal for food use. Also, napin, a major canola protein, limits its applications in foods due to its allergenicity (Wanasundara et al., 2016). Even though the use of canola protein is limited in human foods, sustainable production, availability, and low cost promote the use of canola proteins in non-food applications such as delivery systems (Bandara et al., 2018). Likewise, owing to the unique functional properties of the proteins, different proteins have been used to encapsulate a broad range of active compounds. 𝛼-Lactalbumin was conjugated to chitosan using electrostatic interactions through their opposite chargers to encapsulate resveratrol. Resveratrol was mainly loaded to 𝛼-Lactalbumin-chitosan nanoparticles by hydrophobic interactions and H bonding (Liu et al., 2020). Ultrasonication and pH-shifting combined treatment was used to partially unfold the structure of soy protein to expose more hydrophobic moieties. However, ultrasonication takes a relatively long period to increase surface hydrophobicity. Therefore, pH shifting with ultrasonication has been used for this process where protein is exposed to extreme alkaline or acidic conditions and followed by neutral pH. This process leads to partial unfolding and refolding of proteins, respectively. Using this strategy, soy protein-polysaccharide nanoparticles were formed, and hydrophobic resveratrol was encapsulated, resulting in smaller particle size and hydrophobicity than untreated soy protein (Fang et al., 2021). Also, glycosylating of proteins with polysaccharides is another way of protein modification to overcome the issues associated with native protein structure during encapsulation. By this, functional properties of proteins such as solubility, gelation, emulsification, foaming, and film-forming can be improved (Zhang et al., 2018). Vitamin C encapsulated vitamin gummies were prepared using casein gels to enhance vitamin C’s stability and control the degradation. According to FTIR spectra, H bonds between casein and vitamin C were confirmed. 92% of encapsulated vitamin C was retained for ten weeks, while only 79% for unencapsulated vitamins. Also, simulated studies confirmed the sustainable release of vitamin C encapsulated in casein (Yan et al., 2021). Gliadin is a hydrophobic protein characterized by excellent mucoadhesive properties that are ideal for encapsulating hydrophobic and amphiphilic drugs. The hydrophobicity of gliadin is due to the disulfide bonds. The mucoadhesive properties increased the residence time of encapsulated drugs in the GI tract. However, pH, salt, and heat can cause instability of gliadin nanoparticles due to aggregation. The addition of gum arabic stabilized the gliadin nanoparticles due to the predominant H forces between gliadin and gum arabic at pH five and hydrophobic forces at

2. Protein and lipid as carriers Proteins are excellent raw materials for delivery applications due to their functional properties such as surface activity, emulsification, foaming, water-binding, gelation, and antioxidant activity, facilitating the easier modification of proteins through various chemical and physical techniques (Maviah et al., 2020). Animal proteins and plant proteins are the two major classes of proteins used to encapsulate bioactive compounds (Fathi et al., 2018). Gelatin, collagen, albumin, casein, elastin, milk, and whey proteins are animal proteins used for nanocarriers. Zein, pea, gliadin, and soy proteins are examples of plant proteins widely used in nanocarriers (Maviah et al., 2020; Verma et al., 2018). Gelatin is a promising carrier for bioactives due to its desirable properties such as biodegradability, biocompatibility, and non-toxicity (Elzoghby, 2013). In terms of chemical structure, gelatin can be easily modified or cross-linked to create delivery systems. It was reported that gelatin structure has ∼13% positively charged moieties and ∼12% of negatively charged moieties due to the presence of positively charged arginine and lysine and negatively charged glutamic and aspartic acids, respectively (Hathout & Omran, 2016). Also, leucine, isoleucine, methionine, and valine amino acids make ∼11% of the gelatin chain. All these multifunctional carboxylic and amino groups present in the gelatin facilitate the gelatin interactions with other molecules. Consequently, 2

pH 7 (Wu et al., 2018). 𝛽-carotene was encapsulated in whey proteinbased nanocapsules prepared using 5, 10, and 15% ethanol solutions. According to the results, ethanol caused the protein to unfold and expose the protein’s hydrophobic core, facilitating the interaction between protein and hydrophobic 𝛽-carotene (Rodrigues et al., 2020). As well, whey protein-based microparticles were used to successfully encapsulate curcumin which is unstable at physiological pH and poorly water-soluble. Bioaccessibility of curcumin was increased when they were encapsulated in whey protein compared to free curcumin (Ye et al., 2021). In terms of lipid-based delivery systems, oleic acid (Elmasry et al., 2018; Meghani et al., 2018; Park et al., 2015; Tran et al., 2013), lecithin (Chuacharoen & Sabliov, 2016; He et al., 2015; Jain et al., 2012; Tang et al., 2015), linolenic acid (Yadav et al., 2019), and linoleic acid (Pucek et al., 2017) are some of the commonly used lipid-based substances in delivery systems. These lipid-based substances are obtained from the sources such as flaxseed (Kaithwas et al., 2011), canola oil (Mokhtari et al., 2017), soybean seeds (Peng et al., 2014), clove oil, and other essential oils (Wan et al., 2018). Wan et al. (2018) have discussed that essential oils’ antimicrobial and preservative properties make them a potential source for food delivery systems. Mhule et al. (2018) reported that oleic acid is a potential stabilizer in liposomes and magnetic nanoparticles. Further, the authors (Mhule et al., 2018) have mentioned the properties of oleic acid such as biocompatibility, penetration enhancing property in transdermal delivery systems, and antibacterial activity increase its use in delivery applications. As reported by Perez-Ruiz et al. (2018), lecithin has a wide range of applications in delivery systems due to its biocompatibility and stabilizing properties. Further, it is used to prepare various nano vehicles such as micelle, liposomes, microemulsions, and nanoparticles. The inherent property of self-assembling in aqueous environments due to the amphiphilicity makes lecithin an attractive nano-carrier. Also, lecithin plays a significant role in maintaining membrane fluidity and enhancing the absorption of active compounds (Jabri et al., 2018). Linoleic acid is an essential molecule that cannot be synthesized in the human body, and it can reduce the risk of arteriosclerosis (Yang et al., 2018). As Fang et al. (2017) explained, the attention towards linoleic acid from scientists is increasing due to its in vitro antiproliferative effects and in vivo antitumor effects. The authors have proved that linoleic acid is effective in delivery systems in increasing circulation time and the therapeutic efficacy of loaded drugs. Liposomes are globular structures of lipid bilayers with a 50– 1000 nm diameter. The lipid bilayer is made from phospholipids which have a hydrophilic (polar) head and lipophilic (non-polar) tail (Daraee et al., 2016). The ability to deliver hydrophilic, lipophilic, and amphiphilic compounds is a major advantage of liposomes (Isailović et al., 2013). Maritim et al. (2021) encapsulated glibenclamide in liposomes. They evaluated the drug loading, liposome stability, and drug release affected by the location of the encapsulated drugs in liposomes, type of lipid (saturated, unsaturated), acyl chain length, preparation method, phase transition temperature, and phospholipid to cholesterol ratio. The hydrophobic model drug glibenclamide was loaded in the hydrophobic bilayer. It could also be loaded in the hydrophilic core of the liposome as it had a pH-dependent lower solubility in water. Based on the results, a longer lipid chain, lower cholesterol ratio, higher lipid saturation promoted the drug loading capacity in both core and bilayer. On the other hand, shorter lipid chains and lower lipid saturation enhanced the drug release rate. Interestingly, higher cholesterol levels increased the drug release for drugs (hydrophobic) encapsulated in bilayer while it reduced the drug release for drugs (hydrophilic) encapsulated in core (Maritim et al., 2021). Lipid nanoparticles have received increasing interest as a novel and promising carrier system for bioactive compounds (Gaber et al., 2017; Shishir et al., 2018). SLNs and nanostructured lipid carriers (NLCs) are the two main lipid nanoparticles (Katouzian et al., 2017). Couto et al. (2017) encapsulated hydrophilic vitamin B2 in SLNs, which were formed using fully hydrogenated canola oil (FHCO), sodium lauryl sul-

fate (SLS) (surfactant), and polyethylene glycol (PEG) (stabilizer). Vitamin B2 is easily absorbed, and it is not stored in the body due to its hydrophilic nature. Therefore, vitamin B2 should be continuously given to replenish its required levels. Encapsulation of vitamin B2 in SLNs slows down its absorption by controlled release. Furthermore, SLNs act as a protective cover for vitamin B2 by protecting them from light degradation (Couto et al., 2017). Meikle et al. (2021) fabricated cubosomes using lipid nanoparticles to encapsulate antimicrobial peptides that are susceptible to proteolysis and lack specificity. The encapsulation efficiency of the antimicrobial peptides was increased by optimizing the electrostatic charge of the lipids. Encapsulation in cubosomes enhanced the antimicrobial activity of peptides against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa due to the protection of peptides from enzymatic digestion and the ability to provide a platform for target delivery. Likewise, oleic acid-based NLCs (Huguet-Casquero et al., 2020), solid and liquid-based lipid nanocarriers from Compritol 888 ATO and squalene (Chaudhari et al., 2021), hollow solid lipid micro and nanoparticles (Yang & Ciftci, 2020), and double emulsions (from corn, soy, sunflower, or rapeseed oil) (Tian et al., 2020) were used to encapsulate oleuropein, plant-based bioactives, fish oil, and oligomeric proanthocyanidin respectively. 3. Techniques for manufacturing lipid-protein conjugate-based delivery systems In the delivery systems, lipid-protein conjugation is performed using several techniques. For example, chemical conjugation, desolvation, electrostatic coating, high-pressure homogenization, and lipid film hydration methods are widely used (Elmasry et al., 2018; Gaber et al., 2017). Fig. 1. Shows the different techniques that have been used in recent literature to conjugate lipids and proteins in drug, genes, and nutraceutical delivery applications. An amide bond is formed between the carboxyl group of the lipid and the amine group of the protein during chemical conjugation. The main difference between chemical conjugation and other methods is the formation of a covalent bond in chemical conjugation, where the other methods are based on interactions such as electrostatic interactions (Kabary et al., 2018), hydrophobic interactions (Peng et al., 2014), and hydrophilic and hydrophobic interactions in emulsions (Chen et al., 2020). Carboxylic functionality of the lipids is activated before forming the amide bond to facilitate the high efficiency of conjugation between protein and lipid. As protein can self-polymerize due to the presence of both amine and carboxylic groups, activation of carboxylic functionality of lipids is preferred before the conjugation (Gaber et al., 2017). Pooja et al. (2016) introduced an amino group to solid lipid nanoparticles using stearyl amine to facilitate the conjugation with the carboxylic group of the wheat germ agglutinin. In the same study, stearic acid was used to introduce a carboxylic group to solid lipid nanoparticles to facilitate the conjugation with amino groups of the wheat germ agglutinin, where the conjugation efficiency was increased from 11.45% to 74.1% . Tran et al. (2013) conjugated gelatin and oleic acid using EDC/NHS reaction where monoethanolamine (MEA) was used to activate oleic acid. Due to the contrary solubilities of gelatin and oleci acid, it is difficult to achieve conjugation in water without activate the oleic acid. The carbodiimide-coupling method is commonly used to form this amide bond. In this method, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide/ N-hydroxysuccinimide (EDC/NHS) reaction is commonly used (Elmasry et al., 2018; Gaber et al., 2017; Meghani et al., 2018; Pooja et al., 2016). In a recent study, EDC/NHS was used to conjugate gelatin and oleic acid together (Park, Vo, Kang, Oh, and Lee, 2015). In this work, they prepared gelatin-oleic conjugates in a water-ethanol cosolvent followed by the reaction of 2-mercapto ethanol with remaining EDC to prevent the crosslinking between amine groups and carboxylic groups present in the gelatin (Park, Vo, Kang, Oh, and Lee, 2015). Except carbodiimide coupling, thiol-maleimide coupling where proteins and 3

Fig. 1. Illustration of widely used techniques for lipid-protein conjugation. lipids are conjugated through thioether linkages of thiolated proteins and maleimide-derivatized lipids (Thöle et al., 2008), and Staudinger ligation method also come under the chemical conjugation method. In the Staudinger ligation method, an azide and a triphosphine are reacted together to form an amide bond. One of the major advantages of this reaction is that the process occurs at room temperature without any catalyst (Zhang et al., 2009). Xu et al. (2011) synthesized receptor-targeted liposomes by Staudinger ligation. They used transferrin as the source of protein, which was activated with p-azidophenyl isothiocyanate. The reaction between dioleoyl phosphatidylethanolamine and 2-(diphenylphosphino) terephthalic acid 1methyl-4-pentafluorophenyldiester produced the activated lipid. Finally, activated transferrin and synthesized liposomes were coupled together by Staudinger ligation. This method is simple and specific, producing relatively stable reaction intermediates. Therefore, this method has great potential for future applications in drug delivery systems. Desolvation is another method used for lipid-protein conjugation (Jain et al., 2012). The authors prepared gelatin-coated hybrid lipid nanoparticles using a two-step desolvation method in this study. First, gelatin was dissolved in distilled water, and high molecular weight (HMW) gelatin was precipitated by adding acetone as the desolvating agent. Then the distilled water was used to redissolve the precipitated HMW gelatin. Next, the drug was dissolved in dimethyl sulfoxide, and lecithin dissolved in methanol was mixed. Once the methanol was evaporated, drug-loaded lipid-protein conjugates were precipitated using acetone (Jain et al., 2012). In another investigation, Tang et al. (2015) used the desolvation method to prepare lipid-albumin nanoassemblies. In their study, drugs and lecithin were dissolved in anhydrous ethanol, and that solution was dropwise added to an aqueous albumin solution with constant stirring. Then the resultant solution was sonicated, and rotary evaporation technique was used to remove anhydrous ethanol. Finally, the samples were centrifuged and filtered to obtain drug-loaded lipid-albumin nanoassemblies (Tang et al., 2015). Proteins have negative or positive charges when their pH values are above or below the isoelectric point. In the electrostatic coating method,

oppositely charged lipids can be conjugated to proteins by electrostatic coupling (Gaber et al., 2017). Chen et al. (2016) synthesized albumincoated liposomes through electrostatic interactions. The liposomal solution of the drug was prepared using chloroform, but the chloroform was evaporated at the final step. After that, the resultant lipid film was hydrated in ultra-pure water and extruded through polycarbonate filters. The albumin coating was done by adding the albumin solution (pH = 3) dropwise to the liposomal solution. The liposomal solution had a strong negative charge of -43.5 ± 2.5 mV (pH = 7.4) due to the added drug of indocyanine green. The pH of albumin was adjusted to 3, leading to a positive charge. Thus, albumin and lipid could be easily conjugated due to electrostatic interactions. This method has been reported in many other studies as well (Cardoso et al., 2007, 2008; Faneca et al., 2008; Ilarduya et al., 2006; Nakase et al., 2005; Pan et al., 2008; Piao et al., 2013; Wang et al., 2012; Weecharangsan et al., 2009). High-pressure homogenization can be introduced as another lipidprotein conjugation technology. He et al. (2015) used the high-pressure homogenization method to conjugate lecithin and albumin dissolved in methylene chloride and distilled water, respectively. These two solutions were passed through a high-pressure homogenizer under 5000– 10,000 Psi. Fats were broken down into small droplets due to the applied high pressure. The droplets were stabilized by forming a surrounding protein layer that prevented the coalescence of the droplets (Min et al., 2003). Lipid film hydration is another conjugation technique that is used to conjugate proteins and lipids. Ruttala and Ko (2015) used this technique to encapsulate albumin-paclitaxel nanoparticles within liposomes. First, lipids were dissolved in chloroform, and the chloroform was removed by rotary evaporation in the next step. The albumin-paclitaxel nanoparticles prepared using a desolvation technique were then added to dried cationic lipid film before incubation for 4 hrs at room temperature while mixing intermittently. Finally, the suspension was extruded through a 200 nm-porous membrane. The blank liposomes had a 24-mV positive charge before encapsulating the albumin-paclitaxel. However, after the encapsulation, the charge increased to about 4 mV. This result 4

explains the compensation of the positive charge of blank liposomes by negatively charged albumin-paclitaxel nanoparticles. This type of conjugation can also be classified under the electrostatic coating category.

images of nanoparticles prepared without using phospholipids showed a fairly homogeneous internal structure with a roughly spherical shape. The conjugates that were prepared using phospholipids still showed a spherical shape. However, a core-shell internal structure could be observed in their TEM images. This is mainly due to the ability of phospholipids to self-assemble into multilayers (Chen et al., 2019). Based on the TEM images, the authors proposed that gliadin formed the core of the nanoparticles while phospholipid formed the multilayer shell. Elmasry et al. (2018) have stated that conjugation of oleic acid to gelatin did not change the spherical, smooth, compact, and compact morphology of gelatin particles obtained from TEM images. Further, the authors reported that the dark inner layer presented in the TEM images in gelatin-oleic nanoparticles has resulted from the hydrophobic interactions between oleic moieties and the binding effect of glutaraldehyde. Measuring drug release profiles provides information about the controlled release and creating cytotoxicity by drugs. According to Ruttala et al. (2017), encapsulated drugs in albumin nanoparticles have low colloidal stability, leading to the rapid elimination of the drug from systemic circulation. Conjugation of albumin with a lipid bilayer improves the stability of nanoparticles. Conjugation of transferrin to the lipid bilayer of the albumin nanoparticles further reduced the release rates of encapsulated drugs, thereby limiting the toxicity. Possible reasons for controlled release could be the action of lipid bilayer as a protective cover and the formation of a high molecular mass transferrin layer around the nanoparticle. As the transferrin receptor is overexpressed in proliferating tumor cells, targeted delivery of the drug could be obtained by conjugation of transferring to the lipid bilayer (Ruttala et al., 2017). Also, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) are used as characterization methods in many studies to evaluate the changes in functional groups, driving forces for the formation of conjugates, and physical state of the encapsulated active compounds. These methods can be used to compare the physical and chemical properties between conjugates and single carrier (protein or lipid) systems (Dai et al., 2017; Wang et al., 2018; Wei et al., 2019).

4. Characterization of the developed lipid-protein conjugate-based delivery systems Particle size is one of the most critical factors determining stability, encapsulation efficiency, controlled release, bio-distribution, mucoadhesion, and cellular uptake of encapsulated active compounds (Danaei et al., 2018). Pawar and Pande (2015) formulated oleic acidcoated gelatin nanoparticles impregnated gel to deliver zaltoprofen and observed increased entrapment efficiency with a decrease in particle size. The authors suggested this is due to the increase in surface area that provides more space for the binding drug due to the decrease in particle size. Furthermore, the authors have shown that crosslinker (glutaraldehyde) concentration and agitation speed affect the particle size of conjugates. Based on a study done by Dai et al. (2019), the zein to rhamnolipid ratio affected the particle size of nanoparticles that were produced to encapsulate curcumin. Also, temperature, ionic strength, and environmental pH affected the particle size. The same results were obtained by another study, in which the gliadin to phospholipid ratio affected the particle size of conjugates. Under a low level of phospholipid concentration, particle size decreased due to the inhibition of aggregation of gliadin molecules and reduction in surface tension. The particle size increased with the increase in phospholipid concentration due to the reduction of surface activity or absorption kinetics due to the phospholipid self-association and the formation of more lipid bilayers inside the nanoparticles that result in larger particle size (Chen et al., 2019). Park et al. (2015) showed the effect of the drug loading method on particle size. Their study showed a significant difference in particle size between the incubation method and the in-process drug adding method. The incubation method acquired a smaller size due to the stabilization of structure by drug penetration into some pores of the nanoparticles and absorption of drugs into the outer layer of nanoparticles due to the physical absorption or hydrogen bonding. However, in the in-process adding method, conjugates showed a reduced solubility, like a saltingout effect when the drug was added to the solution. As a result, it led to aggregation and resulted in larger particle sizes. Gaber et al. (2017) have discussed the impact of the hybridization method on particle size. The covalent bonding of protein onto the lipid surface of lipid nanoparticles does not significantly increase the particle size of lipid nanoparticles. On the contrary, the electrostatic interaction method increases the particle size of nanoparticles as a result of hybridization (Gaber et al., 2017). Zeta potential is a measurement of the stability of the particles against aggregation. The Zeta potential of lipid-protein conjugated delivery systems has been reported in many as it is the most common method of determination of stability of the delivery system. The increase in zeta potential results in electrostatic repulsion between the droplets that prevent aggregation and enhance stability (Chen et al., 2020; Li et al., 2020). In general, a stable suspension should have a high zeta potential value (below -20 mV). According to Park et al. (2015), the zeta potential of oleic acid-gelatin conjugates was below -20 mV, while gelatin nanoparticles generally report low zeta potential between 0–10 mV. The high zeta potential of conjugates resulted from the reaction of the amino groups of gelatin with oleic acid. This reaction leads to reduce the cation charges on the surface of conjugates. The carboxylate anions of gelatin decided the surface charge of the conjugates as it is far greater than the charge of remaining amino groups. Therefore, conjugation with oleic acid stabilizes the particles with anionic surface charges (Park et al., 2015). The most common method of evaluating the differences in structure and morphology of the lipid-protein conjugated delivery systems are scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The impact of the gliadin to phospholipid mass ratio on the morphology of conjugates could be observed from the TEM. TEM

5. Applications of lipid-protein conjugate-based delivery systems 5.1. Drug delivery Nanoscale drug delivery systems have provided better solutions for most of the issues related to traditional cancer therapy, including reduced drug solubility, chemoresistance, systemic toxicity, narrow therapeutic indices, and poor oral bioavailability. The development of amphiphilic polymeric vesicles is a new delivery platform in cancer studies to improve the efficiency and bioavailability of drugs (Pooja et al., 2016; Ruttala & Ko, 2015). Furthermore, attaching ligand molecules such as peptides, antibodies, sugars, and aptamers to the micelle shell, which has specificity for biomarkers overexpressed on cancer cells, improves the tumor selectivity and overall therapeutic efficiency of cancer treatments (Kalaydina et al., 2018; Khan et al., 2020; Tabarzad & Jafari, 2016). Therefore, lipid-protein conjugated delivery systems have been widely used for the targeted delivery of drugs (Kratz, 2008; Pooja et al., 2016; Ruttala & Ko, 2015; Tang et al., 2014). Conjugation strategies have also increased drugs’ bioavailability and reduced systemic toxicity (He et al., 2015; Jain et al., 2012). Based on the available literature information, many applications can be found in the area of drug delivery compared to nutraceuticals, genes, micronutrients, and foliage fertilizer delivery and are summarized in Table 1. Fig. 2. is an illustration of the highlights of the 5.1 section. Amphotericin B is an excellent treatment for systemic lifethreatening fungal infections and leishmaniasis. However, the therapeutic efficacy of amphotericin B is limited due to poor water solubility, poor intestinal permeability, and instability at gastric pH (Radwan et al., 2017). Additionally, dose-related side effects of amphotericin B, which cause nausea, fever, and nephrotoxicity, reduce the efficient use of am5

Natural bioactive compounds Lipid hybrid microparticles 1.09 ± 0.21 𝜇m (in scaffolds) Gelatin Cholesterol EDC/NHS chemical conjugation Nanoemulsion 219.7 ± 2.1 nm Rice bran protein Soybean oil Lipopeptide

Not reported Peptide- (Peptide derived from SARS-CoV-2 Spike Glycoprotein) Layer-by-layer coated lipid nanoparticles Nanoparticles 250.5 nm Nanoparticles Bioactive compound Refs. Curcumin Sustainable release of curcumin to prevent the post-engraftment complications

Li et al. (2021) Homogenization Quercetin Increased stability, bioavailability, and cell permeability of the bioactive compound Chen et al. (2020) bromoacyl tetra-ethylene glycol-cholesterol Chemical conjugation

Bioactive Peptide

Lipopeptides inhibit the fusion of virus and host cells by blocking the structural rearrangement of the spike glycoprotein of the virus. The Cholesterol group anchors the peptide on the cell membrane of the virus

Outlaw et al. (2020) Lactoferrin Phosphatidylcholine Electrostatic interaction 185 nm ± 2.6 Gelatin Oleic acid EDC/NHS chemical conjugation Berberine and rapamycin Sesamol

Reduced cytotoxicity, enhanced therapeutic efficacy, targeted co-delivery of drug Targeted delivery of sesamol by increasing skin permeation Kabary et al. (2018) Elmasry et al. (2018) Zein Lecithin Transferrin

Phosphatidylcholine and phosphoethanolamine High-pressure homogenization Chemical conjugation Lutein Liposome 216.5 ± 50 nm (25 °) 196.3 ± 7.09 nm 𝛼- Mangostin

Enhanced lutein stability against photo-and thermal degradation Enhanced penetration through the blood-brain barrier Chuacharoen and Sabliov (2016) Chen et al. (2016) Nanoparticles 185 nm Bovine serum albumin

Soybean lecithin Hydrophobic interactions Coumarin Enhanced nanoparticle stability and long circulation Peng et al. (2014) Synthetic bioactive compounds Nanoparticles ∼240 nm Gelatin Oleic acid EDC/NHS chemical conjugation

Doxorubicin Targeted delivery of doxorubicin Meghani et al. (2018) Nanoparticles Human serum albumin

Electrostatic interaction Phosphatidylethanolamine Glyceryl monostearate EDC/NHS chemical conjugation Indocyanine green/ ICG Paclitaxel Targeted delivery of ICG and enhanced stability Chen et al. (2016)

Solid lipid nanoparticles 121.45 ± 0.85 nm 152.9 nm Targeted delivery and increasing bioavailability of paclitaxel Pooja et al. (2016) Liposome ∼1 𝜇m Gelatin Soybean phosphatidylcholine Lipid film hydration

Paclitaxel Enhanced physical stability against lyophilization stress Guan et al. (2015) Nanoassemblies 107.5 ± 3.2 nm Albumin Lecithin Desolvaion Paclitaxel, Borneol Targeted delivery, increased anti-tumor efficacy and bioavailability of paclitaxel

Tang et al. (2015) Nanoparticles impregnated gel Nanoparticles 247.1 nm Gelatin Oleic acid Physical mixing Zaltoprofen Enhanced skin permeation 171 ± 1.4 nm (genipin 10 mg) Gelatin Oleic acid EDC/NHS chemical conjugation

Irinotecan hydrochloride

Increase the hydrophobic interactions among gelatin molecules to synthesize stable, amphiphilic carriers Pawar and Pande (2015) Park et al. (2015) Liprosome (lipid-protein nanocomplex) Nanoparticles 110 nm

Bovine serum albumin Egg yolk lecithin Desolvation–ultrasonication Paclitaxel Increased drug entrapment, reduced cytotoxicity Tang et al. (2014) 182.3 ± 11.7 nm Albumin Lecithin High-pressure homogenization

Teniposide Reduction of systemic toxicity and enhanced antitumor activity He et al. (2015) Nanoparticles Below 300 nm Gelatin Oleic acid Folic acid Aqueous solvent-based method using monoethanolamine activator

Paclitaxel Targeted delivery, controlled release, and reduced side effects Tran et al. (2013) Polymer-lipid hybrid nanoparticles 253 ± 8 nm Gelatin Lecithin Two-step desolvation method Amphotericin B Increase the oral bioavailability of amphotericin B

Jain et al. (2012) 6 Wheat germ agglutinin lectin Food Hydrocolloids for Health 2 (2022) 100054 Objective of conjugation Delivery system T. Dissanayake, X. Sun, L. Abbey et al. Table 1 Applications of lipid-protein conjugation in drug delivery systems.

T. Dissanayake, X. Sun, L. Abbey et al. Food Hydrocolloids for Health 2 (2022) 100054 Fig. 2. Highlights of the lipid-protein conjugates-based delivery systems for drugs.

photericin B (Nishi et al., 2007). Jain et al. (2012) formed a lipidprotein conjugate delivery system for amphotericin B using lecithin and gelatin. Coating with gelatin supported the further stabilization of the drug, which was encapsulated within the lipid. Furthermore, gelatin coat provided additional protection in the gastrointestinal fluids and increased the structural integrity of the delivery system. These conjugated nanoparticles were formed by a simple manufacturing process based on a two-step desolvation method promoting commercial production. Gelatin has been selected as a protein due to its biocompatibility, biodegradability, low immunogenicity, and high availability at a lower cost. Lecithin has been selected as the lipid due to its high encapsulation efficiency, proven efficiency in the delivery of amphotericin B, and high compatibility. However, slight sensitivity to the harsh gastrointestinal environment could be observed in these hybrid nanoparticles. The developed hybrid nanoparticles resulted in controlled drug delivery and less hemolytic toxicity than free drug, micellar solution of amphotericin B, and liposomal formulation of amphotericin B. Corneal transplantation is the replacement of dysfunctional cornea as a solution for corneal blindness. However, this can result in reduced corneal transparency and visual impairment due to post-engraftment complications such as postoperative inflammation. Li et al. (2021) fabricated a gelatin scaffold with curcumin-loaded lipid-PLGA (poly(lacticco-glycolic acid) hybrid microparticles to address that issue due to the anti-inflammatory, anti-angiogenic, and antioxidative effects of curcumin. Curcumin-loaded lipid-PLGA microparticles were obtained by evaporating the organic solvent that was used to prepare oil-in-water emulsion of cholesterol, PLGA, and curcumin. Lipid-PLGA microparticles were conjugated to gelatin using EDC/NHS crosslinking chemistry. Results showed that incorporation of curcumin-loaded nanoparticles into gelatin scaffold was an effective technique to support the transplantation and prevent post-engraftment complications. Most interestingly, the incorporation of lipid-PLGA microparticles or curcumin did not significantly scarify the transparency of the gelatin scaffold. In a recent study, rice bran protein was used as a stabilizer for quercetin encapsulated soybean oil nanoemulsions (Chen et al., 2020). The rice bran protein increased the zeta potential to higher values due to the increasing repulsion between the proteins. In addition, the emulsifying ability of the rice bran protein was significantly enhanced at the pH of 9 with the increasing amount of rice bran protein. The results were attributed to the interactions between hydrophobic moieties of the rice protein and oil droplets and the interactions of the proteins and the aqueous phase during ultra-high-pressure homogenization. Furthermore, MTT assay results revealed that nanoencapsulation reduced the

damage to cells from quercetin due to the controlled release of quercetin by stabilized nanoemulsion system (Chen et al., 2020). Paclitaxel (PTX) is an anticancer drug that has a high potential to inhibit glioma cell growth. However, the multidrug resistance (MDR) of glioma tumor cells is a major challenge in glioma chemotherapy. One of the major causes for MDR is the overexpression of P-glycoproteins on the tumor cells, which expel the PTX. Therefore, PTX has a low accumulation in the tumor cells (Tang et al., 2015). On the other hand, the poor aqueous solubility of paclitaxel reduces its bioavailability, which very often results in treatment failure (Jibodh et al., 2013). Although the supply of P-glycoprotein inhibitors is a better solution for the MDR of tumor cells, inhibitors exhibit systemic toxicity (Lee, 2010) and low aqueous solubility (Tang et al., 2015). Tang et al. (2015) have coencapsulated paclitaxel and borneol in lipid-albumin nano assemblies to overcome the issues mentioned above related to paclitaxel. Borneol acts as a P-glycoprotein inhibitor and enhances the drug’s action in tumor cells (He et al., 2011). Receptor-mediated transcytosis and the enhanced permeation and retention effect of albumin help the paclitaxel to reach tumor cells. Albumin binds to specific receptors called glycoprotein 60 before paclitaxel enters the tumor cells (Yardley, 2013). Tang et al. (2014) showed that lecithin-albumin nano assemblies are a biocompatible delivery system for targeted delivery of paclitaxel. Pooja et al. (2016) developed a paclitaxel delivery system to enhance anticancer activity against lung cancer cells. In their study, wheat (Triticum aestivum) germ agglutinin lectins conjugated solid lipid nanoparticles (SLNs) were prepared to create a drug delivery system with enhanced biocompatibility, high drug load capacity, targeted delivery, controlled drug release, and improved bioavailability. In this system, lipid was used as the hydrophobic drug carrier, while lectins helped to increase the retention time of the drug in the intestine to enhance the bioavailability by binding to the N-acetyl-D-glucosamine and sialic acid present on the cell surfaces throughout the entire length of the intestine (Pusztai et al., 1993). Further, wheat germ agglutinin enhanced the targeted delivery by binding to the carbohydrate moiety of the glycoproteins and glycolipids, which are overexpressed in cancer cells (Gorelik et al., 2001). Fig. 3. shows the targeted delivery of drugs using lipid-protein conjugates where proteins bind to the specific biomarkers overexpressed on the cancer cells. Park et al. (2015) investigated a new method to synthesize gelatin-oleic nanoparticles using carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction. They then encapsulated the irinotecan hydrochloride model drug in the gelatin-oleic nanoparticles. As a result, hydrophobic interactions between the gelatin molecules were increased due to the 7

Fig. 3. Illustration of proteins binding to the biomarkers over expressed on cancer cells and drug encapsulated lipid nanoparticles entering the cancer cell.

conjugated oleic acids. The resulting delivery system was proposed as a promising carrier by the authors for encapsulation of anti-cancer drugs. Another interesting study by Meghani et al. (2018) led to the design of clickable gelatin-oleic nanoparticles to deliver doxorubicin model drug through an active site-specific targeting. Dibenzocyclooctyne was used as the clickable material to functionalize the gelatin-oleic nanoparticle by targeting the azide-modified sialic acid precursors on the tumor cell surface. Transdermal delivery is a better solution for the low oral bioavailability of drugs. Previous studies have shown less chemotherapeutic toxicity and high drug bioavailability of transdermal administration (El-Houssiny et al., 2015). As a breast cancer medication, ElMasry et al. (2018) encapsulated sesamol in gelatin-oleic acid conjugates to deliver the sesamol to breast carcinoma cells through the skin. In this research, gelatin has been used to encapsulate sesamol, and its efficient internalization, localization, and endocytic uptake caused high cytotoxicity to carcinoma cells (Coester et al., 2006). Oleic acid has been used to conjugate gelatin, a leading penetration enhancer in transdermal applications. Oleic acid can exist with stratum corneum (SC) lipids that present in the outermost layer of the skin (van Smeden & Bouwstra, 2016). The coexistence of more solid SC lipids and fluid oleic acid at physiological temperatures enhances the diffusion of drugs by the phase-separation transport mechanism. In this mechanism, permeable interfacial defects are increased in the SC lipids, which reduces the diffusional path length or resistance (Ongpipattanakul et al., 1991). The work of Elmasry et al. (2018) showed a significantly higher permeation of oleic acid-conjugated gelatin nanoparticles through the skin of mice compared to conventional gelatin nanoparticles and sesamol aqueous solution. Indocyanine green (ICG) is a near-infrared (NIR) contrast agent that is approved for use in medical diagnosis by the United States Food and Drug Administration (FDA) (Polom et al., 2014). However, the application of ICG in tumor imaging is limited due to aggregation-induced self-quenching in aqueous media, easy degradation by exterior light, oxidants and high temperature, and binding to plasma protein leading to subsequent rapid clearance by the liver (Yaseen et al., 2009). Therefore, potential carrier systems should be used for ICG to increase stability, protect from plasma protein binding, and enhance the circulation time. Chen et al. (2016) have suggested a human serum albumin-coated liposomal delivery system to address this issue. Kraft and Ho (2014) have stated that liposomal ICG has proved the ability of optical imaging of

sentinel lymph nodes, vascular permeability, angiogenesis, and solid tumors. However, the inherent instability of liposomes at the presence of serum proteins and payload loss limit the efficient use of liposomes as delivery systems (Shishir et al., 2018). Albumin has many desirable characteristics that can benefit efficacious delivery systems, such as the ability to accumulate in inflamed tissues and solid tumors, lack of inherent toxicity and immunogenicity, biodegradability, and ready availability (Elsadek & Kratz, 2012). Therefore, albumin coating can be introduced to improve the effective use of liposomes. For instance, the study done by Chen et al. (2016) showed remarkable enhancement in NIR fluorescence properties, tumor targeting, and stability of ICG loaded albumin-coated liposomes compared to control ICG solution. A study was done by Kabary et al. (2018) to co-deliver rapamycin and berberine in lactoferrin-hyaluronic coated lipid nanoparticles. The objective of the study was to enhance the anti-tumor efficacy of lung carcinoma cells. The lipid nanoparticles have been characterized to deliver both drugs/bioactives with a high drug payload. However, burst and premature drug release reduced the bioactivity, mainly due to the particle crystallization and drug expulsion from lipid cores. Furthermore, the RES clearance reduces the circulation of lipid nanoparticles, and therefore, surface modifications are done to lipid-delivery systems to control the release of the drug (Kabary et al., 2018). Layer-by-layer (LbL) self-assembly is a flexible and simple surface modification method based on the electrostatic deposition of oppositely charged polyelectrolytes on the nanocarrier surface (de Villiers et al., 2011). For the LbL technique, hydrophilic polymers, including proteins and polysaccharides, are mainly used due to their capability of RES clearance, enhanced blood circulation time, and enhanced permeation and retention based passive diffusion through interstitial tumors (de Villiers et al., 2011). Furthermore, the targeting ability of the nanoparticles is increased by targeting the moiety of the layering material. For instance, lactoferrin is a cationic protein that can bind to transferrin receptors and LDL receptors over-expressed on various cancer cells (Elzoghby et al., 2015). Hyaluronic acid is an anionic polysaccharide that has an inherent ability to bind with CD44 receptors overexpressed on tumor cells, including lung cancer cells (Leung et al., 2010). A recent study by Kabary et al. (2018) showed that coating nanoparticles with hyaluronic and lactoferrin enhanced drug cytotoxicity against A549 lung cancer cells due to increased cellular internalization through CD44 receptors overexpressed by tumor cells. In terms of therapeutic activity, coated nanoparticles exhibited approximately, 88% reduction in microscopic

Fig. 4. Highlights of the gene delivery by lipid-protein conjugates. lung foci number and a 3.1-fold reduction in the angiogenic factor compared to the control mice group (untreated group). In summary, anticancer activity against lung cancer cells was superiorly enhanced by hyaluronic-lactoferrin-coated nanoparticles.

Zhu et al., 2019; Zuvin et al., 2019). Fig. 4. summarizes the content covered in the 5.2 section of the review. Particular attention should be given when genes are encapsulated in carrier systems, as they can cause adverse side effects. The designed gene delivery system should have compatible physiochemical properties, including high solubility, stability, and small size. This can be achieved by chemical and physical modifications of biopolymers used for encapsulation. Also, they should protect the encapsulated gene from adverse physiological conditions and deliver it to target sites using targeting ligands. Another important factor is escaping from RES clearance and nontoxicity to blood and normal cells. Finally, the delivery systems should be able to produce continuously with a satisfied shelf-life during storage (Eftekhari et al., 2019). Fidgetin-like 2 protein should be downregulated in the wound healing process to increase the rate of cell moment. siRNA is used to knock down this protein and accelerate the cell moment, an important phenomenon in wound healing. However, siRNA instability in serum and sensitivity to nuclease degradation reduced its therapeutic efficacy. On the other hand, the cellular internalization of RNA biopolymers is reduced by their large molecular weight, high negative charge, and stiffness (Tezgel et al., 2020). siRNA was encapsulated in NLC before being loaded into collagen scaffolds to overcome the aforementioned issues. However, encapsulation in NLC limited the first burst release of naked siRNA loaded in collagen. Also, cytotoxicity of cryostructurates loaded with only NLC was significantly reduced when loaded into collagen, probably due to the reduction in surface charge of NLC as resulted by complexation with collagen (Tezgel et al., 2020). Hall et al. (2021) designed a peptide-lipid associated multicomponent nanodelivery system to deliver siRNA to prevent the overexpression of proteins in cancer cells. siRNA encapsulated nanoparticles were prepare using four types of lipids (1,2-Dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP chloride salt), cholesterol, phosphatidylcholine). The main role of peptides in this delivery system is facilitating cellular internalization due to their ability to penetrate cells. Moreover, the conjugation of siRNA with peptides through easily breakable bonds facilitates

5.2. Gene delivery Gene therapy is a relatively recent procedure used to genetically modify a patient’s cell function (Ramamoorth & Narvekar, 2015). Gene therapy has become a standard clinical intervention for treating several health issues, including cancers, cardiovascular diseases, infectious diseases, inner ear disorders, dermatological, ophthalmologic, and neurological pathologies. Gene therapy is not limited to DNA delivery but also for small interfering RNA (siRNA), antisense oligonucleotides, and micro-RNA. RNA interference-based medicine is a growing area for treating human diseases. For instance, siRNA’s ability to induce the silencing of a target gene by RNA interference (Valero et al., 2018) is an area studied by researchers. In gene delivery systems, viral vectors are replaced by the non-viral vectors as the latter are bio-safe, less pathogenic, inexpensive, and can easily be produced. However, their transgenes’ low efficiency of delivery and transient expression limit their applications in gene delivery systems. Lipid nanoemulsions, SLNs, lipoplexes, and peptide-based vectors are common non-viral natural delivery systems (Ramamoorth & Narvekar, 2015). Conjugation of lipid and protein is a successful approach in biopolymer vectors to enhance the efficient delivery of genes (Gaber et al., 2017). Gene co-encapsulation with suicide genes in protein associated liposomes (Faneca et al., 2008), siRNA delivery by protein-associated lipid-based vectors (Cardoso et al., 2007), and modification of lipid gene carriers by proteins for tumor-targeted transfection (Wang et al., 2012) are some of the lipid-protein conjugation applications in gene delivery. Gene transfection is the procedure where foreign nucleic acids are introduced to cells to produce genetically modified cells (Kim & Eberwine, 2010). The excellence of lipid-protein conjugation in gene transfection efficiency has been used to treat many chronic diseases (Leung et al., 2010; 9

the release of the gene once it reaches the target site. The study’s results showed the successful encapsulation and controlled release of siRNA in the designed delivery system and safety profiles in several human cells (Hall et al., 2021). A crucial element of gene therapy is their ability to kill a high percentage of tumor cells, even with a low percentage of transfected cells (Faneca et al., 2007). Faneca et al. (2008) coencapsulated vinblastine and HSV-tk suicide gene in human serum albumin (HAS)-associated lipoplexes to increase the lipoplex biological activity and inhibit the mitosis of cancer cells. The conjugation of HSA to lipoplexes is a better strategy to enhance the transgene expression in different types of cells, even in the presence of serum (Faneca et al., 2007). They prepared HSA-lipoplexes by incubating liposomes and proteins for 15 mins followed by further incubation with plasmid DNA solution for 15 mins. Coating lipoplexes with albumin enhanced the in vivo transfection efficiency by avoiding the undesired interactions with serum components. Furthermore, albumin supported lipoplexes to bind to cellular surfaces and the internalization via endocytosis. Consequently, the transfection of the suicide gene was significantly enhanced by the HSA. Thus, there is a significant synergistic effect on antitumoral activity by combining vinblastine with HSV-tk/GCV gene loaded with HSA-associated lipoplexes (Faneca et al., 2008). Liposomes are widely used gene carriers due to unlimited vector size, greater carrier capacity, and ease of large-scale production (Belfiore et al., 2018). However, their application is limited by low colloidal stability, inefficient controlled delivery, fast elimination, and lack of targeted delivery (Sriraman & Torchilin, 2014). Peptide groups have been introduced into liposomes to increase the targeting activity and in vivo half-life. On the other hand, peptide liposomes have a highly desirable level of biocompatibility and a high degree of interaction between delivery systems and the cells. This property is ascribed to their cationic surface charge, which ultimately enhanced their uptake by endocytosis (Zhao et al., 2015). An investigation was done to evaluate the toxicity profiles of lipid head groups by Zhu et al. (2019). This study compared a lipid with 14 carbon chains and tri-ornithine head group (peptide-based lipoplexes) with quaternary ammonium lipid 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) in terms of toxicity. It was found that the cytotoxicity of peptide-based lipoplexes against cancer cell lines was weaker than lipoplexes with quaternary ammonium lipids at low dosages. This finding confirms the biocompatibility of peptide-based lipoplexes. Furthermore, they evaluated the potential of cationic peptide-based liposomes for gene delivery. LucsiRNA gene silencing and tumor inhibition by IGF-IR-siRNA gene in vivo were evaluated in mice injected with Luc-A549 cells. It was noted that lipoplexes with tri-ornithine head groups and lipoplexes with ammonium head groups were compared. Cationic peptide-based lipoplexes efficiently delivered IGF-IR-siRNA compared to lipoplexes with quaternary ammonium lipids. The result of the study confirmed the suitability of cationic peptide-lipoplexes as gene delivery systems compared to quaternary ammonium lipoplexes. It further showed the correlation between lipid head group structure and toxicology.Surface modifications with specific ligands to facilitate receptor-mediated pathways are crucial in gene carriers (Zauner et al., 1998). Also, nuclear localization signals (NLS) enhance the nuclear uptake of genetic materials as it is crucial to deliver genes to the nucleus for proper integration into DNA and expression (Lo et al., 2012). Glucocorticoid receptor (GR) is a nuclear receptor that binds with ligands and forms receptor-ligand complex, which facilitates the translocation of DNA into the nucleus by dilating nuclear pores (Ma et al., 2009). According to Wang et al. (2012), synthesized dexamethasone (dexa) conjugated cationic SLNs (i.e. dexa-conjugated 6-lauroxyhexyl ornithinate (LHON)) for the enhancement of nuclear localization of enhanced green fluorescence protein plasmid (pEGFP) gene. The surface of the pEGFP was modified using transferrin, an iron-binding glycoprotein. Compared to normal cells, more transferrin receptors are present in tumor cells (Bellocq et al., 2003). Therefore, it is suggested that transferrin conjugation to LHON facilitates the delivery of genes into tumor

cells. In the study done by Wang et al. (2012), transferrin was conjugated to polyethylene glycol-phosphatidylethanolamine (PEG-PE) before conjugation with dexa-LHON as PEG-PE, which made it stable, longcirculating, and actively targeted nanocarriers (Lukyanov et al., 2002). During the procedure, transferrin-PEG-PE conjugates were continuously coated on the surface of gene-loaded SLNs via electrostatic interactions. The gene delivery ability was evaluated using HepG2 tumor-bearing mice. The study results showed a remarkably higher transfection efficiency for the surface modified SLNs than the unmodified nanoparticles. Based on the results, the authors have indicated that transferrin and dexa act as highly active targeting ligands to enhance the delivery system’s cell and nuclear targeting ability. RNAi has a higher therapeutic potential for many reasons compared to non-specific drugs and virus therapy. Due to higher specificity to targeted proteins, RNAi does not exhibit adverse side effects. Moreover, the high stability of double-stranded siRNA and effective knockdown at lower concentrations for longer periods improve the therapeutic efficacy. Further, the action of the RNAi takes place in the cytoplasm, and the accessibility to the cytoplasm is easier than the nucleus, which is a major obstacle to plasmid DNA delivery (Brantl, 2002). However, incomplete gene suppression and undesirable side effects due to non-specific in vivo entry into non-target cells are challenges in RNAi therapy. Therefore, delivery systems are introduced to deliver siRNA to target sites and pass the extracellular barriers (Young et al., 2016). Cardoso et al. (2007) formulated a transferrin-associated lipidbased vector to deliver siRNA. This was done by preparing cationic liposomes using 1,2-dioleoyl-3(trimethylammonium), propane (DOTAP), and cholesterol. The cholesterol demonstrated a high level of transfection when it was associated with a cationic lipid. This is because cholesterol enhances the biological stability of lipoplexes by reducing the destabilization of such lipoplexes in the presence of serum (Crook et al., 1998). Cardoso et al. (2007) studied the therapeutic potential of c-Jun siRNA-loaded transferrin-lipoplexes against glutamate toxicity in the HT-22 neuronal cell line. The transferrin was conjugated to lipoplexes by pre-incubating it with cationic liposomes before mixing with siRNA solution. In brief, the findings of their study showed that transferrinassociated lipoplexes reduced the toxicity and enhanced the specific gene knockdown activity compared to conventional lipoplexes. Weeke-Klimp et al. (2007) prepared lactoferrin coupled stabilized plasmid lipid particles (SPLPs) to target hepatocytes. Lactoferrin was used as a hepatocyte-specific targeting ligand, covalently bound to the SPLP surface. In this study, the authors evaluated lactoferrin’s ability as a targeting moiety to deliver DNA encapsulated in SPLPs to the hepatocyte as there are high affinity Ca2+ binding sites on the hepatocytes for lactoferrin to bind (David & McAbee, 1997). According to the results obtained from the in vitro study, liver uptake of SPLP was significantly enhanced due to the coupling of lactoferrin. Additionally, many lactoferrin-SPLPs were uptaken by hepatocytes in the liver, but in vivo transfection activity could not be observed. According to WeekeKlimp et al. (2007), this observation is due to the high stability of lactoferrin-SPLPs after cellular uptake, and therefore, lactoferrin-SPLPs were unable to release enough plasmid. Thus, although the authors have hypothesized that lactoferrin would contribute to the destabilization of lactoferrin-SPLPs, it did not happen. Magnetofection is a term used to describe the transferring of genes bound to magnetic nanoparticles (MP) to target sites using a magnetic field. Generally, they are coated with a cationic polymer for DNA binding (Zuvin et al., 2019). In a study by Pan et al. (2008), a novel vector for luciferase and green fluorescent protein (GFP) reporter genes was formulated using transferrin-associated lipid-coated magnetic nanoparticles. Transferrin was incubated with polyethyleneimine (PEI)-DNAcationic lipid-coated MP at room temperature for 15 mins to conjugate transferrin to form a complex. Transfection efficiency was evaluated after incubation for 15 min and 4 hr. In the 15-min incubation treatment, magnetic vectors showed more than 300-fold transfection activity compared to non-magnetic vectors. Thus, the incorporation of transfer10

Fig. 5. Summary of the issues faced by nutraceuticals and the role of lipid-protein conjugates in addressing them.

rin contributed to further improvement of the transfection efficiency of genes. Some of the applications of lipid-protein conjugation in gene delivery systems are summarized in Table. 2.

of chemical structures that contains one or more aromatic rings with two or more hydroxyl groups. They can be categorized as flavonoids, phenolic acids, and polyphenolic compounds, where flavonols, flavanones, flavonols, flavones, anthocyanins, and isoflavones belong to the group flavonoids. Due to their complex chemical structure, solubility, size, degree of polymerization, and conjugation with other compounds, bioavailability can be reduced significantly (de Araújo et al., 2021). An emulsion gel system of soy protein and olive oil was fabricated by Muñoz-González et al. (2021) to encapsulate polyphenol extract to successfully encapsulate gallic acid, flavanol monomers, catechin, epicatechin, and procyanidins. Alginate was used as a gelling agent to stabilize the gel. This system was able to trap the phenolic compounds in the gel, and composition was analyzed by High-Performance Liquid Chromatography. On the other hand, this system is also a good source for healthy proteins, mono and polyunsaturated fatty acids due to soy protein and olive oil. Also, the addition of polyphenols reduced the gel strength of emulsion gel, most probably due to the chemical interactions with the matrix. However, it can positively affect the posterior bioavailability of entrapped compounds (Muñoz-González et al., 2021). Resveratrol and curcumin exhibit a variety of bioactivities; therefore, their applications in food supplements and pharmaceutical products are high. However, low oral bioavailability, instability at physiological pH, insolubility in water, slow uptake by cells, and rapid metabolism inside cells reduce in vivo therapeutic efficacy of curcumin. Like curcumin, resveratrol also faces some issues such as poor bioavailability, poor stability, poor water solubility, and rapid metabolism inside the body (Peng et al., 2018). Liu et al. (2018) synthesized core-shell nanoparticles using zein-epigallocatechin gallate conjugated (zein-ECGCs) and a rhamnolipid coating to improve the water-dispersibility, chemical stability, and bioaccessibility of encapsulated curcumin and resveratrol within nanoparticles. Another report also showed a highly positive potential of hydrophobic proteins in improving water dispersibility and chemical stability of polyphenols by encapsulation (Patel et al., 2010). However, such delivery systems are usually unstable due to aggregation caused by relatively high surface hydrophobicity. The issue of aggregation can be managed by coating with emulsifier molecules (Patel et al., 2010). Another issue is when polyphenols are encapsulated in hydrophobic protein delivery systems resulted in poor bioaccessibility. Such poor bioaccessibility of encapsulated nutraceuticals can be enhanced by mixing with digestible lipid droplets, which form micelles in the intestinal fluids with released nutraceuticals from protein nanoparticles (McClements & Xiao, 2014). Previous studies have

5.3. Nutraceutical delivery In general discussion, nutraceuticals can be defined as components of foods that provide medical or health benefits due to their bioactivities such as antioxidant, anti-bacterial, anti-hypertensive, anti-cancer, anti-hypercholesterolemia, anti-inflammatory, and anti-coagulation. In addition, nutraceuticals can be categorized into dietary fiber, probiotics, prebiotics, polyunsaturated fatty acids, an antioxidant vitamin, polyphenols, and spices (Verma & Mishra, 2016). Fig. 5. summarizes the content covered in the 5.3 section of the review. Fish oil shows tremendous health benefits by preventing diet-related health issues such as heart diseases, cancers, and rheumatoid arthritis. However, the unpleasant smell, poor aqueous solubility, and oxidative instability prevent its potential benefits. Numerous nanodelivery systems have been developed to address the issues mentioned above by improving the bioavailability of fish oil (Li et al., 2020). Li et al., (2020) stabilized the processing and digestibility of fish oil using a nanoemulsion system consisting of soybean protein isolate (1%, 2%, 3%, 4%, 5%) phosphatidylcholine. The increased percentage of soybean protein isolate significantly reduced nanoemulsion particle size and polydispersity index (PDI). According to the authors, lower soy protein isolate concentrations were insufficient to cover the oil droplets completely, leading to larger oil droplets due to aggregation. However, further increased protein concentration to 5% resulted in increased particle size and PDI due to the formation of submicelles of excessive protein aggregates. Moreover, fish oil oxidation encapsulated in soybean protein isolate- phosphatidylcholine nanoemulsions was lower than in Tween 20 emulsions. This can be explained by the higher ability of proteins to inhibit lipid oxidation than small-molecule surfactants. Also, due to the high surface charge on the soybean protein isolate- phosphatidylcholine nanoemulsions compared to Tween 20 emulsions, they exhibited higher ionic strength stability that is explained by the masking of the charge neutralization effect of Na+ ions by large electrostatic repulsion between droplets (Li et al., 2020). Polyphenols are a group of secondary plant metabolites produced by plants involved in plants’ defense mechanisms to protect them from oxidative stress, UV radiation and attract pollinators. This broad and heterogeneous group of secondary plant metabolites has a wide range 11

Cardoso et al. (2007) Weeke-Klimp et al. (2007) Ilarduya et al. (2006) Nakase et al. (2005) Enhanced gene silencing at minimum toxicity by targeted delivery Enhanced transfection of the gene (only observed in in vitro) Enhanced antitumor activity transfection efficiency Enhanced tumor growth inhibition

Cardoso et al. (2008) Enhanced gene silencing activity Electrostatic interactions Transferrin Transferrin Lactoferrin Transferrin Transferrin Lipoplexes Lipoplexes Lipoplexes Lipoplexes

Electrostatic interactions Chemical conjugation Electrostatic interactions Electrostatic interactions

Chemical conjugation Electrostatic interactions Electrostatic interactions Electrostatic interactions Electrostatic interactions Electrostatic interactions Transferrin Albumin Transferrin Albumin Albumin Transferrin

Lipoplexes Lipoplexes SLNs Lipoplexes Lipoplexes Lipid coated magnetic nanoparticles Lipoplexes Nanostructured lipid carriers Lipoplexes

shown that the co-encapsulation of polyphenols increased bioactivity (Niedzwiecki et al., 2016). Liu et al. (2018) fabricated the hydrophobic core of the nanoparticles by zein-ECGCs with the main objective of stabilizing encapsulated nutraceuticals by the high antioxidant activity of catechin (ECGC). As a summary, lipid-protein conjugated delivery systems can be used to co-deliver contrary soluble bioactives where lipid portion is allocated for the hydrophobic bioactives and remaining hydrophilic bioactives can be encapsulated in the protein portion (Fig. 6.) In another study, rhamnolipid was used to fabricate a delivery system aiming to prevent nanoparticles from aggregation. When the rhamnolipid is absent, thick sediment was observed when zein-ECGCs, dissolved in an ethanol solution, was dropwise added to an aqueous solution. Authors have explained that relatively high surface hydrophobic attractions and weak electrostatic repulsion between nanoparticles cause sedimentation. Rhamnolipids reduce the hydrophobic attraction between nanoparticles by adsorbing the non-polar patches on the nanoparticles to their non-polar parts. Thereby, the enhanced absolute value of the zeta potential increased electrostatic repulsion. Besides, rhamnolipids coated nanoparticles resulted in stable homogeneous colloidal dispersion. Furthermore, nanoparticles exhibited improved chemical stability at pH of physiological conditions and the presence of UV irradiation conditions. Moreover, both curcumin and resveratrol further showed a strong antioxidant activity with remarkable benefits for industrial applications (Liu et al., 2018). The bioaccessibility of curcumin was increased by mixing curcuminloaded zein nanoparticles with curcumin-free digestible lipid nanoparticles (Zou et al., 2016). The main advantage of such a delivery system is harnessing the synergy between protein nanoparticles and lipid nanoparticles. In that case, zein nanoparticles were prepared by an antisolvent precipitation method to achieve a high loading capacity and good chemical stability. Then, the digestible lipid nanoparticles prepared by high-pressure homogenization (microfluidization) were mixed with curcumin-loaded zein nanoparticles. The key goal of having lipid nanoparticles was to increase the bioaccessibility of curcumin by the formation of mixed micelles in the intestine, which can solubilize and transport the hydrophobic curcumin. Zou et al. (2016) combined the effective activities of protein (zein) and lipid nanoparticles into one delivery system. A relatively high positive zeta potential (+20 mV) was recorded for zein nanoparticles as it had a pH value below the isoelectric point (pH=4). Conversely, the net charge of lipid nanoparticles was close to zero. In the end, all mixed nanoparticles showed a net charge near zero, which showed the domination of lipid nanoparticles. As explained by the authors, lipid nanoparticles scatter light stronger than protein nanoparticles due to their larger size. Studies showed that lipid nanoparticles enhanced the bioaccessibility of curcumin encapsulated in zein nanoparticles compared to the delivery system in which the lipid nanoparticles are absent (Zou et al., 2016). Vitamin B12 is an essential vitamin that should be ingested with supplements or fortified food (Brito et al., 2018). It promotes human health and lowers the risk of several chronic diseases. Nutraceuticals can be inactivated or decomposed by harsh gastric environmental conditions such as low pH and pepsin enzyme before reaching target sites (Date, 2016). Furthermore, nutraceuticals with a small size with a large surface area can be affected by environmental stressors such as temperature, pH, and ionic strength. (Zhao et al., 2014). On the other hand, mucosal layers act as a steric barrier in the small intestine, reducing the interaction between nanoparticles and epithelial cells (Liu et al., 2019). Lipid-protein composite nanoparticles were prepared with a three-layer structure using whole barley (Hordeum vulgare) protein layer, phospholipid layer, and 𝛼-tocopherol layer to deliver vitamin B12 . The phospholipid layer and 𝛼-tocopherol layers were separated and stabilized by the barley protein layer in a scaffolded manner and a hydrophilic vitamin B12 was encapsulated in the inner aqueous compartment. This structure can overcome most of the shortcomings related to liposomes and double emulsion-based delivery systems including instability and leaking in the gastric environment. On the other hand, it

Anti-luciferase or anti-c-Jun siRNA c-Jun Luciferase IL-12 P53

Cinci et al. (2015) Piao et al. (2013) Wang et al. (2012) Weecharangsan et al. (2009) Faneca et al. (2008) Pan et al. (2008) Enhanced targeted delivery and antibody-based immunotherapy Enhanced transfection efficiency of siRNA Targeted delivery of genes to tumor cells Enhanced cellular uptake and transfection efficiency Enhanced transfection of suicide gene Enhanced transfection efficiency of genes

Tezgel et al. (2020) Zhu et al. (2019) Prolonged gene release, reduced cytotoxicity Reduced cytotoxicity and enhanced gene transfection Food Hydrocolloids for Health 2 (2022) 100054

AF488-siRNA and siERK1 Luc-siRNA and IGF-IR-siRNA Anti-mCRP siRNA phrGFP siRNA pEGFP G3139 HSV-tk GFP/luciferase Mixing with gel Chemical conjugation (carbamate linkers) Refs. Objective of conjugation

Facilitating cellular internalization, controlled release of gene siRNA Gene Conjugation method Lipid nanoparticles Desolvation Protein Linear and cyclic fatty acyl-conjugated and hybrid peptides Collagen Tri-ornithine

Lipid delivery system Table 2 Applications of lipid-protein conjugation in gene delivery systems. Hall et al. (2021) T. Dissanayake, X. Sun, L. Abbey et al. 12 T. Dissanayake, X. Sun, L. Abbey et al. Food Hydrocolloids for Health 2 (2022) 100054

Fig. 6. Co-encapsulation of hydrophobic bioactive compounds in lipids and hydrophilic bioactive compounds in proteins to co-deliver contrary soluble active compounds in a lipid-protein conjugated delivery system.

can efficiently encapsulate hydrophilic nutraceuticals as the interaction is not required between the active compound and the delivery system. Protein conjugation to nanoparticles increases the encapsulation efficiency of vitamin B12 , as well as increases resistance to digestion in the stomach environment and controlled release of vitamin B12 (Liu et al., 2018). However, the results of a previous study that was done by the same group showed instability of such three-layer- lipid-protein composite nanoparticles in the presence of salt due to aggregation. In addition, due to the lack of reinforcement of the coating, core ingredients in nanoparticles have leaked during storage. Also, in simulated intestinal environments, when the pancreatic enzyme presented, degradation of nanoparticles was fast. These shortcomings resulted in poor absorption efficiency. However, Liu et al. (2019) modified the three-layer structure to address the above shortcomings. The basic principle of succinylation is derivatization of 𝜀-amino groups (lysine and arginine) in proteins with succinate. It was found that succinylation increases the surface charge of protein while improving the solubility in water. Besides, this reduces the tryptic digestion of protein as the succinyl-lysyl-peptide bond is resistant to tryptic hydrolysis. Therefore, surface-modified proteins by succinylation were used to design the three-layer structure of nanoparticles. Surface-modified nanoparticles by succinylation (M-NP) showed higher stability in the physiological environment than unmodified nanoparticles (O-NP). During 30 days of storage, in M-NPs, vitamin B12 leakage was only 4.5 ± 0.5%. This result indicated the very little deformation of nanoparticles during storage. In O-NPs, 20% of vitamin B12 leakage was observed on the 14th day, and over half of vitamin B12 was released on the 30th day. Improved stability of M-NPs can be explained by increased nanoparticle surface charge that gives a strong electrostatic repulsion force and reduces the chance of colliding and agglomerating among nanoparticles. Also, the spatial extension of the succinate chain on the nanoparticle surface and the succinate’s crosslinking effect contributes to the improved stability of the nanoparticles. Besides, the gastric release profiles prove the resistance of M-NPs in the harsh gastric environment and sustainable release over a reasonable period (Liu et al., 2019).

426 is a liposomal formulation that is coated with transferrin to encapsulate oxaliplatin. 2B3–101 is a doxorubicin-loaded liposomal formulation coated with glutathione. As reviewed by Moncalvo et al.(2020), Epaxal®, Inflexal-V®, Curosurf®, T4N5 liposome lotion, Hepatic-directed vesicles-insulin (HDV-1), BiphasixTM, and IL-2 liposomes are liposomal delivery systems for encapsulation of proteins accepted for clinical use. However, according to our knowledge, even though the lipid- and protein-based drug delivery systems are available in commercial use, protein-lipid conjugates-based delivery systems are hard to find at the commercial level. Therefore, there is a need for research studies that can fulfill the gap between laboratory level and commercial level use of lipid-protein conjugates for bioactive delivery. 7. Toxicity of protein- and lipid-based delivery systems Regardless of the vast range of benefits of nanodelivery systems, the toxicological and negative clinical effects of the developed delivery systems should be addressed. Nanoparticles play a huge role among nanodelivery systems due to their proven therapeutic efficacy. However, based on the size, shape, charge, chemical reactivity, dosage, and surface area of the nanoparticles, they can damage living cells due to physical and chemical interactions. As a result of physical interaction between nanoparticles and membranes of the living cells, the membrane can be damaged and protein folding capacity and activity of the membrane can be sacrificed. In terms of chemical interactions, oxidative damage is caused by reactive oxygen species (ROS) (Ahmad & Ghosh, 2020). ROS induce several physiopathological conditions such as hypertrophy, apoptosis, necrosis, genotoxicity, fibrosis, carcinogenesis, inflammation, and metaplasia. Production of ROS is further increased by enhanced expression of pro-inflammatory cytokines and activated inflammatory cells by nanoparticles. Therefore, the design of nanoparticles with reduced ROS production is an urgent need using techniques such as coating of nanoparticles with additional materials and designing of ROS scavenging nanoparticles (Yu et al., 2020). Moreover, research studies have shown smart drug delivery systems such as liposomes and micelles cause toxicity to macrophages and U937 cells, DNA damages due to the surface charge of liposomes, and transient immunogenicity in mononuclear phagocyte systems (Hossen et al., 2019). Plant-based proteins have emerged as an excellent source to replace the animal-based proteins in delivery applications due to the limitations of animal-based proteins, including pathogenic infections of contam-

6. Lipid-protein conjugates-based products undergo clinical trials and commercial production Gaber et al. (2017) summarized the protein-lipid-based nanoformulations that undergo clinical trials such as MBP-426 and 2B3–101. MBP13

T. Dissanayake, X. Sun, L. Abbey et al. Food Hydrocolloids for Health 2 (2022) 100054 inated animal tissues, higher cost, and rejection from consumers due to religious beliefs and personal beliefs choices. However, plant-based proteins such as gliadin can trigger the immune response in celiac disease due to the simulation of Th1 and Th17 cells, leading to villous atrophy and malabsorption. With that concern, the administration of bioactives encapsulated in proteins like gliadin should be considered (Malekzad et al., 2017). Even though the delivering hydrophobic bioactives using lipid-based delivery systems play an active role in nanodelivery, there are many concerns over the potential toxicity of these carriers. One of the major concerns is higher penetration through the biological barriers such as the mucus layer and epithelium cells. It is expected the digestion of nanoemulsions in the upper gastrointestinal tract (GIT), but there are some exceptions such as reaching a high amount of undigested lipid droplets to small intestine due to containing indigestible oils, indigestible food components such as dietary fiber, and resistance to digestion in the upper GIT. Moreover, surfactants and alcohols used for the formulation of delivery systems support penetration through biological barriers. On the other hand, these delivery systems can increase the bioavailability of active compounds to a level that becomes toxic. The other side of these delivery systems is they become a reason to dysregulate the metabolism and hormonal functions due to the rapid digestion of lipids and thereby sudden increase in blood lipids. Also, the other ingredients used for nanoemulsions, such as synthetic emulsifiers, can result in adverse effects due to their toxicity and need in high amounts as nanoemulsions have higher surface area than traditional emulsions (McClements, 2021). Moreover, as electrical charge, digestibility, interactions, size, shape, and the native format of the proteins and lipids are changed during the encapsulation, there is a risk for potential toxicity where the reported studies are very limited in this area. Also, the coatings and electrostatically conjugated proteins and lipids become intact in the GIT. They may inhibit the digestibility of the encapsulated active compounds, which leads to potential toxicity (McClements & Xiao, 2017).

future research studies can be designed to develop new food products containing proteins and lipids which can act as delivery systems for nutraceuticals rather than designing them as capsules or supplements. In fertilizer delivery, existing delivery systems such as chitosan can be replaced by lipid-protein conjugates to compare the performance. One of the major issues of fertilizers is their negative impact on the environment. Therefore, natural polymer conjugates can be explored to synthesize environmental friendly fertilizer delivery systems. Furthermore, the gap between laboratory trials of these delivery systems and commercial level production is still not well addressed. Most of the studies have been limited to the laboratory, and there is a lack of clinical trials and fabrication strategies for commercial level production in a reproducible manner. Declaration of Competing Interest All authors do not have any conflict of interest.