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A little more unknown but not less important is the role that lncRNAs play in the adaptive immune response. In humans, the differentiation of naive T lymphocytes to Th1 cells is controlled by lincRNA-MAF-4, which promotes the epigenetic silencing of MAF, a Th2 cell transcription factor. This lncRNA interacts with the LSD1 and EZH2 transcriptional repressors to place the H3K27me3 mark in the MAF promoter and suppresses its expression. The transcription of lincRNA-MAF-4 is exclusive to Th1 lymphocytes and it is not expressed in naive T cells or any other lymphoid lines (Ranzani et al., 2015) . On the other hand, Th2 lymphocytes specifically express a lncRNA, dependent on the Th2 "master regulator" GATA3, called LincR-Ccr2-5 ′ AS. Using a cell transfer model, it was shown that the decrease of LincR-Ccr2-5'AS using shRNA reduced the expression of Ccr1, Ccr3, Ccr2, and Ccr5, as well as the migration of Th2 effector cells to the lung of recipient mice (Hu et al., 2013) . In memory CD4 + T cells, the production of IL-2 in response to TCR signaling depends on the activation of the nuclear factor of activated T cells (NFAT) (Dienz et al., 2007) . This process can be regulated by NRON, a lncRNA that acts as a repressor, reducing NFAT levels in the nucleus. For this, NRON forms a complex with importin-beta1 and LRRK2, controlling the nucleocytoplasmic traffic of the NFAT (Figure 1D ; Willingham, 2005; Liu et al., 2011) . |
Traditionally, immunological memory has been a characteristic reserved only for adaptive immunity cells. However, several studies have shown that innate immune cells, are capable of generating a type of immune memory known as trained immunity or innate immune memory, in response to prolonged exposure of microbial components, thus being able to remember transcriptional responses and even inherit these memories to their progeny (Kleinnijenhuis et al., 2014; Kaufmann et al., 2018; Hole et al., 2019) . For instance, macrophages derived from trained hematopoietic stem cells (HSCs) are epigenetically reprogrammed and as a result can strongly express immune genes, increasing their ability to resolve an infection (Kaufmann et al., 2018; Mitroulis et al., 2018) . The development of this type of immune memory is accompanied by a stable accumulation of epigenetic marks on promoters of multiple immune genes allowing the characteristic priming of innate memory (Hole et al., 2019) . Although, it is not very clear how these marks are specifically accumulated on immune genes of trained cells, a recent study shows an active participation of a group of lncRNAs, called Immune-gene priming lncRNAs (IPLs), in the trained immunity . |
On the other hand, several immune genes that are distant from each other (from a one-dimensional perspective), but functionally related, can be spatially grouped into threedimensional chromatin microdomains called TAD (topologically associating domains), allowing their proximity and coregulation. Interestingly, many TADs integrate regions that encode lncRNAs, which are actively transcribed along with co-regulated genes (Fanucchi and Mhlanga, 2019; . In this context, IPLs are described as priming mediators of immune genes located in the same TAD that contains them. These lncRNAs are transcribed within these microdomains in order to direct histone modifying enzyme complexes to the set of co-regulated gene promoters, located in the same TAD, inducing their priming by epigenetic labeling. A good example is the upstream master lncRNA of the inflammatory chemokine locus (UMLILO). This IPL contacts the TAD of ELR+ CXCL chemokine genes (which contains the genes CXCL8, CXCL1, CXCL2 and CXCL3), recruiting the WDR5-MLL1 complex to direct it toward the promoters of the genes present in the TAD and catalyze the epigenetic priming, placing the H3K4me3 brand ( Figure 1E ). In mice, the TAD that groups these chemokines lacks an IPL, therefore the expression of these chemokines cannot be trained. Surprisingly, the insertion of UMLILO in the TAD of murine macrophage chemokines resulted in the training of Cxcl genes, providing strong evidence that lncRNA-mediated regulation is essential for the establishment of trained immunity . |
The study and identification of lncRNAs as active elements of gene regulation during the immune response adds a new level of complexity to its analysis. Its ability to dialogue with several proteins, allow them to control aspects ranging protein traffic to remodeling of the chromatin architecture. Despite the existence of several reviews that include the role of lncRNAs in immunity, we consider it important to highlight the role that they play in establishing trained immunity, a feature of the immune system that has even been recently considered as a tool for reducing susceptibility and severity of SARS-CoV2 infection (Netea et al., 2020) . Until now, the genetic approaches used by suggests that the "memory implantation, " require the local expression of the immune-gene priming lncRNA (IPL). |
If modulation on epigenetic regulators can affect a large number of genes and therefore exhibit unwanted effects, the use of lncRNAs could provide the specificity that is required. |
Understanding the factors that dictate the folding state of a given lncRNA, as well as the identification of structural motifs involved in the formation of multicomponent-complexes, may contribute to the design of new therapies. Based on the above, we infer that future advances in the study of lncRNAs could focus on their active participation in the establishment of trained immunity, which could be the beginning for the development of a new generation of immunomodulators, which point to the priming of innate immunity cells, making it more efficient against an immune reaction, as well as helping reverse immunotolerance states. Supplementing the knowledge about lncRNAs with the use of local expression tools (Xu et al., 2019) , could bring us closer to obtaining the precision we are looking for. |
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. |
Though discovered in 1939 in bovine milk, lactoferrin (Lf) was reported to be an iron-containing protein [1] . Then after, its structure and chemical properties were detailed in a study published in 1960 [2] . Subsequently, Lf was found to be related to the superfamily of iron-binding glycoproteins, namely transferrins [3] . Beyond this point, Lf emerged as a focus in many research disciplines, aiming to discover its multitude functions. In this regard, we carried out a search on PubMed library to assess the progress in Lf research, in particular, Lf-based nanoparticles in cancer therapy. Our search results showed an increasing pattern of research on Lf role in cancer therapy, since emerged as early as 1992 (Fig. 1) . Furthermore, the rising progress to the field of nanomedicine by 2005 dragged the researchers' attention towards Lf and its potential application as a drug nanocarrier, and more specifically, the implications of Lf nanoparticles in cancer therapy [4] . Notably, Lf research patterns reached its maximum interest in 2018, with about 21 published articles discussing various roles of Lf in cancer therapy, and 32 research papers about Lf nanoparticles of which 11 articles focusing on cancer therapeutics. Since Lf is a natural protein present in the milk, the chances of eliciting any adverse immune reaction would be minimal [5] . Primarily, the particles fabricated based on Lf protein prepared by mild methods that do not involve any chemical reactions. Studies of our laboratory and other research groups on Lf NPs have shown excellent safety properties even after systemically administered at high doses concentrations with the liver and hematological biochemical parameters are retained [6] . |
with two ferric ions. Apo-Lf has an open conformation and is susceptible to proteolysis while holo-Lf has a closed conformation and is more resistant to proteolysis [9, 11] . Human milk Lf is linked to poly-N-acetyl-lactosaminic glycans in each lobe while bovine Lf contains α-1,3-linked galactose moieties in the terminal non-reducing location. These glycans increase the stability of Lf against proteases and acidic pH [9, 12] . |
Initially, Lf interacts with sulfated proteoglycans at the cell surface and then binds specifically with membrane receptors to stimulate ERK1/2 and PI3K/Akt pathways in the cells. Lf membrane internalization receptors include low density lipoprotein related proteins (LRP1/CD91, LRP2/Megalin), transferrin binding receptors (TFR1, TFR2), as well as ferritin and ferroportin required for iron transfer. The expression of receptors is commonly higher at the surface of cancer cells due to their high metabolic rate [13, 14] . In addition, Lf can enter the cells via charge-based interaction where its positive charge enables its interaction with negatively charged cell surface glycosaminoglycans [15] . Meng et al. developed Lf-conjugated Ntrimethylated chitosan NPs that have displayed significantly higher cellular uptake into 16HBE and SH-SY5Y cells compared with the negatively charged PLGA NPs [16] . The cationic NPs were easily attracted to cells because of the electrostatic interactions between the negatively charged cell membranes and positively charged NPs. An additional Lf receptor is intelectin 1 (Omentin-1), which is a lectin expressed in intestinal epithelia responsible for Lf uptake [17] . Moreover, the intestinal cells also express iron absorption receptors such as divalent metal ion receptor (DMT1) receptors that enhance intestinal uptake of Fe-Lf [18] . Furthermore, Lf binds to Toll-like receptor 4 (TLR4) that is responsible for Lf-mediated induction of NF-kB pathway and C-X-C-motif cytokine receptor 4 (CXCR4) that facilitates Lf-mediated stimulation of Akt signaling [19, 20] . |
After oral dose of bLf, the absolute oral bioavailability was nearly 1% because of its protein nature [21, 22] . In addition to its hydrolysis by pepsin, ionization of the amino acids responsible for holding Lf structure is changed by the stomach low pH leading to changes in its secondary and tertiary structure [21, 23] . Next, Lf is further completely broken down by the small intestine proteases (trypsin, chymotrypsin, amino and carboxyl peptidase). Other studies found that Lf was partially degraded by trypsin into fragments of different sizes, where the larger fragments (>~30 kDa) showed resistance to proteolysis [21, 23] . In contrast, Lf was found to survive enzymes in the small intestine of adult rats [21] . Moreover, holo-Lf can survive pepsin digestion in the stomach if the acidity is not enough [24] . Intact Lf molecule reached peripheral blood from the intestine 10 to 20 min after intragastric intubation and localized in different tissues [22] . After bLf resists the intestinal enzymatic degradation, it binds to LPR receptors on M cells in payer's patches and absorbed into blood and tissues in an immunoreactive form . Takeuchi et al. showed that intraduodenally administered bLf enters the body through the intestinal lymphatic pathway and reaches the whole body through the thoracic lymph fluid [21] . Overall, gastric-mediated degradation of Lf is controversial. A clinical study conducted by Troost et al. found that Lf is systematically absorbed through the gastrointestinal tract (more than 60%), in its intact form, after ~ 30 min. The pH range (4.0-7.0) that allowed the absorption of Lf was higher than the pH needed for digestion with pepsin (1.5-2.0) [25] . However, another clinical trial demonstrated contradictory results [26] . The mechanism of Lf degradation, as well as its degradation products, are still poorly understood. |
Lf has a short half-life in the body estimated to be about 10 min after i.v. administration [21] . According to Peen et al., recombinant human Lf has a half-life of about 12.6 min and completely removed after 7 hr of injection [27] . Once it is transferred by the lymphatic pathway across the intestinal wall, it is rapidly internalized by the liver and excreted into the gall bladder [21] . In the liver, Lf is rapidly cleared from circulation by the action of LRP-mediated internalization by hepatocytes [28] . Intact and fragments of Lf of maternal origin were found in breast-fed infants' urine, so kidneys must be responsible for part of the excretion of Lf [9, 27] . Another way to clear Lf from the circulation and interstitial space is by the phagocytes where Lf is engulfed by receptor-mediated endocytosis. The iron carried by the Lf is then carried by ferritin [9, 27] . With this very short half-life, there is a thought that the Lf carried inside lymphocytes survive for longer time and carry out its function in the body [21] . |
Lf possesses a wide array of different functions including anticancer activity, anti-inflammatory, a potential role in bone health preservation and cognitive function improvement in patients with Alzheimer's disease (Fig. 2) . |
Being an iron-binding glycoprotein, Lf was hypothesized to possess a key role in intestinal absorption of iron across the brush border membrane of human enterocytes owing to the presence of specific receptors for Lf [29] . A recent meta-analysis study concluded that daily oral administration of bLf showed similar efficacy to ferrous sulfate with lower gastrointestinal complications in pregnant women with iron deficiency [30] . On the contrary, using a genetic mouse model of Lf deficiency (LFKO), the ablation of Lf resulted in a mild iron overload [31] . These studies anticipate that the role of Lf in iron absorption is controversial, and that Lf might function by scavenging free iron in the gut, controlling microbial pathogenesis and prevent freeradical induced cellular damage [32, 33] . |
J o u r n a l P r e -p r o o f 3.2.1. Anti-bacterial activity. Lf exerts its antimicrobial action towards a wide array of microbes such as bacteria, fungi, viruses and parasites by two different mechanisms [34, 35] . (a) A bacteriostatic activity mediated by sequestering free iron essential for bacterial growth and proliferation; (b) A bactericidal function, Lf may interact directly, through its highly cationic Nterminus, with the negatively charged lipopolysaccharide of gram-negative bacteria causing cell membrane damage [36] . In addition, supplementation with Lf may decrease the risk of necrotizing enterocolitis by altering the intestinal microbiota in premature infants [37] . A randomized study on H. Pylori positive patients reported that bLf in combination with a 7-day triple therapy was effective with higher eradication rates than other regimens [38] . A specific bactericidal activity was reported for Lf and Lf chimera (synthetic Lf peptides). The Lf bacterial killing mechanism was attributed to membrane disruption followed by intracellular internalization [39, 40] . Then, Lf or Lf chimera would result in alteration of gene expression. For instance, PspA, an antigen which protects pneumococci against host complement system, was down-regulated by treatment with Lf chimera. Whereas, treatment with Lf resulted in decreased expression of luxS gene, and therefore, inhibiting biofilm synthesis [39] . The bactericidal activity of Lf-chimera, could be extended towards their ability to cause perturbation for bacterial cells, leading to programmed cell death type II. Moreover, membrane perturbation of V. Cholera was due to binding of Lf and Lf-peptides to specific sites on V. Cholera membrane [40] . |
Lf showed antiviral efficacy against HIV, HCV, HBV, Influenza viruses, HPV, and poliovirus, by direct binding to the surface of the virus [41] , iron scavenging or by competition for binding to host cells [42, 43] . This is mediated by its ability to activate NK cells and enhance aggregation and adhesion of neutrophils. According to Lang et al., Lf blocks the binding of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) to the host cell [44] . Thus, Lf could prevent the infection of SARS-CoV by inhibiting the early attachment of viral spike protein to the heparan sulfate proteoglycans (HSPGs) at the host cell surface (Fig. 3) . The over-expression of the anti-apoptotic protein Bcl-2 has showed an activity against the viral nucleoprotein, preventing the translocation of the viral components from the nucleus to the cytoplasm [45] . Similarly, it was demonstrated that Lf can inhibit caspase-3 activity, preventing influenza virus-induced apoptosis and hence, it results in sequestration of viral nucleoproteins into the nucleus, preventing virus assembly and propagation in MDCK cells [46] . Additionally, Lf binds to viral III and IIIa polypeptides of Adenovirus causing infection neutralization [47] . An anti-HCV activity was demonstrated by a decrease in the RNA titer with Lf monotherapy; suggesting the effective use of Lf as an adjuvant therapy for Ribavirin and Interferon in HCV treatment [48] . Upregulation of Lf and allocations to HSPGs to prevent initial contact between virus particles and subsequent infection [44] . |
Lf may exert its antiparasitic action through iron scavenging and depletion [49, 50] . Additionally, an amoebicidal activity was explained by binding of apo-Lf to the lipid membrane of the parasite causing its disruption and damage [51] . The spectrum of Lf activity was further extended to plasmodium spp. Lf inhibits the sporozoite invasion by competition on heparan sulfate proteoglycans (HSPG) found on hepatocytes [52] and also inhibits the growth of P. falciparum by generation of oxygen free radicals and damaging the membrane of the parasite [53] . In addition, an antifungal activity of Lf was demonstrated against Candida spp. by competing with the pathogen for the iron [54] . Recently, Lf was shown to act synergistically with lactoperoxide; for the prevention of oral candidiasis [55] . |
Lf was reported to exhibit anti-cancer efficacy via different mechanisms [56] [57] [58] . Table 1 summarizes some of the most promising anti-carcinogenic mechanisms of Lf. GBM hLf Suppression of the proliferation of NMD and FN primary cells as well as U87MG cells by reducing the expression of cyclin D1 and D4. [72] highly metastatic cancer cells PC-3, Mg-63, Hs 578T and MDA-MB-231, characterized by higher levels of V-ATPase compared to the non-cancer cells [73, 74] . The selectivity of bLf compared to other conventional V-ATPase inhibitors is also mediated by suppression of the lysosomal V-ATPase [75] . Moreover, the anticancer apoptotic activity of Lf was shown to be indirectly related to the inhibition of inhibitors of apoptosis (IAP), for instance, Survivin. Survivin is expressed in various cancer cells and inhibits the effect of caspase leading to cancer evade apoptosis. Lf causes down-regulation of Survivin gene expression. The anti-survivin activity was reported by nano-formulated bLf against colon CSCs and tumors (Fig. 4 ). More importantly, Lf up-regulates inhibitors of IAP, HTRA2 and SMAC, which activates the cleavage of caspases, mainly caspases 8, 9, and 3. Therefore, induction of apoptosis in cancer cells via Lf was attributed, mainly, to inhibition of IAPs [76] . Lf also rapidly internalizes into cancer cells and increases the sensitivity of resistant tumors to the action of chemotherapeutics such as doxorubicin (DOX) thus overcoming chemo-resistance [77] . In addition, Lf nanocarriers could increase the expression of cytokines TNF-α, and IFN-γ with anticancer activity. Interestingly, the iron saturation of Lf could be linked to its anticancer efficacy so that the iron saturated holo-Lf exhibited higher anti-tumor efficacy than apo-Lf or native Lf while the mechanism is still unclear [78] . |
The ability of Lf to promote wound healing is well reported through different mechanisms [79] . Talactoferrin (TLf), a recombinant human Lf, helps production of pro-inflammatory cytokines IL-6, IL-8, TNF-α and MIP-1α. It also increases the expression of hyaluronan required for forming granulation tissue, upregulates platelet derived growth factor and promotes keratinocyte proliferation and migration essential for wound re-epithelization, in addition to cellular protection from apoptosis [80] . TLf was found to be safe and effective in Phase II clinical trials in patients with diabetic ulcers [81] . |
Chitosan microparticles (mean size 4.9 µm) were developed via emulsification-solvent evaporation for controlled release of Lf (Table 2 ) [82] . The microparticles demonstrated high Lf loading (16.7%, w/w) together with gradual controlled release of Lf over a period of 24 hr. However, the microparticles may be disintegrated in the gastric acidic conditions due to solubility of chitosan in acidic pH so it was not suitable for oral delivery. Therefore, Lf was encapsulated into calcium alginate microparticles prepared by electrostatic complexation then coated with chitosan. The microparticles maintained their integrity in the GIT at both pH 1.2 and 6.8 which would be useful for the intestinal delivery of Lf [83, 84] . |
To increase its oral absorption, cationic Fe-bLf was electrostatically adsorbed onto calcium phosphate nanocrystals (NCs) and then coated with a thin layer of chitosan. The chitosan-coated NCs were further encapsulated in alginate gel to provide protection against GIT degradation [18] . In the acidic stomach environment, alginate exists in unionized state forming a matrix around the inner core to protect the entrapped Fe-bLf. Reaching the alkaline intestinal pH, the ionized sodium alginate is formed releasing the Lf content. The mucoadhesive nature of FebLf NCs, mediated by the interaction between the positively charged of both iron and chitosan with the negative charged of the mucus, enabled increased internalization in the intestinal tissue layer of the nanocarriers. Moreover, LPR and DMT1 receptors on intestinal cells help the absorption process of the nanocarriers (Fig. 5 ) [14] . The alginate/chitosan-coated Lf NCs displayed promising activity against different types of solid tumors [14, 85] and osteoarthritis [86] . Maximal Uptake was achieved through iron receptors; DMT1 in addition to LRP receptors. Through EPR mediated effect, the NPs reached cancer tissue and subsequently internalized through pinocytosis utilizing Tf and Lf receptors [14] . |
Incorporation of bLf into liposomes was found to improve its anti-inflammatory action following its oral or intra-articular administration by improving its resistance to gastric digestion and hence increasing its intestinal absorption [87] . Moreover, liposomal Lf showed higher cytotoxicity against cancer cells by increasing its intracellular accumulation and protecting from lysosomal or proteasomal degradation [88, 89] . |
PEGylated Lf can be synthesized by direct conjugation between and the free amino moieties of bLf and the branched PEG N-hydroxysuccinimide (NHS) esters. Alternatively, bLf could be coupled to two p-nitrophenyl esters of linear 5 and 30 kDa PEG [90] . Compared to PEG-NHS, slower reaction rates but remarkably higher degradation stabilities could be achieved using PEGp-nitrophenyl active esters. PEGylation of Lf improved its oral bioavailability so that the proteolytic half-life of 20k and 40kDa-PEG-bLf has been increased by 2-fold and 6-fold, respectively compared to unmodified Lf [91, 92] . Intragastric injection of 20k-PEG-bLf at a dose of 30 mg/kg into rat stomach increased its intestinal absorbed amount by 10-fold and prolonged plasma half-life by 5.4-fold relative to unmodified Lf. PEGylated bLf enhanced its hepatoprotective effect against CCl4-induced liver injury by increasing the SOD activities and ROS scavenging in mitochondria, leading to the reduced activity of AST and ALT [93] . The 40k-PEG-bLf displayed enhanced hepatoprotection and improved pharmacokinetics in comparison with both unmodified bLf and 20 k-PEG-bLf conjugates. Liposo. Lf exhibited powerful anti-inflammatory action against CCl4-induced liver injury. |
[89] |
Alginate chitosan calcium phosphate bLf nanocapsules: AEC-CCo-CP-bLf-NCs. |
J o u r n a l P r e -p r o o f |
In this technique, the drug is simply mixed with the protein aqueous solution in the presence of oil phase. When the oil phase was added to the soluble mixtures of drug and protein, the drug was adsorbed to protein molecules forming emulsified aggregates. The complex aggregates were disaggregated by sonication and then cooled resulting in their precipitation with formation of solidified drug-protein particles. Finally, the formed particles are cleaned off oil. Different drugs e.g. temozolamide [96] , fluorouracil (5-FU) [97] , Oxaliplatin [98] and siRNA [6] have been successfully loaded into Lf by this method ( Table 3 ). The Lf NPs prepared using this method offered several advantages including small particle size 60-90 nm and high drug loading (50% loading). moreover, the NPs displayed pH-responsive drug release with higher release at endosomal pH (pH 5 and 6) which can be advantageous for tumor targeted drug delivery. This behavior can be attributed to the conformational change or shrinkage of Lf in that acidic milieu. Since this technique does not need any harsh chemical treatments such as using stabilizing or cross-linking agents, we anticipate that Lf molecules may revert back to their native conformation and regain their receptor binding epitopes [6] . However, some limitations of this technique can arise from the difficulty of oil removal and the effect of sonication on the structural integrity of protein molecules. |
Lf nanoparticles could be prepared by desolvation technique by addition of a miscible organic solvent of the drug to the aqueous protein solution at adjusted pH. When the solution turns turbid, revealing particle formation, a crosslinking agent e.g. glutaraldehyde solution was added to harden the particles. Curcumin-loaded Lf NPs prepared by desolvation exhibited a particle size of 214.7 nm with encapsulation efficiency of 53.7% [99] . The nanoparticles were optimized by varying different factors e.g. protein concentration, solvent ratio, temperature, sonication strength, flow rate, cross-linking agent and pH. Smaller particles were formed by heating the protein at 35-60℃. At high temperature, protein unfolding increased the exposure of the SH groups resulting in increased intra-crosslinking of protein macromolecules thus producing smaller nanoparticles [100] . Generally, desolvation is advantageous to other preparation techniques of protein NPs by its simplicity with no need for harsh conditions including high shearing or heating, that might influence the structural stability of proteins. |
Lf NPs can be prepared by controlled thermal denaturation at 61 and 93℃ corresponding to its two lobes. The hydrophobic domains usually become exposed upon protein unfolding resulting in its aggregation via hydrophobic interaction. The protein nanoparticles prepared by thermal denaturation exhibited high pH stability that can be attributed to the strong hydrophobic bonding formed between protein macromolecules following their unfolding. Moreover, the outer glycan components of Lf extending into the external aqueous medium can enhance the stability of the protein nanoparticles by increasing the steric repulsion. |
Simvastatin and fenretinide were co-encapsulated into TPGS-TAT-modified Lf NPs (177 nm) prepared by heat denaturation-driven protein self-assembly process [101] . An aqueous Lf solution was stirred on the oil bath at 80°C for 10 min followed by dropwise addition of organic solution of both drug and cell-penetrating peptide TPGS-TAT (developed by TAT was conjugated to D-α-tocopheryl polyethylene glycol succinate). In a consequent study, the same thermal denaturation procedure was used for preparation of mannosylated Lf NPs (150 nm) for codelivery of shikonin and JQ1 [102] . Loading of the antioxidant polyphenol cichoric acid to Lf by heating to 95℃ at pH 7 resulted in formation of NPs with a diameter of about 67 nm with higher antioxidant capacity than free cichoric acid [103] . This technique can be considered as a green one that does not require toxic cross-linkers or organic solvents. |
Being glycoprotein rich in cysteine with 14 disulfide bonds, Lf has a great tendency to form nanoparticles (NPs) via albumin-bound technology (Nab). Homogenization of protein solution induces the formation of a protein layer coating the drug particles where the S-S bonds were broken and new bonds were formed due to the local heat generated because of the high shearing. Gambogic acid and oleanolic acid (OA) were loaded into Lf nanoparticles with size about 100-200 nm by Nab technology [104, 105] . The solution of OA, in mixture of methylene dichloride and ethanol (7:3), was cooled to 25°C and then slowly added to the aqueous Lf solution. The two solutions were mixed and homogenized at 50 MPa for three cycles followed by evaporated at 45°C under reduced pressure then freeze-dried without adding any cryoprotectant. In addition, the hydrophilic structure of LF make the particles readily redispersed in aqueous solution. |
Polyelectrolyte complexes (PEC) were formed based on electrostatic interactions between positively charged Lf, with higher isoelectric point (pI ≈ 8.5) than most of other proteins (pI ≈5) and negatively charged polysaccharides in aqueous solutions. Aqueous solutions of Lf and polysaccharide can be mixed at ambient temperature to form electrostatic nanocomplexes followed by heating up to 92°C above the thermal denaturation temperature of Lf to promote protein aggregation and particulate formation [106] . For example, Lf/N-succinyl chitosan/galactomannan nanocomplexes were formed by their simple mixing at 1:2:3 and 1:3:2 ratios prior to thermal treatment [107] . Alternatively, Lf NPs with a diameter of 200-400 nm were first formed by thermal denaturation followed by electrostatic complexation with negatively charged polysaccharides e.g. alginate, carrageenan, or pectin [108] . Reversal of the particle surface charge from positive to a negative value indicated successful coating by a layer of the polysaccharide. Yan et al. has used heat-denatured Lf/pectin PEC NPs as a carrier for curcumin [109] . Curcumin was released in a pH-dependent manner showing higher release at acidic pH. This pattern can be explained by the weakened Lf/pectin electrostatic attraction at acidic pH due to protonation of pectin carboxylic groups. The encapsulated curcumin showed higher antioxidant capacities through DPPH radical scavenging ability. Lf can also form electrostatic nanocomplexes with anionic polysaccharides without need for further heating. In our laboratory, novel nanocomplexes were developed based on the electrostatic complexation between positively charged Lf and negatively charged chondroitin sulfate [110] . The hydrophobic phytomedicine ellagic acid, converted into water soluble nanosuspension, was co-encapsulated together with water soluble cytotoxic drug DOX into the hydrophilic Lf-ChS nanoparticles. As the pH of Lf solution decreases far below its isoelectric point, Lf macromolecules become strongly positively charged resulting in formation of a strong and stable nanocomplex at pH 3.5 with smaller size 138.2 nm. Similarly, Lf-hyaluronic acid-EGCG ternary nanocomplexes were formed via hydrophobic and electrostatic interactions as well as hydrogen bonding [111] . |
The structure of Lf makes it capable to chemically conjugate with various hydrophobic moieties or drugs. Utilizing the hydrophilic property of Lf in enhancing the water solubility of the hydrophobic drugs where the conjugated structures drive the self-assembly forming nanomicelles in aqueous media. Chaharband et al. has prepared Lf-curcumin conjugate where the carboxylic moiety of Lf was activated via carbodiimide reagent and then coupled to the hydroxyl group of curcumin forming an ester bond [112] . The conjugate of curcumin to Lf macromolecule was self-assembled into nanostructures with a size of 165 nm. By increasing the Cur-Lf molar ratio from 10 to 40, the conjugation ratio was also increased from 0.85 to 4.52, respectively. Similarly, Lf-deferasirox conjugate was prepared via ester bond carbodiimide coupling and selfassembled into nanoparticles with a size of 100-500 nm [113] . In another investigation, DOX-Lf conjugate were prepared also via carbodiimide coupling reaction forming amide bond through activating the carboxylic moiety of Lf and attaching it to the amino group of DOX [77] . In our laboratory, the chemotherapeutic drug pemetrexed was chemically coupled to Lf via carbodiimide coupling. The Lf-drug conjugate was anchored onto the surface of aminated mesoporous silica nanoparticles entrapping the herbal drug ellagic acid [114] . The synergistic combination showed enhanced internalization and cytotoxicity against MCF-7 breast cancer cells as revealed by lower combination index value (CI = 0.885) in comparison with soluble drugs. In addition to carbodiimide coupling, Lf conjugates with the polyphenolic drugs epigallocatechin gallate (EGCG), chlorogenic acid (CA) and gallic acid (GA) were successfully elaborated by free-radical graft copolymerization in aqueous media using ascorbic acid/H 2 O 2 redox pair system [115, 116] . Moreover, Maillard reaction was used for covalent bond formation between carbohydrate (glucose or polydextrose) and Lf-CA conjugate resulting in higher thermal stability [117] . |
Nanosized micelles can be developed by the aqueous self-assembly of amphiphilic co-polymers. In our laboratory, an amphiphilic co-polymer was synthesized by chemical carbodiimide coupling of the hydrophobic corn protein zein to the hydrophilic Lf protein [118, 119] . Upon dispersion in aqueous solution, the chemical conjugate was self-assembled into nano-micelles (276.6 nm) with a low CMC value. This property protected the micelles from dis-assembly after dilution as a result of injection into systemic circulation. Two lipophilic drugs; rapamycin and wogonin were efficiently entrapped into the hydrophobic core of zein-Lf micelles whereas the hydrophilic Lf shell enabled prolonged circulation. The protein amino groups of Lf allowed further crosslinking of the micelles using glutaraldehyde leading to lower particle diameter and higher colloidal stability. In comparison to the commonly used synthetic amphiphilic copolymers (e.g. PLA-PEG), zein-Lf co-polymer is considered GRAS (Generally recognized as safe) material composed of natural protein components with much less cost and toxicity [120] . |
Polymeric oily-core nanocapsules are commonly used for solubilization and controlled delivery of hydrophobic drugs. The NCs are composed of a polymeric shell enveloping an oily core for drug encapsulation. The hydrophobic synthetic polymers (e.g. PLGA or PCL) are among the most commonly used shell forming materials for that type of NCs. However, besides their high cost, numerous toxicity issues have been raised related to their acidic degradation products or immunological reactions. In our laboratory, an oily core composed of Capryol PGMC was used for solubilization of the hydrophobic drugs sorafenib and quercetin [121] . SFB was precomplexed with phospholipid to maximize its incorporation in the oily phase. A surrounding shell of cationic Lf was electrostatically attached onto the negatively charged oily core to form liver tumor-targeted NCs exploiting the Lf interaction with its receptors upregulated by liver cancer cells. To further enhance tumor targeting, lactobionic or glycyrrhetinic acid was chemically anchored to the Lf shell where they bind with ASGP or GA receptors on HCC cells, respectively [121] . |
Inspired by the emerging role of inorganic materials in therapy and diagnosis, hybrid nanocomposites were fabricated by chemical coupling of Lf to various types of inorganic nanoparticles ( [127, 128] . The combination of chemo-and photothermal/magnetic therapy showed enhanced antitumor efficacy. Lf was also used to chemically modify the surface of graphene oxide/Fe 3 O 4 NPs (GO@Fe 3 O 4 NPs) to increase their accumulation into glioma cells [129] . The resultant nanocomposites showed acid-responsive release of DOX with enhanced killing of glioma C6 cells. In another investigation, ultrasmall graphene nanosponge-supported lipid bilayers (40 nm) were co-loaded with docetaxel (DTX) and gasified perfluorohexane (PFH). This nanoplatform acted as a photothermal agent for combined gasification/chemothermotherapy of cancer by releasing both drug and heat under exposure to near-infrared laser irradiation [130] . The surface of nanosponge was further modified with Lf via chemical conjugation to the lipid bilayer. Lf increased the lateral bilayer fluidity and enhanced its penetration into tumor spheroids mediated by transcytosis. Lf-silver nanocomplexes have been reported for their efficient antibacterial activity [131] . For example, Lf was used to modify the surface of silver nanoparticles (AgNPs) resulting in a synergistic anti-biofilm efficacy against S. aureus and P. aeruginosa. [132] . Moreover, the adsorbed Lf layer was found to decrease the cytotoxicity of silver NPs [133] . Quantum dots, as potent fluorescent imaging agents, were chemically coupled to Lf via a tumor cleavable bond to prevent systemic toxicity of its Cd component [134] . The cationic Lf-QD conjugate was used to electrostatically decorate drug-loaded nanocapsules to enhance their tumor accumulation. The QD-induced fluorescence enabled tracing the biodistribution of nanocarriers and their internalization into breast cancer cells. Silica nanoparticles were also coupled to Lf to enhance their brain delivery. The Lf-modified silica NPs (25 nm) exhibited the highest transport across the cerebral endothelial cells via Lf receptor-mediated transcytosis [135] . In addition, Lf was successfully used to modify the surface of ellagic acid-loaded mesoporous silica NPs (MSNPs) to enhance their accumulation into breast tumor tissue. The carbodiimide-activated carboxylic group of Lf was coupled to the amino groups of APTES-modified MSNPs [136] . On the other hand, few studies reported the use of Lf protein to stabilize the highly luminescent gold nanoclusters [137, 138] . Those Lf-gold nanocomposites might have future applications in tissue imaging and cancer theranostics. |
By virtue of its receptors overexpressed by cancer cells and some other tissues such as brain, Lf was increasingly exploited to modify the surface of drug nanocarriers. Two main strategies have been investigated for the design of Lf surface-modified nanocarriers; covalent conjugation and electrostatic complexation. Chemical conjugation was utilized using various coupling reactions such as carbodiimide and maleimide thiol coupling methods. High yield and conjugation efficiency was obtained when carbodiimide and maleimide thiol coupling were used for conjugating Lf to SLNs [139] and PEGylated liposomes [140] with conjugation efficiency of 71.02% and 74%, respectively. In addition, Lf was coupled to the surface of poly(propylene imine) dendrimer using N-c-maleimidobutyryl-oxysuccinimide ester (GMBS) as a linker. This heterobifunctional cross-linking agents inhibit the polymerization that can be obtained using homo-bifunctional cross-linking reagents e.g. dimethyl suberimidate [141] . |
Many challenges including the tight junctions between brain endothelial cells, their low pinocytic activity, presence of efflux pump systems and inactivating enzymes hamper efficient delivery of drugs and genes to the brain across the blood brain barrier (BBB) [142] . Lf receptors including LRP1 and LRP2 (megalin) are well expressed in glioma cells, brain microvessels and neurons (Fig. 6) [143] . In addition, Lf bears positive charge in physiologic pH, which facilitates its uptake into negatively charged brain capillary endothelial cells (BCECs). Therefore, Lfmodified nanocarriers are internalized by receptor-and adsorptive-mediated endocytosis into BCECs and glioma cells. In comparison with Tf, Lf-conjugated dendrimers for gene delivery showed 2.2-fold increased uptake and 2.3-fold increased brain gene expression in BCECs with lesser accumulation in liver, spleen and kidney [144] . In contrast to Tf, the lower plasma concentration of endogenous Lf, its positive charge as well as unidirectional transport of Lf across the BCEC monolayer might result in higher brain accumulation of Lf-conjugated vector [145, 146] . Chan and coworkers have recently investigated the vasculature of U87-MG glioblastoma xenograft model [147] . The authors found that 97% of the NPs could enter the tumors through an active trans-endothelial process rather than the commonly known passive extravasation transport mechanism through the inter-endothelial gaps. This suggested mechanism may account for the entry of Lf-modified nanocarriers to brain through the brain capillary endothelial cells. The NPs may bind to endothelial cells followed by their transport into the tumor through vesicles and transcellular channels. Therefore, this mechanism can further strengthen the concept of Lf-mediated transcytosis, and its role in enhancing penetration into deep tumor tissues. Lf-targeted huperzine A-loaded nanoemulsion has shown higher accumulation in hCMEC/D3 brain cells via transcytosis compared with untargeted nanoemulsion [16] . Furthermore, kumari et al. has confirmed that Lf NPs accumulated in brain tissue and delivered high temozolamide concentrations to tumor through transcytosis across BBB reaching the brain tumor site [96] . 6 . Schematic illustration of the brain targeting mechanism of Lf. |
Lf-conjugated PEG-PCL polymersomes (POS), with around 40 Lf molecules per POS, were coloaded with DOX and Tetrandrine (Tet) ( Table 5) . Although particles had 220 nm diameter which increases the mononuclear phagocytic system (MPS) clearance, the full PEGylation protected it and increased their circulation time [148] . Notably, Lf modification increased the concentration of POS more in right hemisphere compared to the un-modified ones, while Tet, as MDR inhibitor, enhanced their cellular uptake by decreasing cellular resistance thus resulting in the smallest tumor volume and longest survival time. In another investigation, Lf-modified procationic liposomes (PCLs) loaded with DOX, showed longest survival time on Wistar male rats inoculated with C6 glioma cells compared with control group. Lf-conjugation increases cellular uptake into glioma C6 cells; while PCLs uptake occur via adsorption-mediated endocytosis solely, Lf-PCLs uptake occur via receptor-and adsorption-mediated endocytosis [149] . To further enhance the penetration of Lf-modified nanocarriers, tLyP-1, a tumor-homing peptide, was co-administered with Lf-modified paclitaxel (PTX)-loaded polyethylene glycolpolylactic acid (PEG-PLA) NPs. tLyP-1 enhanced the tissue penetration of NPs via the neuropilin-1-dependent (NRP-1) internalization pathway, especially because NRP-1 receptors are specifically expressed on endothelial cells of tumor blood vessels [150] . While Lf modification improved cellular uptake of the NPs relative to unmodified NPs. In addition to drug delivery, the cationic nature of Lf enabled both high binding capacity of nucleic acids as well as endosomolytic activity mediated by proton sponge effect that contributed in escape of Lf NPs from endolysosomal compartment into the cytoplasm where siRNA can bind with RNA-induced silencing (RISC) complex. As an efficient combination therapy for glioblastoma multiforme (GBM) was enabled by development of Aurora Kinase B (AKB) siRNA-loaded Lf NPs, to enhance BBB crossing, co-administered with TMZ treatment [6] . AKB was found to by overexpressd in malignant glioma where it induces degradation of the tumor suppressor gene p53 thus triggering tumor growth. As a result, the combined TMZ treatment and AKB silencing has significantly improved the survival of treated mice from 14 to 33 days. Lf could be also used to enhance brain accumulation of nanoparticles through intranasal route based on the high expression of LfR on the nasal epithelial cells and olfactory linings. Both Lf and hyaluronate were coupled to the surface of acid-responsive lenalidomide-conjugated FePt alloy nanoparticles. While hyaluronic acid enhanced brain tumor targeting by its CD44 binding ability, Lf facilitated the olfactory uptake through enhanced mucosal and nasal penetration, which led to brain accumulation of the nanoconjugates. Also, LfR are overexpressed on the surface of glioma cells, thus increasing their specific uptake by cancer cells. In addition to this direct intranasal transport, Lf-modified nanoconjugates absorbed into blood circulation will most likely target brain delivery since LfR are overexpressed on the surface of brain endothelium [151] . The acidic microenvironment of lysozome resulted in cleavage of the pH sensitive hydrazone bond between drug and NPs to trigger the drug release. The NPs efficiently generated reactive oxygen species (ROS) due to release of Fe and Pt which increased its cytotoxicity against U87MG cells. |
The Lf receptors are found to be upregulated by neurons involved in the pathophysiology of Parkinson and Alzheimer's diseases [152, 153] . Both diseases are characterized by iron overload in specific areas in the brain. Since Lf plays an important role in iron metabolism, its therapeutic effect in Parkinson Disease (PD) and Alzheimer has been studied as metal chelator [154] . In case of PD, Lf is overexpressed in activated microglia where it plays an important neuroprotective role. This was evident for both apo-(iron-free) and holo-(iron-saturated) Lf. The suggested Lf neuroprotective mechanisms could be mediated through the increase of the mitochondrial J o u r n a l P r e -p r o o f transmembrane potential, indicating better mitochondrial activity [155] . Another hypothesis for the neuroprotective activity of Lf is iron-dependent, where Lf suppressed transferrin receptors and divalent metal transporter, leading to enhanced iron internalization [156] . Further, Lf is associated with upregulation of brain-derived neurotrophic factor (BDNF), in addition to activation of the pathway of regulated protein kinases-mitogen activated protein kinase (ERK/MAPK). Interestingly, Lf protects dopaminergic neurons in the nigrostriatal area by decreasing levels of α-synuclein through upregulation of hypoxia-inducible factor 1α [157] . In addition to its intrinsic activity, Lf and borneol were used together to modify the surface of dopamine-loaded nanoparticles (Lf-BNPs) for enhanced permeability and striatum-specificity for treatment of PD [158] . The NPs could alleviate the 6-hydroxydopamine-induced striatum lesion in rats by effective delivery of dopamine after intranasal administration. Similarly, dopaminergic SK-N-SH cells were protected against rotenone-induced neurotoxicity in dopaminergic neurons using Lf nanoparticles encapsulating curcumin prepared by sol/oil chemistry [159] . The use of genes such as glial cell line-derived neurotrophic factor (GDNF) encoding gene showed a great promise in PD therapy due to the stimulatory effect of GDNF on dopaminergic neurons [160] . In order to achieve successful brain delivery, hGDNF was encapsulated in Lffunctionalized PAMAM nanoparticles. Lf-modified NPs showed the highest GDNF expression compared to saline and Tf-modified nanoparticles. Compared to viral vectors, higher expression of hGDNF was achieved by continuous administration of Lf-modified NPs in chronic rotenoneinduced model. This extended administration enhanced locomotor activity, decreased loss of dopaminergic neurons in addition to improved levels of monoamine neurotransmitters in PD rats [161] . |
Increased iron in Alzheimer disease is associated with oxidative stress, reactive oxygen species and nitric oxide synthase production. Lf has an anti-oxidative effect as it acts as iron chelator in the brain to restore iron hemostasis. It also has anti-inflammatory activity, leading to decreased levels of TNF-α and IL-6 [162] . These results were further confirmed in a three-month clinical trial, with decreased serum amyloid β, p-tau, and MAPK1 [163] . Wang et al. found that the intranasal administration of Lf led to a reduction in β-amyloid deposition through activation of αsecretase which increased the metabolism of amyloid precursor protein. This led to amelioration in cognitive function and learning ability in treated animals [164] . Recently, Bartolome et al. found that salivary Lf could be a promising biomarker for non-invasive early detection of Alzheimer's disease, as it was shown to have specifically lower salivary levels [165] . Lf-polyamidoamine dendrimeric nanoparticles (PAMAM-Lf) were designed for the efficient delivery of rivastigmine (RIV) to Alzheimer's model. Administration of Lf-PAMAM NPs in Alzheimer's animal model enhanced the brain bio-availability of RIV and increased AChE activity resulting in higher Ach level and improved memory deficit than unmodified dendrimers [166] . Application of the hydrophilic peptide, neuron growth factor (NGF), for management of Alzheimer's disease is a feasible strategy. However, delivering therapeutic peptides to the brain faces many challenges such as degradation in serum and failure to bypass BBB. To deliver NGF across BBB, Lf-decorated liposomes were developed. The NGF Permeability across HBMECs/HAs monolayer showed higher penetration for Lf/NGF liposomes compared to nontargeted liposomes and free NGF [167] . In another hand, the viability of Aβ-insulted SK-N-MC cells was increased upon incubation with Lf/NGF-liposomes. Similarly, encapsulation of another neuroprotective peptide, S14G-humanin, into Lf-modified polymersomes inhibited the overexpression of caspase-3 and Bax and enhanced cholinesterase activity. As a result, they efficiently protected the hippocampuses of rats against learning and memory deficits triggered by using amyloid-β25-35 [168, 169] . |
Lf was proposed as a safe alternative with powerful antitumor activity specifically against colon cancer. First, the anti-inflammatory effect of Lf on colon cancer was widely reported in literature. In Lf knockout mice, NF-ĸB was upregulated resulting in an aggravated inflammatory response as evidenced by the increased levels of the nuclear p65 protein expression, p65 + cell numbers, and IKKα/β. Furthermore, the pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were found to be upregulated, which can cause AKT and mTOR deregulation and subsequent tumor progression [180] . Lf may also induce apoptosis in colon cancer cells by several pathways including induction of caspase 3/7, activation of p53 tumor suppressor gene, and enhancing expression of the pro-apoptotic genes; TP5313, PMAIP-1, and SFN thereby promoting subsequent cell death in HT-29 cells [57] . Ingestion of Lf was correlated with an elevation in the activity of NK cells, and increases in CD16 + /CD56 + subset of NK cells in the blood, which was consistent with regression of colorectal polyps. bLf was concluded not to induce activation/inhibition of immune system but rather increase the responsiveness of the immune system [181, 182] . In addition to its antiinflammatory and immunomodulatory effects, bLf was shown to have antioxidant effect by increasing the transcription of antioxidation related genes: GCLC and GCLM, involved in glutathione synthesis and scavenging of reactive oxygen species (ROS) [57] . Selenium-saturated bovine Lf (Se-bLf) showed a promising anti-oxidative property in HT-29 cells treated with 250 µM of H 2 O 2 , by increasing the activity of the enzymes glutathione peroxidase, glutathione stransferase and glutathione reductase showing a protective effect against cancer development [183] . Nano-formulations of bLf were found to confer a promising efficacy against colon cancer; owing to its both colon targeting and antitumor effects. Lf can target cells colon cancer cells and may internalize itself utilizing these receptors regulated by intestinal epithelial cells [57] . Liposomal bovine Lf was able to inhibit the expression of TNF-α mRNA expression in colorectal RKO and RCN-9 cells resulting in a lower inflammation, as demonstrated by reduced numbers of neutrophils associated with suppression of colorectal polyps [184] . In a study by Kanwar et al., alginate coated, chitosan conjugated, calcium phosphate, Fe saturated bLf nanocapsules (AEC-CCo-CP-Fe-bLf NCs) with a size of 396.1 nm were effective against triple positive cancer stem like Caco-2 cells [14] . These nanocarriers were able to localize at the tumor site by EPR effect and then internalize utilizing LRP1, transferrin and Lf receptors in addition to iron receptors: DMT1, ferroprotein, and ferritin receptors, which were believed to play a major role in the absorption of Fe-bLf NCs [185] . These NCs showed enhanced efficacy compared to Fe-bLf NCs; due to better efficient delivery of Lf by the coating polymers, chitosan and alginate. Additionally, treatment of Caco-2 bearing mice with the NPs has downregulated anti-apoptotic factors including Survivin and Bcl-2, while upregulated pro-apoptotic Bax, Fas, caspase-3 and caspase-9 [14] . Consequently, modification of the surface of AEC-CCo-CP-Fe-bLf nanocapsules with LNA (EpCAM and nucleolin)-aptamer enhanced tumor targeting via binding to the cell surface receptors; EpCAM and nucleolin and hence translocate into the nucleus [186] . The uptake of the targeted NCs into Caco-2 cells was enhanced by 2.84 folds through receptor mediated internalization. The mice treated with both non-targeted and targeted NCs showed a significant decrease in the tumor volume by 6.8 and 2.91 folds, respectively. The nanocapsules were then modified by addition of zinc ferrite to a solution of apo-Lf in the presence of sodium bicarbonate to produce zinc ferrite saturated bovine Lf (Zn-Fe-bLf) which was then encapsulated in a similar way into alginate-coated chitosan nanogel. The antitumor activity of the Zn-Fe-bLf NCs 24 nm was enhanced as demonstrated by dramatic reduction in tumor volume in human xenograft colonic adenocarcinoma to 0.10 mm 3 compared to the vehicle NCs (52.28 mm 3 ) and control group (93.51 mm 3 ). Application of magnetic field and photothermal laser after treatment with Zn-Fe-bLf NCs resulted in further tumor reduction. Moreover, the presence of Zn may improve CT scanning signal intensity of the NCs compared to Fe-bLf revealing efficient multimodule nanotheranostic properties [185] . Lf NPs could also be used as a carrier for chemotherapeutic drugs such as oxaliplatin and 5-Flourouracil to enhance their targetability to colon cancer. The NPs exhibited enhanced cytotoxicity against COLO-205 cells as shown by lower IC 50 (1.08 and 0.98 µg/ml for Lf-5FU and Lf-oxalo NPs, respectively) compared to free drug (IC 50 of = 2.27 and 2.22 µg/ml for free 5FU and free oxaliplatin, respectively). The nano-formulations showed a 7-fold decrease in AFC (structural deformity which turns into colon adenocarcinoma) compared to a 3-fold decrease by the free drugs in azoxymethane induced AFC in Wistar rats [98] . |
Lf binds specifically to LRP and asialoglycoprotein receptors (ASGP-R) that facilitate its internalization into hepatocellular carcinoma (HCC). Therefore, modification of PEGylated liposomes with Lf improved their uptake into ASGPR-positive cells including HepG2, BEL7402, and SMMC7721 but not in ASGPR-negative cells [187] . DOX-loaded Lf-PEGliposomes exhibited higher anticancer efficacy in HepG2 bearing mice relative to free drug. For combined HCC therapy, we have prepared Lf-coated oily core nanocapsules for synergistic delivery of sorafenib and quercetin [121] . The dual targeted lactobionic acid/Lf-NCs exhibited enhanced uptake into HepG2 cells and higher anti-tumor effect in HCC mice model compared to free drugs. For oral targeted HCC therapy, Lf-coated gliadin NPs co-encapsulating celecoxib and diosmin showed a synergistic anticancer action due to their common inhibitory effects on COX-2, NF-κB and TNF-α [188, 189] . The high stability of Lf-GLNPs is probably due to surface coating of GL-NPs by Lf providing both hydrophilic surface and charge-based stabilization. In addition, the glycan chains of Lf may add steric stabilization mechanism. The Lf-coated NPs exhibited enhanced internalization and improved anti-tumor efficacy against HepG2 cells at 48 h and HCC-bearing mice. Lf-targeted gliadin NPs demonstrated a remarkable inhibition of NF-κB and TNF-α expression compared with the positive control. |
After oral administration, the small size FebLf NCs (80 nm) induced apoptosis of claudin-low, triple-negative MDA-MB-231 breast cancer cells through mitochondrial depolarization, downregulation of the apoptosis inhibitory proteins; survivin, and livin as well as PI3K followed by activation of caspase-3 [18] . By virtue of their superparamagnetic properties, FebLf NCs can be considered as suitable cancer nanotheranostic agent for MRI contrast imaging and magnetic guided cancer therapy. In another investigation, Lf-coated DOX-loaded porous magnetite nanospheres (PMNSs) were developed for synergistic chemo-magnetic field photothermal therapy of breast cancer [190] . The injected Lf-DOX-PMNSs significantly into 4T1 bearing mice suppressed the proliferation of 4T1 cells and tumor weight by activating both the extrinsic pathway of apoptosis via upregulation of the TNF-α mRNA and enhancing the intrinsic Bax mRNA expression. The Lf coating layer not only enhanced the drug targeting to tumor site but also prolonged its circulation by forming protein corona that can evade clearance by RES and showed acid responsive drug release at tumor microenvironment. |
In addition to its intrinsic anticancer effect, Lf was exploited as a hydrophilic shell of amphiphilic micelles co-encapsulating two water insoluble drugs rapamycin and wogonin within its hydrophobic core composed of the plant protein zein [118] . In addition to its active tumor targeting properties, the brush-like hydrophilic Lf corona increased the micellar stability, inhibited their opsonization and prolonged their systemic circulation leading to maximized accumulation at tumor site by the EPR effect. In a consequent study, oleic acid-coated Fe 3 O 4 NPs were encapsulated together with dasatinib in the micellar hydrophobic zein core [119] . Attributed to their superparamagnetic characteristics, the micelles showed enhanced cytotoxicity against MDA-MB-231 cells upon application of external magnetic field. In another approach, Lf was electrostatically anchored onto the surface of zein nanospheres co-loaded were the hydrophobic aromatase inhibitor exemestane and the herbal flavonoid luteolin [191] . Lfmodified NPs enhanced their targeting to breast cancer cells resulting in improved cytotoxic effect against MCF-7 and 4T1 breast cancer cells with 2.5-fold higher selectivity index SI to cancer cells rather than normal cells compared to the free drugs. Another interesting application of holo-Lf is its ability to relief of hypoxia in TME of solid tumors. Hypoxia is known to reduce the efficacy of radiotherapy by weakening the radiationinduced cell DNA damage. The presence of Fe in the structure of holo-Lf can induce decomposition of H 2 O 2 to oxygen thus alleviating the hypoxic TME. Through its oxygen generation capacity, Lf-PEGylated DOX-loaded liposomes showed synergistic anticancer effect and increased survival rate of mice when combined with 4 Gy X-ray radiation in breast cancer bearing mice which could be confirmed by photoacoustic imaging [78] . Lf NPs were also used to enhance the efficacy of breast cancer photodynamic therapy by encapsulation of photosensitizer (PS) Chlorine e6 (Ce6). The NPs inhibited the aggregation of the hydrophobic Ce6 in aqueous media thus improving its efficacy to generate ROS [192] . Under light irradiation, the NPs triggered a remarkable cell death in SK-OV-3 and MDA-MD 231 cells 4 folds higher than that of free Ce6. |
Lf receptors were found to be upregulated on the bronchial epithelial cells therefore, Lf-modified lipid nanocarriers were used for the targeted delivery of PTX to lung cancer therapy [193] . Lftargeted lipid NPs showed enhanced cytotoxicity against BEAS-2B human bronchial epithelial cells, relative to non-targeted NPs and free PTX. After intravenous administration into rats, the Lf-NPs showed higher amounts of drug accumulated in lung tissue relative to non-targeted NPs. Beside the use of Lf-targeted nanocarriers for systemic therapy of lung cancer, the inhalable Lftargeted nanocomposites have emerged as a promising alternative to avoid the systemic toxicity and enhance deep lung deposition [194] . For this purpose, different Lf-targeted NPs were prepared for synergistic lung cancer treatment and then transformed into inhalable nanocomposites via spray-drying with suitable pulmonary inert carriers. Examples of those nanocarriers include; (i) HA/Lf layer-by-layer coated lipid nanoparticles co-encapsulating rapamycin-phospholipid complex and berberine-sodium lauryl sulfate hydrophobic ion pair [195] , (ii) Lf-chondroitin electrostatic nanocomplex co-loaded with DOX hydrochloride and ellagic acid nanocrystal soluble form [110] , and (iii) Lf/chondroitin layer-by-layer functionalized monoolein liquid crystalline NPs co-entrapping pemetrexed and resveratrol (Fig. 7) [196] . All those nanocarriers exhibited favorable physicochemical properties such as small size, improved drug release pattern, improved uptake and enhanced cytotoxic effect against A549 lung cancer cells. for deep lung deposition, those nanocarriers were mixed with drying aids such as mannitol, maltodextrin, leucine or their combinations prior to spray-drying to form inhalable nanocomposites. The inhalable powder demonstrated favorable aerosolization efficiency (MMAD of 2.68-3.28 µm and FPF of 55.5-89.58% %) revealing efficient particle deposition into deep lung tissues. After reaching lung tissues, the carriers dissolved releasing the nanoparticles followed by internalization into lung cancer cells by virtue of their Lf, HA, or chondroitin-based receptor mediated endocytosis. In lung cancer bearing mice, all the prepared inhalable nanocomposites displayed powerful anti-cancer efficacy by reducing the lung weight, decreasing the number and diameters of lung adenomatous foci, downregulating the level of proliferative and angiogenic markers and increasing the apoptotic markers compared to intravenously administered nanocarriers. [196] . |
Lf demonstrated direct cytotoxic action against highly metastatic PC-3 prostate cancer cells via inhibition of V-ATPase and subsequent reduction of the ability of those cells to acidify the TME. Moreover, Lf induced phenotypic changes of DU-145 and LNCaP reducing their invasive properties. Lf also downregulated the expression of steroid hormone receptors (e.g. estrogen receptor ERα and, progesterone receptor PR) and increased the expression of oncosuppressive miRNAs (e.g. miR-133a and miR-200b) in DU-145 cells [197] . As a targeting ligand for prostate cancer cells, Lf modification of diaminobutyric polypropylenimine dendriplexes enhanced the delivery and transfection of the encapsulated plasmid DNA encoding TNFa, TRAIL or IL-12 to prostate cancer cells after intravenous administration [198] . The Lf-targeted dendriplexes resulted in much stronger suppression of PC-3 and DU145 tumors in comparison with Tf-modified dendriplexes. Conjugates of DOX to iron saturated Lf (Fe-bLf-DOX) showed higher cytotoxicity against DU145 prostate cancer cells and lower toxicity against non-cancerous RWPE-1 cells compared to iron free Apo-bLf-DOX and free DOX [77] . Besides it increases uptake into cancer cells via interaction with additional receptors, the iron acts as an essential effector of DOX induced cytotoxicity. As a result, Fe-bLf-DOX exhibited superior efficacy in decreasing Bcl-2 expression levels associated with elevated PTEN expression. After internalization into the endo-lysosome, DOX was released upon cleavage of the covalent amide bond between drug and the bLf protein by the hydrolytic enzymes and the acidic environment thus resulting in increased nuclear accumulation of DOX. Therefore, the Lf-DOX conjugate could be considered as DOX prodrug while including a functional carrier bLf with cytotoxic nature thus causing decrease in the LC 50 of DOX by more than 4 folds. Another interesting property of Lf conjugate is inhibiting the efficacy of membrane efflux pumps in efflux DOX out of cells resulting in higher retention of drug in the prostate cancer cells [77] . Thus, bLf-DOX conjugate induced both down-regulation of P-gp as well as the bypass of P-gp [77] . |
Modification of the surface of curcumin-loaded magnetic polydiacetylene nanocarriers (PDNCs) with Lf significantly improved their brain accumulation by enhancing BBB penetration resulting in powerful suppression of orthotopic brain tumor and increasing the animal survival by about 30%. At the same time, the NPs ability to exhibit both self-responsive fluorescence and MRI contrast imaging upon cellular internalization enabled cell trafficking and imaging-assisted cancer therapy. Thus, the Lf-targeted magnetic PDNCs can be promising theranostic application for targeting gliomas [170] . Another promising imaging probe for cancer, highly fluorescent CdTe quantum dots (QDs) were conjugated to Lf-targeted nanocapsules. The oily core of nanocapsules co-solubilizing celecoxib and honokiol was electrostatically coated with anionic chondroitin sulfate layer followed by a cationic Lf-QDs conjugate corona [199] . The covalent bond formed between Lf and QDs prohibited the systemic release of Cd into circulation and guarantee the site-specific release of QDs only at tumor sites. The QDs luminescence was quenched in vitro upon conjugation with Lf (OFF state) due to electron/energy transfer mechanism. Upon intracellular uptake into MCF-7 cells, luminescence was restored (ON state) due to detachment of surface-bound ligands from QDs in cytosolic environment confirm and monitor intracellular uptake of our nanoparticles that was confirmed using confocal microscopy. In vivo, tumor tissues of Lf-QDs-CS-NCs treated mice group showed the highest fluorescence intensity compared with liver and kidney that proved their effective localization in the tumor tissue. |
Recently, Lf and RGD dimer dual-modified extremely small (13.4 nm) gadolinium oxide nanoparticles were prepared as highly efficient theranostics of GBM. The NPs successfully crossed the BBB due to its small diameter and Lf binding to its receptors then enter brain tumor cells by RGD2-integrin αvβ3 interaction, and thus can be used for MRI-guided radiosensitization of brain tumors [125] . The NPs exhibited high contrast MRI as indicated by extraordinary relaxivities thus showing much better signal enhancement for T1-weighted MRI of tumors than commercial Gd-chelates. |
One of the most interesting properties of Lf is its ability to reach the nucleus. On this regard, Lf has great potential for macromolecular delivery to cytoplasm and nucleus such as nucleic acids (siRNA, mRNA, shRNA and DNA) and gene editing machinery (Cas9 and sgRNA). Furthermore, by virtue of its cationic charge, Lf can bind to RNA and DNA and activates transcription. It was reported early in 1992 that Lf can easily traffic to the nucleus of K562 cells in temperature-dependent manner [200] . In 5637 bladder carcinoma, U87MG, MCF7, NIH3T3 and HeLa cells, a pentapeptide enriched in basic amino acids (Gly-Arg-Arg-Arg-Arg), derived from the N-terminal portion of human Lf was internalized within 10 min into the nucleus and mainly nucleoli [201] . Nuclear localization of Lf was also observed in human THP-1 monocytic and bovine rectal epithelial cells [202, 203] . |
In addition, a cell penetrating peptide derived from lactoferricin termed as L5a CPP (RRWQW) was shown to efficiently deliver DNA into the nucleus of human A549 lung cancer cells without induction of cytotoxic effect [204] . Compared to Tf, Lf was shown to be apically internalized into Caco-2 intestinal epithelial cells and then localized to the nuclei while Tf was internalized from the basolateral side and localized in the cytoplasm [205] . Nuclear trafficking of Lf was shown to be mediated by non-endocytic mechanism in some studies, which offers an easy way to escape from endosomes and late lysosomes for functional delivery of nucleic acids to cytoplasm and nucleus. More sophisticated investigations are required to fully understand the nuclear targeting mechanism of Lf and its nanoparticulate form in different cell lines to make the best use of Lf for wide therapeutic applications. |
Being a key component in the mammalian innate immune system, Lf plays an essential role in the host defense against pathogens and inflammation where its deficiency may precipitate infection recurrence [206] . The cationic nature of Lf contributes to its ability to bind to surface molecules found on immune cells, triggering signaling cascades leading to immune cells activation and maturation [207] . Bovine Lf exerts its immunostimulatory effect through its immunogenic forms which are formed by enzymatic digestion in the GIT [208] . Oral Lf is thought to send signals through receptors on the epithelium of the intestine and immune cells as macrophages and lymphocytes [22] . Moreover, the absorbed Lf can prime the immune system where an enhanced cytokine release was demonstrated including cytokines related to early (TNFα, IL-6, IL-10) and adaptive (IL-12p40, IFN-γ, IL-4) immunity. Overall, two possible explanations may account for the immunomodulatory effects of Lf: a) Immune cells express Lf receptors. Therefore, a receptor-mediated interaction between Lf and immune cells or indirect uptake of Lf by immune cells, results in the priming of immune system, b) Secondly, Lf may help educate the immune cells present in the GIT microenvironment. The immune cells will be transported via the lymphatic system to lymph nodes or spleen [209] . A study by Mulder et al. showed that oral supplementation of bLf in humans provided immunomodulatory effect through T-cell activation and anti-oxidant effect [210] . Moreover, Lf was shown to contribute to inflammatory response by granulocyte accumulation and activation, inducing phagocytosis and augmentation of natural killer cells activity (Fig. 8 ) [211, 212] . Being an innate immunity component, the immuno-modulatory activity of Fe-bLf dramatically augmented the anti-tumor efficacy of Fe-bLf-DOX in prostate cancer bearing mice [77] . This was demonstrated as reduced serum levels of the tumor promoting cytokines IL-5, IL-6 and IL-17 and elevated levels of IFNγ and TNFα as well as GM-CSF, CCL17 and CCL4 responsible for triggering intratumoral infiltration of cytotoxic T cells and macrophages. Lf nanocarriers were also used for remodeling of the immunosuppressive tumor microenvironment. The surface of Lf nanoparticles was modified with TPGS-coupled TAT cell penetrating peptide to enhance the BBB penetration of simvastatin and fenretinide drug combination. Interaction of Lf with LRP-1 receptors expressed by brain capillary endothelial cells (BCEC) and U87 glioma cells enhanced BBB permeation while TAT peptide increased the intracranial tumor penetration by 1.5 folds higher than that of un-modified Lf nanoparticles [101] . Simvastatin was reported to remodel TME and functional plasticity of TAMs whereas fenretinide induces ROS production and also can reverse the pro-tumoral M2-macrophages to the anti-tumoral M1 TAMs and reduce angiogenesis. The nanoparticles showed powerful synergistic cytotoxic effect against glioma cells in vitro and inhibited the tumor growth in vivo. Mechanistically, the dual drug loaded NPs succeeded to remodel since they effectively repolarized M2-TAM to M1-TAM as demonstrated by downregulating CD206 (TAM2 biomarker) and increasing the expression of iNOs and STAT1 (TAM1 biomarkers). The nanoparticles also improved the infiltration of cytotoxic NK cells into the tumor as revealed by overexpression of the NK cell marker NKp44. Moreover, it increased ROS production which in turn induced mitochondrial apoptosis. Another example of application of Lf NPs in TME immune-reprogramming includes the use of mannosylated Lf nanoparticles for combined delivery of shikonin and JQ1 for both metabolic and of TME [102] . The nanoparticles targeted mannose and LRP-1 receptors overexpressed by TAMs and cancer cells, respectively. The dual drug loaded nanoparticles could remodel the TME via different pathways. First, the NPs elicited antitumor immunity mediated by SHK ability to induce immunogenic cell death. As a result, the nanoparticles enhanced maturation of dendritic cells and hence increased the amount of tumor infiltrating CD8+ T cells [102] . Moreover, the SHK-loaded NPs suppressed cancer glucose metabolism through inhibition of pyruvate kinase M2 resulting in reduced lactate production and hence repolarizing M2-TAMs. On the other hand, JQ1 can decrease PD-L1 expression on cancer cells and downregulate Foxp3 and CTLA-4 in the tumor-infiltrating regulatory T cells (Tregs) so the NPs dramatically reduced the percentage of the intratumor infiltrating Tregs. |
The anti-HIV drug Indinavir (IDV) suffers from poor entry into brain due to efflux by the Pglycoprotein expressed in BBB. Chemical coupling of Lf to the surface of indinavir-loaded nanoemulsion (Lf-IDV-NEs) has enhanced its BBB penetration and increased its residence time in brain [213] . As a result, after administration into rat model, Lf-coupled nanoemulasion remarkably increased the brain concentration of IDV compared to free drug. Encapsulation of the antiretroviral drug Zidovudine (AZT) into Lf NPs for oral delivery resulted in improved pharmacokinetics (4 fold increase in AUC and 2-fold increase in Tmax and t 1/2 ), higher efficacy and 2-fold lower organs related toxicities compared to free drug while keeping the antiviral activity unaltered [214] . Lf nanoparticles co-encapsulating the anti-HIV-1 drug Efavirenz and the anti-microbial drug curcumin (ECNPs) were used as a vaginal microbicide for multipurpose prevention technologies (MPT) [215] . After topical application in rats, the NPs could deliver higher drug amounts in vaginal lavage while showed significantly lower drug absorption in vaginal tissue and plasma relative to its free form. An anti-retroviral drug combination (zidovudine, efavirenz and lamivudine) was encapsulated into Lf NPs to improve their absorption and hence increase their anti-HIV efficacy [216] . The NPs showed pH-dependent drug release behavior with very low initial burst effect while most of the entrapped drugs were released at pH 5 with minimal toxicity to the erythrocytes and less tissue-related inflammation. |
Lf exhibits favorable modulatory effects on bone cells and enhances bone growth thus it can be used as a bone growth factor for bone regeneration and bone engineering. Addition of Recombinant human Lf (rhLf) to the culture media of NHOst human osteoblast cells resulted in increased level of alkaline phosphatase (ALP), a marker for osteogenic activity, and enhanced ERK1/2 activation, which is a part of proliferative signaling. Treatment of piglet with rhLf has increased the serum calcium and bone mineral density compared to control piglets [217] . Treatment of MC3T3 osteoblast precursor cells with rhLf for 24 hr resulted in increase in the anti-apoptotic Wnt5a together with an increase in Akt phosphorylation, while down-regulated caspase 3, which is a marker of cell apoptosis. Therefore, tyramine-coupled rhLf was enzymatically cross-linked using horseradish peroxidase and hydrogen peroxide into injectable hydrogel for encapsulation of MC3T3 cells. A remarkable increased expression of ERK (a proliferation marker) and RunX2 (a differentiation marker) was observed in the encapsulated MC3T3 cells. The cells maintained their differentiation ability as revealed by expression of collagen I protein, and osteocalcin after 21 days post culture in mineralization media [218, 219] . A number of studies suggested that Lf-induced osteogenic activity and proliferation of osteoblasts may be mediated by binding with TGF-β receptors and stimulating TGF-β canonical pathways, or by activation of MAPK pathways [220] . Due to its antiapoptotic effect on osteoblasts and suppression of osteoclastogenesis, Lf can be considered as anabolic factor for anti-osteoporotic therapy [221] . Different studies found that hydroxyapatite nanocrystals (HA) and Lf had a synergistic effect on bone homeostasis. To increase its bioavailability and prolong its action, Lf was electrostatically adsorbed onto HA nanocrystals forming a single protein layer well exposed to cells. Lf/HA combination resulted in several beneficial effects [222] . This includes enhancing the expression of RUNX2 mRNA, Osterix and IBSP, common markers of osteoblast differentiation confirming a synergistic effect on osteogenic differentiation and formation of bone matrix. Moreover, Lf reduced the apoptotic effect of HA on osteoblasts, suppressed the stimulatory effect of HA on osteoclasts and stimulated osteoblasts to produce osteoprotegerin (OPG) that reduce the activity of osteoclasts [86] . |
J o u r n a l P r e -p r o o f |
Despite the promising role of Lf as a drug nanocarrier and tumor-targeting ligand with anticancer and immunomodulatory functions, some challenges have to be overcome prior to clinical translation. First, the cationic nature of Lf can cause toxicity and adverse inflammatory response [223] . Positively charged NPs are known to suffer from higher non-specific uptake and rapid clearance from blood by macrophages of liver, spleen and lung compared to anionic and neutral particles [224] . Mitochondrial damage and cell necrosis were observed after injection of cationic liposomes or chitosan NPs mostly mediated by inhibition of the cation-binding site of Na + /K + -ATPase [225] . Moreover, high expression levels of the inflammatory cytokines e.g. IL6, CXCL2 and CCL2 were detected in lung, spleen and liver of rats after administration of cationic micelles and liposomes [223] . We anticipate that the effect of positive charge of Lf macromolecules can be alleviated via its electrostatic complexation with polyanionic polysaccharides to form nanocomplexes with negative or low positive charge according to the complexation ratio. Another approach might involve the formation of multi-stage nanocarriers via encapsulation of the cationic Lf NPs within negatively charged or PEGylated phospholipid bilayers. This approach avoids the toxicity of the released cationic Lf NPs into circulation while makes use of their positive charge in enhancing their uptake into cancer cells via charge-mediated transcytosis. |
The positive charge of Lf can also be beneficial for endosomal escape particularly for gene delivery. Second, the short half-life of Lf after systemic administration due to the action of proteolytic enzymes and its oral susceptibility after oral intake may lead to inadequate delivery. Many effective strategies have been successfully implemented to prolong its half-life and increase its oral stability including PEGylation, encapsulation or chemical crosslinking [90, 226] . Finally, although the reported safety and biocompatibility of Lf, the use of bovine Lf might induce possible immunogenicity with undesired reactions. Antibodies against bovine Lf have been detected in the serum of mice treated with Lf-coated nanocapsules [134] . Therefore, the use of recombinant Lf can overcome this limitation (discussed below). |
Bovine Lf (bLf), is commercially available and well tolerated form of Lf with similar biological functions to human Lf but with lower cost. Based on the results of several toxicological studies on bovine milk derived Lf (cMDLf), including an acute, a 4 week, a 13-week, and two chronic oral toxicity studies in rats, as well as hypersensitivity, allergenicity and mutagenicity studies, it can be concluded that cMDLf does not produce adverse effects, not mutagenic and unlikely to be a clinically relevant allergen or the primary causative agent of any immunologically driven hypersensitivity consumption. bLf is recognized by the US Food and Drug Admission and the European Food Safety Authority as GRAS (generally recognized as safe) component for numerous indications [227] [228] [229] . Table S1 lists some of the Lf-based products available in the pharmaceutical market. Many studies and clinical trials have demonstrated the safety and tolerability of orally administered rhLf in treatment of non-small cell lung cancer and renal cell carcinoma [5, 75] . We have summarized the most important outcomes of the clinical trials based on Lf products for various applications (Table S2) . Talactoferrin (TLf) is a recombinant human Lf which is intended for oral administration. Production of recombinant human Lf may proceed using different expression systems, including fungi, animals, plants and viruses. Nevertheless, recombinant human Lf, produced by Aspergillus awamori, has been directed toward therapeutic uses [230, 231] . More prominently, TLf has shown to modulate the immunity in various pathological conditions. For instance, TLf was demonstrated to induce INF-γ dependent activation of NK cells and CD8+ cells in the gut associated lymphoid tissue (GALT), Peyer's patches as well as systematically. In addition, the subsequent effect was further extended towards inhibition of neu + tumors [232] . Moreover, TLf showed a comparable antitumor effect to human Lf and bovine Lf in colon and liver cancer cells, through induction of apoptosis, chemokine C-C motif ligand 20 (CCL20) secretion and TGF-β1 expression [233] . Furthermore, it was shown that topically, TLf enhanced the release of key inflammatory mediators, such as IL-8, IL-6, TNF-α and macrophage inflammatory protein-1 alpha, which are responsible for tissue repair, and subsequently wound healing [81] . In addition to recombinant human Lf, a lot of research is directed to Lf-derived peptides. The peptides Lf (1-11), Lactoferricin and Lactoferrampin (268-284), are the three major peptides derived from Lf showing pharmacological actions: antibacterial, antiviral, antiparasitic, antifungal and anticarcinogenic activities. It worth noting that Lactoferricin B, corresponding to residues 17-41 of bLf, was reported to exert more potent antimicrobial activity than Lf [234] . Table S3 elucidates biological functions of Lf peptides. Being chemically modified, LTX-315 is a 9-mer cationic peptide derived from bovine Lf and possess an anti-tumor activity, especially against solid tumors [235, 236] . The mechanism of oncolytic property resides in the activation of local anti-tumor immunity, alone or combined with chemotherapeutic agents. In this regard, LTX-315 was shown to promote the infiltration of CD4+ and CD8+ cells to the tumor sites, in addition to a reduction in myeloid derived-suppressor cells (MDSC) and CD4+ regulatory T cells. The modulation of the tumor micro-environment exhibited by LTX-315 paved the way to a more pronounced anti-tumor activity when combined with DOX [236] . Furthermore, it was demonstrated that mitochondrial permeabilization through Bcl-2 family protein dependent pathway, was an additional anti-tumor mechanism of LTX-315 [237] . |
The diagnosis of drug-associated interstitial lung disease (ILD) involves three elements: clinical suspicion, differentiation from other parenchymal lung diseases using computed tomography (CT) and other clinical tests for alternative disease, and a compatible histological pattern. This review article will discuss the radiological evaluation of a patient with non-small-cell lung cancer (NSCLC) suspected of suffering from drug-associated ILD, the diagnosis and management of drug-associated ILD, and the development of a diagnostic algorithm designed to distinguish gefitinib ('Iressa')-associated ILD from other forms of parenchymal disease. |
The onset of drug-associated ILD during therapy for advanced NSCLC usually occurs within a few weeks of the start of treatment (Thomas et al, 2000; Gupta et al, 2002; Kudrik et al, 2002; Read et al, 2002) . Indeed, retrospective analysis of the first 152 patients in Japan to experience gefitinib-associated ILD showed that 475% of cases occurred within 3 months, with the majority of these occurring within 4 weeks. |
The symptoms of drug-associated ILD, as with all forms of the condition, include rapidly developing breathlessness and a dry and unproductive cough, together with fever (Thomas et al, 2000; Gupta et al, 2002; Kudrik et al, 2002; Read et al, 2002) . Such symptoms are nonspecific and can occur with a large number of common illnesses often associated with NSCLC, or they may be due to cancer progression or lung cancer therapy. These patients are also prone to pneumonia and many have radiation-related injury as a result of prior treatment. Cardiovascular causes of the symptoms, such as fluid overload, congestive heart failure and pulmonary embolus, are not uncommon. Differentiation of drug-associated ILD from these illnesses is difficult and the diagnosis is usually made by exclusion. Highresolution CT (HRCT) is recommended on first suspicion of ILD to provide an assessment of the parenchymal nature of the cause of symptoms. |
The radiological manifestations of drug-associated ILD, although heterogeneous and nonspecific, enable many of the alternative causes of symptoms in advanced NSCLC to be excluded. There is no specific radiological pattern of parenchymal change connected with drug-associated ILD. Furthermore, in the early stages of disease, patients with symptoms secondary to drug reaction may have a normal chest radiograph. |
High-resolution CT allows a more precise assessment of the presence, pattern and distribution of parenchymal and airway abnormalities than a chest radiograph. It has the advantage over lung biopsy of providing an overall view of the extent and pattern of parenchymal involvement rather than being limited to a small region, which may not be representative of the overall pattern of disease. However, there is limited information on the correlation between the findings on HRCT and histological patterns in drug-associated lung disease. Data based on a small number of cases suggest that the different histological patterns of drug reaction are not reflected by distinctive HRCT findings (Cleverley et al, 2002) . *Correspondence: Professor NL Müller; E-mail: [email protected] Despite these limitations, it seems reasonable to approach the radiological manifestations of drug-associated lung disease by the use of the underlying histological pattern (Ellis et al, 2000; Myers et al, 2003) . Using such an approach, the most common manifestations can be classified into diffuse alveolar damage, hypersensitivity pneumonitis, organising pneumonia, nonspecific interstitial pneumonia (NSIP) and eosinophilic pneumonia (other less common drug reactions are not discussed here). Any one histological pattern can be caused by a number of different drugs. Furthermore, similar histology is found in other conditions that are not associated with drug use, such as idiopathic interstitial pneumonias, viral, bacterial and fungal pneumonia, pulmonary haemorrhage or leukaemia and collagen vascular disease. Occasionally, HRCT may demonstrate findings that are highly specific for the diagnosis, including increased attenuation in amiodarone lung and areas of fat attenuation in lipoid pneumonia. |
Diffuse alveolar damage is characterised histologically by the presence of alveolar airspace and interstitial oedema, hyaline membrane formation and proliferation of type II pneumocytes (Rossi et al, 2000; Cleverley et al, 2002) . In relation to drug-associated pulmonary disease, it occurs most commonly with cytotoxic agents such as bleomycin and, less commonly, with aspirin and narcotics (Rossi et al, 2000; Cleverley et al, 2002) . The corresponding radiological features are also found in adult respiratory distress syndrome (ARDS). The chest radiograph shows bilateral patchy or homogeneous airspace consolidation involving mainly the middle and lower lung zones (Rossi et al, 2000; Cleverley et al, 2002) . High-resolution CT demonstrates extensive bilateral ground-glass opacities and dependent areas of airspace consolidation (Rossi et al, 2000; Erasmus et al, 2002) ( Figure 1A ). |
A number of drugs may result in hypersensitivity pneumonitis, including methotrexate, cyclophosphamide and antidepressants such as fluoxetine and amitriptyline (Ellis et al, 2000) . The radiological and HRCT findings are identical to those seen in hypersensitivity pneumonitis secondary to the inhalation of organic dust and consist of bilateral ground-glass opacities and/ or small poorly defined centrilobular nodular opacities (Ellis et al, 2000; Cleverley et al, 2002) . The majority of patients also demonstrate lobular areas of air trapping, although this is less Figure 1 High-resolution CT images demonstrating radiology of drug-associated ILD. (A) A 77-year-old man with diffuse alveolar damage secondary to amidarone; note the extensive bilateral ground-glass opacities, airspace consolidation and bilateral pleural effusions. (B) A 36-year-old woman with hypersensitivity pneumonitis secondary to sertraline; note the extensive bilateral ground-glass opacities and lobular areas of air trapping (arrows). (C) A 69-year-old man with BOOP-like reaction to amiodarone; note the mild reticulation and bilateral areas of consolidation and ground-glass opacities in a predominantly peribronchial distribution. (D) A 47-year-old man with NSIP reaction to bleomycin; note the extensive bilateral ground-glass opacities with mild superimposed reticulation. (E) A 47-year-old man with eosinophilic pneumonia reaction to dilantin; note the patchy bilateral areas of consolidation involving the peripheral regions of the upper lobes. apparent than in extrinsic allergic alveolitis to airborne agents (Ellis et al, 2000) ( Figure 1B ). |
Organising pneumonia, also known as bronchiolitis obliterans organising pneumonia (BOOP)-like reaction, has been reported most frequently in association with methotrexate, cyclophosphamide, gold, nitrofurantoin, amiodarone, bleomycin and busulphan (Cleverley et al, 2002; Erasmus et al, 2002) . The chest radiograph shows patchy bilateral areas of consolidation, masses or nodules, which may be symmetric or asymmetric (Müller et al, 1990; Ellis et al, 2000) . In a few patients, the disease manifests with a lone mass. On HRCT, areas of consolidation often have a predominantly peripheral or peribronchial distribution (Müller et al, 1990; Ellis et al, 2000) ( Figure 1C ). |
Nonspecific interstitial pneumonia is one of the most common forms of drug-associated pneumonitis. Nonspecific interstitial pneumonia is characterised histologically by homogeneous alveolar wall thickening by fibrous tissue and mononuclear inflammatory cells. The reaction is seen in association with a variety of drugs, the most common being methotrexate, amiodarone and carmustine (Erasmus et al, 2002) . The corresponding radiographical and HRCT findings usually consist of patchy or diffuse ground-glass opacities (Rossi et al, 2000; Erasmus et al, 2002) ( Figure 1D ). On disease progression, there may be evidence of fibrosis with development of a reticular pattern and traction bronchiectasis. In some patients, the fibrosis is patchy in distribution and predominantly peribronchovascular, a pattern most commonly seen in patients receiving nitrofurantoin. Late chemotherapy lung may predominate in the upper and lateral parts of the lung. |
Eosinophilic pneumonia is characterised histologically by the accumulation of eosinophils in the alveolar airspaces and infiltration of the adjacent interstitial space by eosinophils and variable numbers of lymphocytes and plasma cells. Peripheral blood eosinophilia is present in p40% of patients. Eosinophilic pneumonia secondary to drug reaction is seen most commonly in association with methotrexate, sulphasalazine, para-aminosalicylic acid, nitrofurantoin and nonsteroidal anti-inflammatory drugs. Chest radiography and HRCT show bilateral airspace consolidation, which tends to involve mainly the peripheral lung regions and the upper lobes (Rossi et al, 2000; Cleverley et al, 2002 ) ( Figure 1E ). |
Alternative diagnoses to drug-associated ILD in NSCLC include progression of the cancer, infection, radiation-related lung injury, fluid overload, congestive heart failure and pulmonary embolus. Additionally, some lung cancer patients may develop BOOP or other steroid-responsive inflammatory disorders that cannot be clearly related to drug therapy. Other possible causes of dyspnoea that do not give infiltrates include comorbid diseases such as chronic obstructive pulmonary disease (COPD) and aspiration of food and saliva (particularly in patients with vocal cord paralysis or brain metastases). |
In the USA, pulmonary embolism is particularly common in patients with lung cancer, with as many as 20% of patients estimated to develop a deep vein thrombosis or pulmonary embolism during the course of their disease (Lieberman et al, 1961; Sack et al, 1977; Lee and Levine, 2003) . |
Most episodes of pneumonia in patients with lung cancer are due to bacteria. This is particularly the case when risk factors of neutropenia, endobronchial lesions, underlying COPD and aspiration are present. Although opportunistic infections are not common, fungal infections should be considered if the patient has received a high dose of corticosteroids. Viral infections with herpes simplex, cytomegalovirus or respiratory syncytial virus may also rarely result in pneumonia in patients who have received highdose corticosteroids or very intensive chemotherapy. |
The development of lung fibrosis following radiation therapy is well documented and is usually confined to the radiation port (Abid et al, 2001; Aviram et al, 2001) . With the use of threedimensional radiation portals, however, resulting infiltrates from radiation may not result in the traditional straight-edged infiltrate and may be more difficult to distinguish from other entities. |
Patients with lung cancer who present with respiratory failure should undergo systematic investigation. Pulmonary function tests, such as measurement of forced expiratory volume in 1 s, carbon monoxide diffusing capacity and measurement of arterial oxygen saturation with pulse oximetry, are commonly used. These tests ascertain the type of defect, for example, obstructive or restrictive ventilatory defects, which helps establish the cause, such as an exacerbation of any underlying obstructive airways disease versus interstitial disease. They also indicate the severity of the disease that helps determine the need for further assessment and treatment. |
A standard chest CT scan is commonly performed to exclude a diagnosis of disease progression or pulmonary embolism. However, as discussed previously, obtaining high-resolution cuts is very helpful (HRCT) if drug-associated ILD is suspected. |
Bronchoscopy is useful to evaluate some NSCLC patients with dyspnoea to assess extension of the cancer and to exclude an opportunistic infection using bronchioalveolar lavage. However, the value of a bronchoscopy in establishing the diagnosis of drugassociated ILD is less clear, since bronchioalveolar lavage is not specific for drug-associated disease, and biopsies obtained by transbronchial biopsy are small and often do not yield enough tissue to make this distinction. Open lung biopsy is rarely performed in NSCLC patients with respiratory distress since most patients have advanced disease with a limited prognosis. Furthermore, procedures requiring surgery are not usually believed to arrive at a definitive diagnosis of drug-associated ILD. |
Patients with mild symptoms or pulmonary function abnormalities (such as a decrease in diffusing capacity of o20% from baseline or no change in oxygen desaturation during exercise), or with transitory or slight radiographical infiltrates are monitored using pulmonary function tests, symptoms and usually CT scans. Diagnostic evaluation and treatment for drug-related lung disease is considered in those patients who experience dyspnoea at rest or on mild exertion, have a X20% decrease in carbon monoxide diffusing capacity, or experience oxygen desaturation at rest or during exercise. Patients whose radiographical infiltrates are extensive or progressive are also considered for therapy. |
Retrospective analysis of the adverse-event reports from patients diagnosed with ILD following gefitinib treatment is also difficult as there is often limited or heterogeneous clinical information, no pathology result and no access to the results of radiological investigations. |
A diagnostic algorithm has therefore been developed to assess the accuracy of the reports of drug-associated ILD among Japanese patients receiving gefitinib. This approach to differential diagnosis used an algorithm developed to aid early diagnosis of drugassociated ILD in clinical practice. |
The algorithm used both radiological and clinical evidence to exclude alternative diagnoses, such as infection, tumour progression, heart failure and pulmonary embolism, to arrive at a presumptive diagnosis of drug-associated ILD (Figure 2 ; Table 1) . Patients were categorised based on the strength of evidence supporting the diagnosis of drug-associated ILD (category 1, good supporting evidence for ILD; category 2, limited supporting evidence for ILD; category 3, no supporting evidence for ILD). |
In the first assessment, adverse-event reports from Japanese patients receiving gefitinib for NSCLC were evaluated using the algorithm, with available clinical information and radiographical reports but without access to chest radiographs or HRCT films. These findings were then compared with those of a second assessment, conducted by an independent panel of expert radiologists and physicians. The panel assessed the radiological and clinical findings for the same patient population using a set of standardised criteria (Cleverley et al, 2002) . |
The initial 152 reported patients with NSCLC in Japan who had experienced adverse events involving the lungs while receiving treatment with gefitinib were included in the first assessment of the algorithm. Of these, 135 were included in the second assessment because radiological examinations, including 47 with CT imaging, were available. Approximately 20% of these patients (23 out of 135) were considered by the expert panel not to have drug-associated ILD, highlighting the difficulty in diagnosing drug-associated ILD in patients with NSCLC. |
These results were then compared with those obtained using the algorithm. A large proportion (17 out of 23) of patients not considered by the panel to have drug-associated ILD had been categorised as having 'good' or 'limited' supporting evidence for drug-associated ILD. The initial algorithm based on the terminology used in radiology reports, without considering differential diagnosis criteria, was not adequate. Infection, heart failure and tumour progression can be differentially diagnosed and excluded using additional radiological clinical data. |
Therefore, the algorithm is now used to enable the clinician to make a diagnosis having first undertaken all the necessary steps in the clinical examination and investigation. This approach is applied to the prospective nested case -control study to investigate the relative risk and risk factors for ILD in NSCLC patients in Japan treated with and without gefitinib. An independent case review board will review each reported case of ILD using the information gathered from the algorithm. |
General principles in the management of acute respiratory distress in patients with lung cancer are influenced by the multiple causes of respiratory failure that are associated with cancer, lung cancer therapy and the presence of comorbid disease. In addition, the diagnosis may remain uncertain, even when invasive procedures are performed, and empirical therapy for the likely causes is frequently given. Finally, respiratory failure in patients with cancer results in high mortality (Groeger et al, 1999) requiring aggressive assessment and therapy. |
There are no firm guidelines for the treatment of drug-associated ILD and therapy tends to be on an empirical basis. Withdrawal of the drug suspected of causing the ILD is the first step in treatment. For patients in respiratory failure, high-dose methylprednisolone (250 mg four times a day i.v.) for several days is commonly used. If the patient responds, then the dose is reduced (0.5 -1 mg kg À1 day À1 orally) for several weeks before being gradually tapered. For patients in respiratory distress, methylprednisolone (1 mg kg À1 day À1 or 60 mg day À1 ) is commonly used, again with gradual dose reduction. Low-dose methylprednisolone (10 -20 mg) is prescribed for patients with mild radiographical or pulmonary function abnormalities, particularly if oral corticosteroids are contraindicated (White and Stover, 1984; Baughman et al, 1994; American Thoracic Society, 2002) . |
Immunosuppressive agents such as azathioprine have been used as steroid-sparing agents in the treatment of drug-associated ILD, particularly in chronic cases of bleomycin-associated ILD (Maher and Daly, 1993) . These agents are useful for patients in whom corticosteroids cannot be tapered or who cannot tolerate corticosteroids. It is also advisable to avoid combining agents associated with ILD, such as bleomycin and possibly mitomycin, with other agents that cause lung damage, such as oxygen and radiation, as this may result in worsening of the lung damage. If radiotherapy is indicated in a patient who has experienced mild bleomycin toxicity, then concomitant low-dose corticosteroid may minimise any further lung damage. Following resolution of the drug-associated ILD, some patients are susceptible to exacerbations on subsequent insults (e.g. during a respiratory infection) and may require further treatment. |
In cancer patients, drug-associated ILD is most commonly observed during mitomycin, paclitaxel, docetaxel or gemcitabine therapy. Table 2 outlines the types of injury reported with these agents and their response to therapy (Buzdar et al, 1980; Chang et al, 1986; Goldberg and Vannice, 1995; Rivera et al, 1995; Ramanathan and Belani, 1996; Bookman et al, 1997; Merad et al, 1997; Piccart et al, 1997; Semb et al, 1998; Vander Els and Miller, 1998; Dunsford et al, 1999; Thomas et al, 2000; Fogarty et al, 2001; Read et al, 2002; Barlesi et al, 2004) . Acute pneumonitis, both interstitial and noncardiac pulmonary oedema pattern, has been observed with mitomycin. Response to high-dose methylprednisolone has been reported within 24 -48 h of therapy; however, approximately 60% of patients developed ongoing and persistent lung disease (Rivera et al, 1995) . Chronic pneumonitis, similar to bleomycin-type pneumonitis, has also been reported during mitomycin therapy, which responded to prednisone therapy (Buzdar et al, 1980; Chang et al, 1986) . As with bleomycin, it has been suggested that oxygen therapy be avoided during mitomycin therapy, although the evidence for this is not as established as that for bleomycin (Klein and Wilds, 1983) . |
Infusion hypersensitivity is very common during paclitaxel therapy. Pretreatment with an antihistamine, a corticosteroid and an H2 blocker largely prevents or ameliorates this reaction (Bookman et al, 1997) . Mild interstitial pneumonitis with transitory infiltrates has also been reported; observation or lowdose corticosteroids resulted in a good response, even when this occurred in conjunction with radiotherapy (Goldberg and Vannice, 1995; Ramanathan and Belani, 1996) . Fluid retention syndrome during docetaxel therapy is very common and is associated with dyspnoea. This reaction can be prevented by dexamethasone (Piccart et al, 1997; Semb et al, 1998) . Interstitial pneumonitis and ARDS-like pattern have also been reported with docetaxel, particularly when it is used in combination with radiotherapy or gemcitabine. Both reactions respond well to corticosteroid therapy; however, when docetaxel is used in combination with radiotherapy or gemcitabine, the response to corticosteroid therapy can be variable to poor, with some deaths reported (Merad et al, 1997; Dunsford et al, 1999; Read et al, 2002) . |
Self-limiting dyspnoea is common during gemcitabine therapy. Additionally, many patients experience a mild capillary leak throughout the lung, similar to that seen in ARDS. However, these patients are asymptomatic and have normal pulmonary function; therefore, gemcitabine therapy can be continued without the need for corticosteroids. Gemcitabine-associated ILD has also been reported and the response to corticosteroid therapy is variable. As with docetaxel, more deaths due to ILD have been reported when gemcitabine is used in combination with other therapies (radiotherapy or chemotherapy) (Vander Els and Miller, 1998; Dunsford et al, 1999; Thomas et al, 2000; Fogarty et al, 2001; Barlesi et al, 2004) . |
In the management of patients with NSCLC, it is often necessary to treat patients with an agent that has been previously associated with ILD. In this scenario, it is important to consider the patient's previous response to that therapy, the severity of the lung damage and the respiratory distress, the presence of fibrosis and the previous response to corticosteroid therapy. For some patients, the benefits may outweigh the risks and therapy may be re-instituted with concomitant low-dose prednisone (10 -20 mg day À1 ); however, for some patients and/or agents the potential for causing further lung damage may be too great. |
In summary, lung cancer patients receiving systemic therapy frequently develop dyspnoea and infiltrates. It is difficult to make a specific diagnosis in most cases because of the difficulty of performing invasive procedures in this patient population. Radiological assessment, and HRCT in particular, can play a key role in establishing a diagnosis of drug-associated ILD; however, in the vast majority of cases, the radiological manifestations of drugassociated pulmonary injury are nonspecific, making an accurate diagnosis difficult. Corticosteroids are indicated for suspected drug-associated ILD; however, the outcome is variable unless the patient develops respiratory failure, in which case the mortality is high. |
As a result, clinicians are reluctant to withhold corticosteroid therapy if there is any indication of drug toxicity, further complicating the diagnosis. However, once a patient responds to corticosteroid therapy, the decision to re-institute a drug suspected of causing ILD is made on an individual basis. In this article we described an algorithm developed to assess the incidence of drugassociated ILD in Japanese patients receiving gefitinib for NSCLC. Such an algorithm, once validated, may be a useful tool in the differential diagnosis of ILD in patients with cancer and help clarify some of the apparent discrepancies in the incidence and reporting of ILD. Buzdar et al (1980) , Chang et al (1986) , Goldberg and Vannice (1995) , Rivera et al (1995) , Ramanathan and Belani (1996) , Bookman et al (1997) , Merad et al (1997) , Piccart et al (1997) , Semb et al (1998), Vander Els and Miller (1998) , Dunsford et al (1999) , Thomas et al (2000) , Fogarty et al (2001) and Read et al (2002) . a Even when paclitaxel is used in conjunction with radiotherapy. b When docetaxel is used in combination with radiotherapy or gemcitabine, the response is variable to poor. |
This appendix aims at discussing some mathematical details of the economic-epidemiological study presented in the main text by also o¤ering di¤erent perspectives about the second-best optimal policies against COVID-19. In doing so, it considers the normative analysis of 1) joint control programmes under the assumption under the baseline scenario discussed in the main text, i.e., = 0 and " = 1, and 2) the possible combinations of the mortality indicator, = f0; 1g, with the welfare criterion, ranging from Benthamite (" = 1) to Millian (" = 0) utilitarianism. |
The dynamics of the SIR subsystem in (8) presented in the main text is independent of the evolution of k. About the stability of the subsystem given by the epidemiological equations in (8) we refer to Theorem 2.1 in Busenberg and van den Driessche (1990) . Regarding the fourth equation, we have that when the SIR subsystem respectively converges to the infection-free equilibrium or the endemic equilibrium, the dynamics of k are asymptotically governed by _ k = xAk `1 (b { c + )k, where{ = 0 and`= 1, or{ = i and`= 1 i , respectively. The result of the proposition follows immediately as in both cases the last equation of the system is Bernoulli-like. |
Normative analysis under = 0, " = 1: joint programmes (lockdown avoidance) |
The normative analysis presented in the main text has concentrated on the existence of possible (second-best) optimal values of 1) the parameter govering the extent of social distancing (q) in the absence of TTI ( = 0), and 2) the tax rate governing the extent of TTI ( ) in the absence of social distancing (q = 1). However, the social distancing and TTI programmes can be combined with the aim at maximising the social welfare function (28) in the main text subject to (8) and taking the locking/unlocking thresholds as given. In this case, results show that at low or moderate TTI e¢ ciency (as measured by ) an internal solution for q exists, meaning that a certain degree of social distancing is anyhow necessary in that case. However, for a su¢ ciently high TTI e¢ ciency, there is always an optimal tax rate ( = max ) allowing lockdown avoidance (q = q max = 1) in both the short (T = 2) and longer terms (T = 10). With our parameter choices, this holds for any ! > 0 and the welfare-maximising tax rate is larger the larger !, as the society is willing to accept a higher tax burden to grasp the bene…ts of reducing COVID-related mortality. Lockdown avoidance may follow two distinct trajectories (Table A1 ). In the short term (T = 2) when ! = ! low the amount of resources collected through (the welfare-maximising) taxation is not su¢ cient to avoid infection prevalence exceeding the locking threshold (T L ) at some stages of the epidemic (see Fig. 5 in the main text for comparison purposes). However, the reduction in epidemiological costs and the corresponding increase in economic bene…ts are larger than those related to the lockdown policy, which is therefore not implemented. When ! = ! high , the resources collected through taxation at the optimum are high enough to almost always avoid prevalence to exceed T L , though maximal welfare is lower than when ! = ! low due to the larger amount of resources the society is willing to collect in that case. As for long-term horizons (T = 10), results are similar to short-term ones, so that the planner chooses not to declare lockdown in either case ! low and ! high though in some phases infection prevalence exceeds the T L threshold. However, the TTI-related expenditure decreases over time due to the reduced number of infections compared to the short term causing a reduction in epidemiological costs and an increase in economic bene…ts. Table A1 . Welfare analysis of joint intervention programmes: ! low = 1=10 7 and ! high = 10 ! low . |
This section o¤ers a broader perspective about the second-best optimal policies against COVID-19 analysed in the main text (Section 5) by considering the possible combinations of the mortality indicator, = f0; 1g, with the welfare criterion, ranging from Benthamite (" = 1) to Millian (" = 0) utilitarianism. Results show (compare Figs. A1 and A2 with Figs. 6 and 7 in the main text) marked robustness for both dimensions. In particular, social welfare still exhibits a hump-shaped pattern with q and regardless of the choice on the pair ( ; "). Qualitatively, this result was not unexpected as COVID-19 mortality has been tuned to crudely mirror real-world mortality in the working population, implying negligible e¤ects on the size of the general population N , which follows an almost constant trend. This implies that the COVID-related welfare e¤ects are not signi…cantly a¤ected by the adopted utilitarian criterion. We pinpoint that the relative weight of the disutility of COVID-19 mortality (! > 0) in Figs. A1 and A2 has been set to obtain the same levels of the second-best optimal values of the control intervention parameters q and . |
(a) (b) Figure A1 . Case = 0. Panel (a): comparison between the patterns of W (q) for " = 0, ! = 6:4 10 6 (blue curve, left axis) and " = 1, ! = 1=10 7 (black curve, right axis). The optimal value is q = 0:36. Panel (b): comparison between the patterns of W ( ) for " = 0, ! = 6:4 10 6 (blue curve, left axis) and " = 1, ! = 1=10 7 (black curve, right axis). The optimal value is = 0:013. |
(a) (b) Figure A2 . Case = 1. Panel (a): comparison between the patterns of W (q) for " = 0, ! = 6:4 10 6 (blue curve, left axis) and " = 1, ! = 1=10 7 (black curve, right axis). The optimal value is q = 0:36. Panel (b): comparison between the patterns of W ( ) for " = 0, ! = 6:4 10 6 (blue curve, left axis), and " = 1, ! = 1=10 7 (black curve, right axis). The optimal value is = 0:012. |
The Coronavirus infectious disease pandemic has exhibited collateral challenges to the healthcare systems globally [1] . By the mid of July 2021, the global cases of COVID-19 had exceeded 171 million with more than 3 million deaths [2] . Total cases in Nepal during that time was 658,778 cases with 9412 deaths [2] . To this date, there lacks an effective antiviral agent against the causative agent, SARS-CoV-2. Therefore, public health measures, such as social distancing, lockdowns, hand hygiene, contact tracing, quarantine, and isolation remain the mainstay of prevention and mitigation measures [3] [4] [5] . |
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