Over the past few decades, long acting injectable (LAI) depots of polylactide-co-glycolide (PLGA) or polylactic acid (PLA) based microspheres have been developed for controlled drug delivery to reduce dosing frequency and to improve the therapeutic effects. Biopharmaceuticals such as proteins and peptides are encapsulated in the microspheres to increase their bioavailability and provide a long release period (days or months) with constant drug plasma concentration. The biodegradable and biocompatible properties of PLGA/PLA polymers, including but not limited to molecular weight, end group, lactide to glycolide ratio, and minor manufacturing changes, could greatly affect the quality attributes of microsphere formulations such as release profile, size, encapsulation efficiency, and bioactivity of biopharmaceuticals. Besides, the encapsulated proteins/peptides are susceptible to harsh processing conditions associated with microsphere fabrication methods, including exposure to organic solvent, shear stress, and temperature fluctuations. The protein/peptide containing LAI microspheres in clinical use is typically prepared by double emulsion, coacervation, and spray drying techniques. The purpose of this review is to provide an overview of the formulation attributes and conventional manufacturing techniques of LAI microspheres that are currently in clinical use for protein/peptides. Furthermore, the physicochemical characteristics of the microsphere formulations are deliberated. Protein and peptide drugs are known to be some of the most effective therapies to elicit a desired therapeutic activity due to their specific interactions with biological targets . HoweverLong-acting injectable (LAI) microspheres have been previously exploited for the delivery of protein and peptide therapeutics owing to their high drug loading capacity and potential to provide prolonged drug release for extended periods. The advantages of LAI microspheres include enhanced stability and bioavailability and improved efficiency and patient compliance . PLGA/PLProtein and peptide therapeutics have been encapsulated in polymer matrixes to create microspheres or micro particles. Biocompatible and biodegradable polymers such as polylactide-co-glycolide (PLGA) and polylactic acid (PLA) are most commonly employed and widely utilized at a clinical level. The Food and Drug Administration (FDA) have approved a number of LAI microsphere formulations because of their proven safety history. Details of some commercially available PLGA/PLA-based microsphere formulations are presented in Although currently available commercial PLGA-based LAI microspheres have often shown success, there are several challenges that limit the broader development and application of LAI microspheres. Peptide and protein instability, poor release kinetics of PLGA, presence of residual solvents in the product, and increased cost of manufacturing are the major issues ,20,21. DThe selection of the PLGA/PLA polymer for the development of microspheres depends on the route of administration, which is specific for a particular drug, the amount of microspheres administered per unit dose, rate of drug release from the microspheres in a day to meet the therapeutic concentration of the specific drug, and the degradation time of the polymer. Although the drug release from the microspheres is faster than the complete degradation of the polymer, the degradation time of the polymer plays a major role in the selection of the suitable polymer . PLGA isThe hydroxyl moiety is usually attached to the growing PLGA chain via an ester bond. In the formation of PLGA with a free acid end-cap, water itself serves as an initiator . Other aThe selection of PLGA in the microsphere development depends on the appropriate selection of PLA and PGA ratio. This will determine the degradation kinetics of the microsphere formulation in the body. The hydrophilicity/hydrophobicity nature of the PLGA matrix depends on the lactide to glycolide ratio. PLA is hydrophobic in nature, whereas PGA is more hydrophilic; hence, PLGAs with a high ratio of PGA has a faster degradation rate owing to their hydrophilic nature, which promotes the absorption or penetration of water molecules into PLGA matrix, causing hydrolysis of polymer chains . The higPLGA polymers with different lactide to glycolide ratios  can exhibit different drug loading and drug release profiles owing to changes in their solubility profiles, degradation characteristics, and interactions between the drug and polymer . The natg) is one of the important physicochemical properties of the PLGA polymer. The Tg of PLGA usually lies above the physiological temperature (37 °C) and increases with the lactide content and molecular weight of the polymer or the addition of compounds such as active pharmaceutical ingredients (API) or plasticizers [The glass transition temperature  [Most of the injectable PLGA microsphere formulations in clinical use utilize a linear PLGA except Sandostatinlu-PLGA) . The numlu-PLGA) . Linear lu-PLGA) .Organic solvents are used to dissolve both the drug and the polymer in emulsion-based microencapsulation techniques. For efficient encapsulation, the organic solvent should meet the following criteria; low toxicity, low boiling point, high volatility, and ability to dissolve the polymer . MethylePrevious studies reported that the microspheres prepared with methylene chloride are more uniform and spherical, while the ethyl acetate-based microspheres appear to be partially collapsed. Moreover, encapsulation efficiency is high for microspheres made with methylene chloride than the microspheres prepared with ethyl acetate as a solvent ,63. The Depending on the boiling point and vapor pressure of the particular solvent, the solvent removal rate also relies on the evaporation of the solvent from the hardening batch, which is controlled by the unstirred boundary layer in the liquid or gas. The volatility of solvent can also impact the solvent removal rate from the microparticles. For a solvent with lower volatility, the solvent removal process can be expedited by increasing the temperature or applying reduced pressure. However, increasing the temperature of the hardening batch may lead to a more rapid flux of the solvent across the oil-in-water (O/W) interface, causing small fractures on the particles, results in increased porosity and lower drug loading ,67. For The toxicity of the solvent is crucial for the regulatory approval of the microsphere product. The maximum residual solvent level in the microsphere product depends on the toxicity of the respective solvent, and their maximum recommended levels are specified in the International Council for Harmonization (ICH) guidelines. Several methods, including elevated temperature drying or final lyophilization of the microspheres, help to reduce the final residual solvent levels to an acceptable value .During microsphere formation, an emulsifier or stabilizer is required to ensure the stability of the emulsion droplet until enough solvent has been evaporated/extracted  from the oil droplet to maintain particle formation . TypicalThe polarity of the organic solvent used in the emulsion system can affect the amount of PVA adsorbed at the interface (polymer–organic solvent–water). With the increasing organic solvent miscibility in water, the residual PVA associated with the microspheres increases. This is due to a higher amount of PVA being portioned into the polymeric phase containing an organic solvent, which is more miscible in the aqueous phase, causing the deposition of the higher portion of PVA on the surface of the microspheres .w/v) of PVA in the bovine serum albumin-PLGA nanoparticles made by the water-in-oil-in-water (W/O/W) double emulsion technique. The highest encapsulation efficiency was obtained at 1% w/v PVA concentration [Drug loading into PLGA microspheres depends on the PVA concentration. The lower the concentration of PVA, the lower the wettability of formed microspheres will be. There is a possibility that a lower concentration of PVA might not be high enough to create a stable emulsion due to leakage of the drug before skin formation of the microparticles. Chitkara and Kumar et al. evaluated three different concentrations  or double emulsion (W/O/W). For poorly soluble molecules, the O/W method is frequently used. This method has the following steps: dissolution of the insoluble drug in an organic solvent containing the PLGA or PLA polymer; emulsification of organic or dispersed phase in an aqueous or continuous phase; extraction of the solvent from the dispersed phase by the continuous phase, accompanied by solvent evaporation, converting the droplets of the dispersed phase into solid particles; and then collection and drying of microspheres to eliminate the residual solvent . This O/In general, organic solvents can be removed from the emulsion system by evaporation to a gas phase or by extraction into the continuous phase. For solvent evaporation, the carrier solvent must be dissolved in the continuous phase before evaporation occurs.The W/O/W double emulsion method is suitable for encapsulating hydrophilic molecules, and the main steps include the formation of primary and secondary emulsions and the removal of organic solvents using a suitable washing/evaporation process. To form a primary emulsion, an aqueous solution containing hydrophilic molecule is added to the solution of polymer previously dissolved in the water-immiscible organic solvent such as methylene chloride, chloroform, ethyl acetate, and then the mixture is emulsified by vortex, homogenization or ultrasonication ,80. PVA The stability of the primary emulsion is crucial for the successful encapsulation of proteins or peptides in the double emulsion technique. Schugenes et al.  studied Coacervation or phase separation is a process by which the polymer solution is separated into two immiscible equilibrium liquid phases: dense coacervate phase concentrated in polymer and a dilute (supernatant liquid) polymer phase. The coacervation technique relies on the decreasing solubility of the coating polymer with the addition of a third component (coacervating agent) to the polymer solution in an organic solvent . CoacervGenerally, the polymer separation or formation of the polymer-rich phase is induced by adding a coacervating agent, typically silicone oil. Depending on the polymer molecular weight, concentration, and processing temperature, the phase separation occurs above the critical concentration of the silicone oil. The forming coacervate droplets are hardened by adding a hardening agent such as hexane or octamethylcyclotetrasiloxane. The main drawback associated with this technique is residual coacervating or hardening agent present in the microspheres, which may cause a problem of reduced biocompatibility . BrieflyThe preparation of microspheres by spray drying has been reported for the encapsulation of proteins to improve the stability of proteins. The spray drying method can overcome the problem of large volumes of solvent contaminated water phase associated with the emulsion based microencapsulation methods. However, spray-drying requires relatively large batch sizes compared to emulsion methods. Therefore, spray drying is often less suitable in the early stage development of microsphere formulation . The sprIn the spray drying method, the aqueous solution of proteins or peptides is dispersed in the organic phase containing a PLGA/PLA polymer to form a primary emulsion. This primary emulsion is atomized in a stream of heated air with a proper inlet/outlet temperature, and then the solvent evaporates instantaneously from the droplets formed to produce microspheres of particle size ranges from 1 to 100 µm depending on the atomizing conditions. The microspheres are collected from an airstream using a cyclone separator. Further, vacuum drying might be required to remove the residual solvents. The entire spray drying process can be operated in closed-loop configurations under aseptic conditions. Various spray drying process parameters, including inlet/outlet temperature, drying flow rate, feed rate, and atomization energy input, influence the physicochemical properties of PLGA/PLA microspheres and thus the stability and release profile of encapsulated proteins/peptides ,95,96. TAlthough the inlet airflow temperature is high, the contact time between the hot air and sprayed droplets is limited. Therefore, when the sprayed droplets and hot airflow proceed through the chamber in the same direction, the droplets will absorb the heat, and their surface temperature will increase. However, in practice, the actual product temperature is about 15 to 25 °C lower than the outlet air temperature. Therefore, it is feasible to use the spray drying technique for the encapsulation of heat-sensitive molecules, such as peptides and proteins, by controlling the spray drying process parameters ,99. The Complex LAI microsphere products have been used to deliver protein/peptide therapeutics over weeks to months in a controlled manner. The LAI microsphere products have complex formulation constituents and complicated manufacturing processing steps . The phyA variety of characterization techniques have been used to determine physicochemical characteristics and ensure consistency in manufacturing processing and product performance of protein/peptide loaded microspheres. These analytical techniques are used to determine the physicochemical characteristics of the microspheres, such as particle size and shape, polymer molecular weight, encapsulation efficiency, in vitro drug release, glass transition temperature, residual organic solvent, moisture content, and porosity. However, despite widespread use of microspheres, no specific guidance or standard method has been developed for in vitro release testing. Some of these physicochemical properties are determined during the microsphere development stage to guide formulation and process development, and some are tested for quality control purposes. A summary of various techniques for testing the physicochemical properties of microspheres is presented in In vitro release testing methods with reproducibility and good discriminatory ability are critical for both quality control purposes and to assist in product development. It is important to understand the release mechanisms and factors that affect the release rate in order to modify the drug release. For LAI PLGA/PLA microspheres, real-time release testing utilizes an extended period, which affects the product batch release time. Therefore, accelerated in vitro release methods that correlate with the real-time in vitro release of microsphere products are essential. Several factors, including temperature, pH, and presence of enzymes and surfactants, can expedite the rate of polymer hydration/degradation and drug diffusion, thereby accelerating drug release from PLGA microspheres ,122. TheDuring the in vitro release of PLGA-based LAI microspheres, water is absorbed by PLGA upon immersion in aqueous media or administration in vivo, and the volume occupied by the water inside the PLGA matrix creates pores. This porous connected network allows drug release as the number and size of water-filled pores increases in the polymer matrix . Also, tDue to the lack of compendial in vitro release methods, various in vitro release testing methods such as sample-and-separate, membrane dialysis, and continuous flow have been widely used for in vitro release testing of microsphere products . Sample The particle size of PLGA/PLA microspheres influences the drug release behavior and injectability of the microspheres. Particle size varies with the different microsphere preparation techniques. The smaller particle size shows an enhanced initial burst release than the large size microspheres due to increased specific surface area. On the other hand, the polymer degradation rate is more prominent with the large microspheres compared with the small ones . The eroEncapsulation efficiency is described as the amount of protein/peptide encapsulated into microspheres following preparation. The physical and chemical properties of the polymer, the solvent used in the encapsulation process, and drug–polymer interactions can influence the encapsulation efficiency of proteins/peptides. It has been reported that microsphere preparation temperature in the solvent evaporation method is likely to affect the encapsulation efficiency. At a low preparation temperature, the immiscibility between the polymer phase and the water phase is increased, leading to the rapid formation of the microsphere wall. On the other hand, at a higher preparation temperature, the solvent evaporation rate is increased, resulting in the quicker formation of the microsphere similar to that observed at the low preparation temperature . It is nPorosity, either on the surface or in the internal polymeric matrix, significantly affects the drug release profiles of the microspheres. The pore size of microspheres can be modulated by selecting the appropriate solvent. The organic solvent with lower volatility produces large pores, whereas a more volatile solvent gives rise to small pores . The polThe Tg of the PLGA microspheres is closely related to the amount of residual solvent in the formulation. While preparing PLGA microspheres, several formulation and process parameters influence the Tg of the polymer, which include but are not limited to PLGA type, drug physicochemical characteristics, residual solvent (s), drying rate, and post-treatment . TypicalThe particle morphology of PLGA microspheres can influence the product performance, particularly the in vitro release profile. A modification in the manufacturing process is known to affect particle morphology . It has Recent progress in the area of PLGA long-acting injectable formulations mainly includes: demonstrating the influence of raw materials and manufacturing variables on the performance of LAI microspheres; exploring biorelevant in vitro–in vivo correlations (IVIVCs) for biodegradable injectable PLGA microspheres; obtaining a better understanding of the impact of properties of PLGA polymers on product performance; developing modeling tools to facilitate the development of generic LAI formulation development as well as bioequivalence guidances for LAI formulations; investigating potential peptide PLGA interactions during product manufacturing and use; and developing an analytical method for separating PLGA polymers. Recent studies aimed to determine the qualitative sameness of PLGA polymers because altered PLGA characteristics have been recognized as a critical factor that may cause performance variation in PLGA microsphere drug products. Similar PLGA polymers from different sources may have different physicochemical properties such as inherent viscosity, Mw, Tg, and blockiness. This could significantly impact physicochemical properties  and consequently the release characteristics of PLGA LAI microspheres. Bo Wan et al.  evaluate®. The in vitro release profiles of naltrexone microspheres were compared to the pharmacokinetic profiles of the microspheres using a rabbit model to develop an IVIVC and evaluate its prediction. The findings showed that the established USP 4 approach was capable of detecting manufacturing process-related performance changes, as well as forecasting the in vivo performance of naltrexone microspheres in the tested animal model. The bi-phasic and tri-phasic release characteristics of naltrexone microspheres, with varied burst release and lag phase, are a significant difference. The evolution of IVIVCs is influenced by these differences in release profiles.IVIVC can be used to predict the in vivo burst release based on the in vitro burst release of microsphere formulations, which helps to predict the in vivo performance of microsphere formulation(s) with no or minimal burst release . It has In recent years, considerable research has been conducted to develop modeling tools to facilitate the development of generic LAI formulation development as well as bioequivalence guidance for LAI formulations. A tool for developing models appropriate for characterizing the complex absorption process and the pharmacokinetic time course of LAI formulations is challenging. The plasma concentration–time profiles following administration of LAI PLGA formulations are often irregular and cannot be interpreted easily with conventional models based on first-order absorption kinetics and lag time. Sam N. Rothstein’s team ,147 demoPLGA is the key component of LAI drug products responsible for providing controlled and sustained drug release. It is important to determine key PLGA characteristics to ensure comparable product performance because of the impact PLGA has on drug release kinetics. John Garner et al.  developeAlthough PLGA/PLA is the most widely used biodegradable and biocompatible polymer in LAI microsphere formulations, the limitations, including incomplete drug release profile and burst release, should be considered while developing an LAI depot of PLGA/PLA microspheres. Among the three conventional manufacturing techniques , emulsion–solvent evaporation is the most applicable method in clinical LAI microspheres. However, in the emulsion–solvent evaporation method, several process parameters such as polymer concentration, phase ratio, surfactant concentration, protein loading, and intensity of homogenization should be optimized because these parameters can affect the characteristics of microspheres such as particle size, release profile, and encapsulation efficiency. By modifying the PLGA characteristics, it is possible to produce a microsphere formulation with desired properties and physiological behavior. Further, it is important to understand and select appropriate processing conditions to guarantee the desired physicochemical characteristics of the LAI microspheres. The high complexity of the manufacturing process, absence of adequate standards, and product-specific regulations hindered the progress of developing generic LAI depots of PLGA/PLA microspheres. Although FDA guidance on LAI microspheres is not complete, the information available from the existing literature might be informative and instructive to bring LAI PLGA/PLA microsphere products to the market in the near future.


Aim: To report the optical characteristics of the fluid in an anterior chamber iris cyst. Method: A 26-year-old male presented with blurring of vision in his right eye for two months, without any other associated ocular complaints. His visual acuity was 6/ 9 on Snellen’s chart. On slit lamp examination, a small translucent pigmented cyst was noted inferiorly in the anterior chamber, struck on to the cornea at 6 o’clock periphery, without any feeder vessel. Anterior segment optical coherence tomography (OCT) revealed a cystic lesion with hyperreflective walls and hypo reflective lumen attached to the cornea, compressing the endothelium.Results: The optical density (OD) of the cyst fluid was determined using ImageJ, an open code Java-based image processing software. The OD of cyst fluid was found comparable to the anterior chamber fluid.Conclusion: Ultrasound biomicroscopy (UBM) and OCT are useful tools for the diagnosis of cystic lesions of the anterior segment. The innovative use of an OCT image and the ImageJ software to determine the optical density of the iris cyst may aid in the diagnosis and follow-up of such cases. Primary iris cysts are clinically classified as epithelial and stromal subtypes. Epithelial cysts arise from layers of pigmented epithelium of the iris. Additionally, based on the location, cysts can be central, middle, or peripheral in relation to the pupil. The iris pigmented cysts can present at the pupillary margin, posterior layers of iris epithelium or free-floating in aqueous or vitreous [2]. Iris cysts have always fascinated ophthalmologists because of rarity of occurrence and mystery about their origin. The aetiology of primary iris cysts is poorly understood, whereas secondary cysts have an antecedent cause like the use of miotics, trauma or surgery [3]. The clinical and morphological features of such cysts are well characterized; however, the optical properties of the contents are not well documented. ImageJ software has been used in retinal images to characterize the optical density of subretinal fluid in various retinal diseases [4]. This report describes for the first time the optical density (OD) of an anterior chamber iris cyst using optical coherence tomography (OCT) and ImageJ software. Iris cysts in the anterior chamber have been well described in literature. These cysts are lined with pigment epithelium and contain clear fluid. They can be free-floating in the anterior chamber or fixed to anatomical structures. Usually, these cysts remain asymptomatic, but can lead to complications due to inflammation, glaucoma, or mass effect. At times, these mass lesions have been mistaken for malignancy, and literature has few reports of enucleation performed for the fear of malignancy . The optical density of the cyst was 21.5, which was comparable to anterior chamber fluid, suggesting clear contents inside the cyst .Anterior segment OCT revealed a cystic lesion in the anterior chamber, with hyperreflective walls and clear hypo-reflective lumen attached to the cornea compressing the endothelium  is usually the recommended modality for the imaging of iris cysts, which displays high reflectivity of the cyst wall; however, OCT characteristics are not well described in literature [7]. As documented in this report, the OCT findings substantiate similar findings, as noted in the UBM studies. The outer wall of the cyst is hyperreflective, with clear hypo reflective lumen. Besides, OCT has advantages over UBM, as it is a rapid, non-contact, and non-operator dependent imaging modality to document the characteristics of the cyst and the surrounding ocular tissues.Primary stromal cysts arise from ectopic surface epithelium, trapped in the iris during embryological development, while secondary iris cysts arise after ocular trauma or surgery [5]. The optical density describes the amount of light received by the sensor while passing through a particular medium. We found that the optical density of the cyst fluid was comparable to the anterior chamber fluid. This innovative method could be used for objective assessment and follow-up of such cases, as any change in the contents would be picked up objectively at the initial stage by the change in OCT characteristics and optical density. In this report, we adopted an innovative approach to characterize the optical properties of the fluid inside the cyst using ImageJ software [6]. Such patients should be advised to undergo a cardiological evaluation, as the same defect in smooth muscles may lead to aortic dissection. The cysts at the margin of the pupil are called floccule and are believed to be caused due to a genetic defect in smooth muscles [7].Treatment of iris cysts depends on the location and symptoms of the individual. The long-term complications of iris cyst include obscuration of the visual axis, corneal decompensation, secondary uveitis, and glaucoma. Treatment options range from observation to fine-needle aspiration, laser , or surgical excision. However, a step ladder approach should be adapted, and treatment should be individualized based on the clinical picture. The OCT seems to be a promising tool for imaging the cyst and optical properties of the fluid on imageJ software may prove to be an objective method, useful for the diagnosis and follow-up of such patients, as it was recently reported in capsular block distension syndrome [Ultrasound biomicroscopy and OCT are useful tools for the diagnosis of cystic lesions of the anterior segment. Innovative use of ImageJ software on OCT image to determine the optical density of the cystic lesion may further characterize the iris cyst, as described in this report. The measurement of the optical density of the contents of the cyst may aid in the initial diagnosis and follow-up of such cases, however further investigations are warranted in larger cohorts of patients.Conflict of Interest statementThe authors declare no conflict of interest. Informed Consent and Human and Animal Rights statementInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the review board of Institute of Naval Medicine, Indian Naval Hospital Ship Asvini, Colaba, Mumbai, India.AcknowledgementsNone.Sources of FundingAuthors have no competing interests to declare. No financial disclosures. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Consent was taken from patient for use of images, health data and OCT scans for the purpose of research and publication. No external funding was received.DisclosuresNone.


For a long time, host cell death during parasitic infection has been considered a reflection of tissue damage, and often associated with disease pathogenesis. However, during their evolution, protozoan and helminth parasites have developed strategies to interfere with cell death so as to spread and survive in the infected host, thereby ascribing a more intriguing role to infection-associated cell death. In this review, we examine the mechanisms used by intracellular and extracellular parasites to respectively inhibit or trigger programmed cell death. We further dissect the role of the prototypical “eat-me signal” phosphatidylserine (PtdSer) which, by being exposed on the cell surface of damaged host cells as well as on some viable parasites via a process of apoptotic mimicry, leads to their recognition and up-take by the neighboring phagocytes. Although barely dissected so far, the engagement of different PtdSer receptors on macrophages, by shaping the host immune response, affects the overall infection outcome in models of both protozoan and helminth infections. In this scenario, further understanding of the molecular and cellular regulation of the PtdSer exposing cell-macrophage interaction might allow the identification of new therapeutic targets for the management of parasitic infection. Entamoeba histolytica (E. histolytica), whose name reflects its ability to lyse host tissue, induces cell death via a caspase 3-like activity in a process which requires cell-to-cell contact . Ve. Vevβ3, parasite .pos and PtdSerneg parasites has been performed in the context of infection with the obligate intracellular parasitic T. gondii, and anti-PtdSer antibodies are frequently detected during infection, the role of PtdSer receptors on phagocytic cells has not been analyzed so far. Infection with the PtdSerpos subpopulation of T. gondii inhibits NO production by activated macrophages [While characterization of PtdSerrophages , an efferophages . A schemIn this review, we have discussed various aspects regarding the modulation of host cell death by parasites and the consequent handling of dying cells by phagocytic macrophages. Several of these are the strategies the parasite has developed to turn host cell death to its own advantage or to acquire an apoptotic-like phenotype to more easily propagate within the host. Furthermore, we have highlighted some of the latest discoveries on the mechanisms of sensing infected and non-infected apoptotic cells by professional phagocytes, with particular attention to the engaged PtdSer-receptors and their overall impact on the course of the infection.Chimeric Antigen Receptors for Phagocytosis (CAR-P) [Recently, a series of preclinical evidence highlighting the role of phagocytosis mediated by macrophages has been in the spotlight, as observed via the use of phagocytosis checkpoint inhibitors in cancer treatment  as well  (CAR-P) , which dN. brasiliensis or S. mansoni. Here, the advantageous effect of macrophage transfer relied on the acquisition of a specific macrophage activation status rather than on their phagocytic capacity. Transfer of macrophages leads indeed to host protection against a lethal N. brasiliensis-induced inflammation, thanks to their capacity to respond to type 2 cytokines acquiring a tissue remodeling profile [Few studies have reported the beneficial effect of macrophage-based cell therapy in parasitic diseases, for example, in the context of infection with helminth parasites  profile . Likewis profile . HoweverMany of the strategies reported in this review are used by parasites which cause diseases classified as Neglected Tropical Diseases (NTDs), such as schistosomiasis, Chagas disease, and leishmaniases, for which novel or improved treatments are urgently needed. Blocking macrophage capacity to uptake infected apoptotic cells might represent an alternative approach to inhibit the survival and propagation of many intracellular parasites. However, blockage of phagocytic receptors on macrophages via, for instance, systemic administration of phagocytic receptor small molecule inhibitors will lead to reduced engulfment of most of the apoptotic cells generated, independent of their infection status. This in turn might limit the macrophage tissue remodeling response naturally resulting from the uptake of non-infected apoptotic cells. Similarly, blocking phagocytosis during helminth infections might suppress the generation of an anti-inflammatory response and therefore prevent tissue regeneration.These downsides hint toward the development of therapeutic strategies aiming at manipulating phagocytosis to promote only the engulfment of selective targets. The use, for instance, of CAR-P technology might in the future unleash the power of phagocytosis, leading to the generation of an immune environment in which macrophages are reprogrammed to taking up only uninfected damaged cells, thus avoiding the detrimental consequences of taking up cells infected, for instance, with intracellular protozoan.In conclusion, understanding the mechanisms behind phagocytosis regulation and further characterizing the molecules driving the macrophage/apoptotic cell cross-talk may open up new venues for the treatment of various inflammatory disease and alternative approaches for the management of parasitic infection and in particular NTDs.


Non-alcoholic fatty liver disease (NAFLD) is a major public health problem worldwide. NAFLD (both simple steatosis and steatohepatitis) is characterized by alterations in hepatic lipid metabolism, which may lead to the development of severe liver complications including cirrhosis and hepatocellular carcinoma. Thus, an exhaustive examination of lipid disorders in the liver of NAFLD patients is much needed. Mass spectrometry-based lipidomics platforms allow for in-depth analysis of lipid alterations in a number of human diseases, including NAFLD. This review summarizes the current research on lipid alterations associated with NAFLD and related complications, with special emphasis on the changes in long-chain and short-chain fatty acids levels in both serum and liver tissue, as well as in the hepatic expression of genes encoding the enzymes catalyzing lipid interconversions. In the last decades, chronic liver disorders have become a major public health problem worldwide. Obesity and metabolic syndrome are well-known risk factors in chronic liver diseases (CLD). Over 400 million people struggle with obesity, and it is estimated that 75% of them will develop non-alcoholic fatty liver disease (NAFLD) . NAFLD eThe global prevalence of NAFLD has been estimated to be 25%, and it is not equally distributed in all parts of the world. In fact, more cases are reported in Western countries and the United States . HoweverLipids are a very heterogeneous group of organic compounds with different functions, e.g., energy storage and the formation and stabilization of intra- and extracellular membranes. Lipids are also precursors of bioactive molecules present in the circulation and they can even regulate genes expression ,11. FattWhereas a significant proportion of FA species can be synthesized by the human body, FAs are also taken up from the diet or even produced by the gut microbiota  . The majStudies based on lipidomics approaches provide essential data on the changes of a wide range of lipid species  and can The lipidomic landscape of NAFLD is not well defined. The analysis of the alterations in hepatic and plasma lipid homeostasis in NAFLD patients could provide essential evidence on the pathophysiological hallmark of NAFLD ,17. SeveIn addition to SFAs, MUFAs were also elevated in liver tissue samples in the course of NAFLD ,21,26,30Additionally, lipidomic analyses revealed a progressive decrease of hepatic PUFAs in parallel to the severity of NAFLD ,30. ThusDiet is another key factor involved in the modulation of the hepatic FAs. Epidemiological studies have shown that patients with NASH claim a diet richer in fat and poorer in complex carbohydrates and protein that the general population . SeveralAs it was mentioned before, the number of non-invasive diagnostic tests for NAFLD is very limited. Screening for markers of liver injury is becoming a standard protocol for patients suffering from metabolic disorders including obesity, insulin resistance, or type 2 diabetes. Thus, the levels of serum transaminases  only indicate damage in hepatocytes, but they are not sensitive enough to serve as a reliable non-invasive test in NAFLD patients ,34,35. TAn early and accurate detection of liver steatosis is of great interest because of the association between NAFLD and cardiovascular events and other metabolic diseases. Today, the quantification of liver steatosis can be performed by several ultrasound techniques. Controlled attenuation parameter (CAP) is the technique available in the FibroScan system  that measures the attenuation of the ultrasound beam as it traverses the liver tissue . CAP is Emerging evidence has proposed the potential use of metabolomic approaches to search for new non-invasive biomarkers of NAFLD . In partThe molecular mechanisms of NAFLD progression are still not completely understood. It is assumed that pathological changes are strictly related to chronic inflammation, insulin resistance, increased intrahepatic TAG accumulation, and de novo lipogenesis (DNL). In NAFLD patients, similar to healthy subjects, lipogenesis increases dramatically in the postprandial state . The conACC and FASN [SREBP-1c gene expression was significantly correlated with ballooning and fibrosis score in NASH patients [The sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors  that activate the synthesis of FAs, TAGs, and cholesterol ,58. Exagand FASN . Remarkapatients .The mechanisms by which alterations in the hepatic mRNA levels of genes involved in lipid metabolism may occur are not well understood. Interestingly, circulating FAs can act in a hormone-like manner on hepatocytes, and some of them  can induce hepatic lipogenesis ,61. ReceThe above discussed NAFLD-related lipid alterations and their molecular mechanism based on the changes of the expression of genes encoding enzymes of lipid metabolism are summarized in PNPLA3) gene polymorphisms are associated with NASH, NAFLD, and even NAFLD-related HCC [PNPLA3 gene is I148M and it has been associated with an increase in hepatocellular lipid retention by altering TAG hydrolysis [PNPLA3 gene is involved in NAFLD development and progression.Patatin-like phospholipase domain-containing 3  may play a role in NAFLD development and progression ,69,70. DShort-chain fatty acids (SCFAs) are the main products of bacterial fermentation of dietary fibers, along with proteins and peptides that have escaped digestion by host enzymes in the upper gut. A growing body of work has identified SCFAs as mediators of diet-induced crosstalk between the microbiome and the host and that these microbial metabolites are important for health . The mosPrimary liver cancer was the sixth most commonly diagnosed cancer and the third leading cause of cancer death worldwide in 2020, with approximately 906,000 new cases and 830,000 death . The mosAs mentioned above, the liver is one of the main organs involved in the regulation of lipid metabolism. Therefore, alterations of liver function are directly associated with disturbances in lipid homeostasis. The biosynthesis of FA is enhanced in HCC and thus could potentially serve as a therapeutic target, together with additional lipid metabolism alterations that promote adaptation to the local environment in HCC . Severe According to many authors, the association between blood and tumor lipidomes is still little known . HoweverOn the other hand, HCC patients have elevated levels of acylcarnitines in their blood compared with healthy subjects ,90. ThisThe above-discussed HCC-related lipid alterations are summarized in Lipidomics based on mass spectrometry is usually an applied technique to record alterations of the lipid profile in many cancer/tumor matrices. Therefore, lipidomic studies have a potential to improve prevention, early detection, and targeted therapy of HCC.NAFLD is associated with lipid alterations both on the level of liver and serum lipid composition as well as on the level of the expression of genes related to lipid metabolism in hepatocytes. A thorough understanding of these disorders creates an opportunity to establish new diagnostic methods and therapeutic goals based on lipid metabolism in NAFLD patients. Furthermore, HCC, a frequent late complication in NAFLD patients, is associated with alterations of lipid metabolism, and the above conclusions also apply to this condition.


Extracellular vesicles (EVs) are kinds of two-layer vesicles secreted by cells. They play significant roles in mediating component exchange between cells, signal transduction, and pathological development. Among them, the tumor-derived EVs (TDEVs) are found related to the tumor microenvironment and cancer development. TDEVs can be designed as a natural drug carrier with high tumor targeting and permeability. In recent years, drug delivery systems (DDS) based on TDEVs for cancer treatments have received considerable attention. In this review, the biological characteristics of TDEVs are introduced, especially the effect on the tumor. Furthermore, the various approaches to constructing DDS based on TDEVs are summarized. Then we listed examples of TDEVs successfully constructing treatment systems. The use of chemical drugs, biological drugs, and engineered drugs as encapsulated drugs are respectively introduced, particularly the application progress of active ingredients in traditional Chinese medicine. Finally, this article introduces the latest clinical research progress, especially the marketed preparations and challenges of clinical application of TDEVs. Extracellular vesicles (EVs) are two-layered vesicles secreted by cells. According to the size and release mechanism, EVs can be divided into exosomes, microvesicles, and apoptotic bodies. As early as the 1960s, when scientists were culturing chondrocytes, they observed that the cells secreted a small vesicle with a diameter of about 100 nm. These small vesicle buds directly from the cell membrane and can induce the formation of hydroxyapatite crystals. Later, researchers collectively referred to the cystic structures released to the outside of cells as "extracellular vesicles." Among them, the name exosomes were first used in the 1980s. A vesicle-like structure substance containing transferrin receptors was discovered during the research process of the maturation of reticulocytes. In 1991, Stein and Luzio named vesicles shed from the membrane of neutrophils as extra nuclear cells. With the gradual deepening of EVs research, people gradually discovered that EVs are not only secreted by certain specific tissues or cells but secreted by all cells. As the winners of the Nobel Prize in Physiology or Medicine in 2013 discovered the regulation mechanism of cellular vesicle transport, in recent years, more and more scientists have devoted themselves to basic research and transformational applications of EVs. This promotes the rapid development of this field in vivo. However, the detection of EVs, as one of the liquid biopsy methods, still has great potential for development There are still many gaps in our research on the physiological functions of EVs. Early research believed that EVs are trashcans of cell metabolic waste, and their release is conducive to maintaining the stability of the intracellular environment. Recent studies have shown that specifically targeted EVs are a way of signal transduction between cells. EVs also play an important role in a variety of pathological processes, including viral infections, cardiovascular diseases, central nervous system-related diseases, tumor occurrence and development, and immunotherapy. Especially in the development of tumors, tumor-derived EVs (TDEVs) have shown a non-negligible role. In the late 1990s, studies found that exosomes derived from tumor dendritic cells could affect tumor growth. TDEVs can mediate component exchange and signal transduction between tumor cells, promote tumor growth, reshape tumor extracellular matrix, change tumor microenvironment, and regulate tumor development. Therefore, TDEVs have great research value. At present, the related research of EVs is limited by the existing experimental methods, and it is not possible to track the physiological and pathological secretion pathways of EVs Cancer is the number one killer of human health, but there is no effective treatment to overcome this difficulty. Therefore, a large number of researches are aimed at innovating cancer treatment drugs. In 2020, the momentum of innovation in the global tumor treatment field is very strong. A large number of innovative tumor drugs have been approved, behind the booming anti-tumor drug R&D pipeline. Among them, EVs occupy a place as a therapeutic carrier of tumor drugs. A search in this field in Pubmed found more than 1,200 articles related to this field of research. Due to the advantages of EVs such as excellent compatibility, better permeability, natural stability, low immunogenicity, and toxicity, the use of EVs to construct a drug delivery system (DDS) as a carrier for therapeutic drugs has shown significant transformational value. In addition, TDEVs, as a carrier of cancer treatment drugs, have shown their prominence in the field of biomedicine. Based on this, we have also read a lot of relevant literature and found that no one has made a good summary in this field. However, the role of TDEVs in tumor recurrence and metastasis makes its clinical application as a tumor therapy drug-carrier also have certain risks. Therefore, how to design the construction of drug delivery systems to maximize the value of TDEVs is a problem to be solved In recent years, research on natural products with cancer therapeutic activity has had a very large impact. Traditional Chinese medicines (TCM), including monomeric active ingredients and compound preparations, have been found to have effective therapeutic effects on tumors and have been widely used in the research of tumor therapeutic DDS. A large number of natural products such as traditional Chinese medicine have the characteristics of high hydrophobicity, low solubility, poor stability, and short half-life. As a result, its bioavailability is low and it is difficult to be widely used in clinical practice. At present, many studies have proved that EVs loaded with traditional Chinese medicine ingredients have enhanced efficacy and improved drug resistance. In addition, TDEVs have a better targeting effect than EVs. Therefore, we hope to find a way to construct a TDEVs vector, so that Chinese medicine can be targeted to play its role in the treatment of tumors This article combines the latest findings of EVs in recent years and reviews the research on TDEVs as tumor therapy drug-carriers. Firstly, the biological characteristics of TDEVs are summarized, especially their biological origin, pharmacokinetics, and their effects on tumors. In addition, the general construction methods of DDS based on EVs are summarized, including separation, drug loading, and engineering modification of EVs. In addition, the application of TDEVs in cancer treatment in recent years is reviewed. It also introduced the promotion of TDEVs in the efficacy of chemical drugs, biological drugs, and engineering drugs, especially the progress in the application of active ingredients in Chinese medicine. Finally, this article introduces the latest clinical research progress, marketing situation, and challenges faced by the clinical application of TDEVs.Figure 1).Exosomes are EVs in the range of 30-100 nm Specifically, the first step in exosome formation is the transformation of endosomes into MVBs. One of the key steps is the formation of ILVs in the late endosomes, which is mainly controlled by the mechanism of the endosomal sorting complex required for transport (ESCRTs) In addition, there are some mechanisms that are not associated with ESCRTs, especially under hypoxic conditions in the tumor microenvironment N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNAREs), and coat protein complexes I and II. Subsequently, MVBs were fused or degraded to the cell membrane. The fusion process was mediated by the G protein-coupled receptor (GPCR) signal pathway, controlled by trap molecules, via phosphorylated 110 serine residues in t-SNARE SNAP23, and through the GPCR signal of the H 1 histamine receptor. Finally, the ILVs in MVBs are released into the extracellular environment in the form of exosomes and enter the systemic circulation Then, when MVBs are formed, they are transported to the cell membrane. The transport process involves a number of signaling proteins such as clathrin, GTPases, soluble Microvesicles (MVs) are 100-1,000 nm in size. In addition to size differences, there are also many differences between microvesicles and exosomes in proteomics and lipidomics. Compared to exosomes, which contain more extracellular matrix, heparin-binding, receptors, immune responses, and cell adhesion proteins, as well as lipids such as glycolipids and free fatty acids, MVs mainly contain endoplasmic reticulum, proteasome, and mitochondrial proteins, lipids including ceramide and sphingomyelin C-linked domain of Rho-associated protein kinase 1 (ROCK-1) and activates the phosphorylated myosin light chain (MLC) of ROCK-1, resulting in myosin interaction. RhoA/Rock signaling pathway is also involved in MVs biogenesis. RhoA is a small GTPase protein in the Rho family that regulates actin tissue and actin contractility and is involved in cytoskeleton regulation. RhoA activates the Rock (RhoA kinase), stimulates the LIM kinase, then inhibits the fibroin, reorganizes the donor cell actin cytoskeleton, and finally leads to the release of MVs The key mechanism for the formation of MVs is the reorganization of the cytoskeleton, which involves the breakdown of proteins associated with the plasma membrane. There are some signal pathways involved in this process, such as the calpain-dependent pathway and Caspase-3-dependent pathway. Calpain-dependent pathway stimulates calcium intracellular flow by an agonist, activates thiol protease and calpain in the cytoplasm to move to the cell membrane. Then they bind with phosphate ester on the membrane and generates calmodulin by calcium-regulated conformational change. Activated calmodulin cleaves α-actin and talin filaments, allowing cytoskeleton proteins to be separated, thus causing MVs release. In addition, Caspase-3 cleaves the Figure 2).Apoptotic bodies (ABs) are EVs produced under conditions of apoptosis. Compared with exosomes and microvesicles, apoptotic bodies are larger, with a diameter between 800-5000 nm. The main marker of apoptotic bodies is phosphatidylserine. Apoptosis is a kind of programmed cell death that does not cause inflammation. A major feature of apoptosis is that apoptotic cells eventually divide into closed apoptotic bodies. Apoptotic bodies seal and store the intracellular substances from dead cells in vesicles, and deliver these substances to other cells that can swallow apoptotic bodies, such as macrophages and tumor cells. However, the distribution of the contents of apoptotic cells to apoptotic bodies is random. Therefore, specific organelles or nuclear contents may or may not exist in a specific apoptotic body. The process of fragmentation of apoptotic cells into apoptotic bodies is conducive to the removal of apoptotic cell debris and plays a very important role in controlling immune stress after apoptosis. However, apoptotic bodies will not leak the contents of dead cells to the surrounding environment, avoiding the triggering of inflammation. Recent studies have shown that the lysis of cells into apoptotic bodies in the late stage of regulation is a highly coordinated and regulated biological process. Since apoptosis is believed to play an important role in the cell cycle and the normal development of the immune system, AB also plays a regulatory role in inflammation, autoimmune diseases, and cancer. Studies have shown that ABs derived from apoptotic bone marrow mesenchymal stem cells can enhance angiogenesis and cardiac function recovery in rats with myocardial infarction in vivo. With this high spatial and temporal resolution in vivo images, EVs can be observed in the manner in which they spread around the donor cell, across the biofilm, and in the organs after entering the great circulation When the EVs is released by the donor cell, it begins its journey inside the body. There are many ways to label and track EVs, such as fluorescence imaging, bioluminescence imaging, nuclear imaging, tomography imaging, and so on. Zebrafish models are often used to show the distribution of EVs in vivo. First, EVs from different cell sources are distributed in different locations. TDEVs accumulate more easily in tumor tissues of tumor-bearing mice, and lung metastatic hepatoma cells tend to be distributed to the lung, indicating that TDEVs have the ability to recognize early tumor tissues Blood kinetic analysis showed that EVs are cleared quickly after they enter the bloodstream and have a half-life of fewer than 10 min In particular, TDEVs can cross the blood-brain barrier (BBB). The BBB is made up of endothelial cells, pericytes, and astrocytes that are close to the brain to fight microbes and other foreign particles outside the brain. TDEVs can break through the complete BBB through endocytosis, and it can also decrease the expression of Rab7 in brain endothelial cells and improve the efficiency of transport, so as to avoid the mechanism of apoptosis of BBB hypophysis cells. TDEVs also contain proteins and nucleic acids that help to complete this process. The microRNA triggered BBB breakdown, miR-181c mediated BBB disruption by down regulating the target gene PDPK1, which regulates the abnormal restriction of actin. The miR-181c reduction of PDPK1 results in the down-regulation of phosphorylated fibroin, which in turn leads to the production of a driven fibroin that initiates the regulation of actin elements in vivo, they are finally taken up by target cells. The uptake process consists of three stages: first, EVs target the receptor, and then, they are internalized by the recipient cells, and finally, EV content is transferred to the recipient cells. EVs can be internalized by recipient cells in three ways: endocytosis, receptor-mediated cell signaling, and phagocytosis After EVs are distributed in vivo. Among them, TDEVs plays the role of communication between tumor cells and other cells. Additionally, they are the vital middle person of cell-to-cell correspondence between tumor cells and stromal cells in the nearby or far-off microenvironment Figure ).EVs are released from the donor cells and are absorbed by the recipient cells through circulation in the body, which transports bioactive substances such as proteins, lipids, nucleic acids, and metabolites from one cell to another TDEVs promote tumor proliferation and growth. TDEVs can activate the cell pathway and induce the proliferation of tumor cells. For example, the surface of EVs derived from melanoma cells carries hyaluronic acid synthase HAS3, which contains a large number of Indian hedgehog homologs (IHH). It can activate the hedgehog signal cascade of target cells, induces c-Myc activation, and regulates the expression of cyclin. This signal transduction of IHH in HAS3-EVs leads to tumor cell proliferation and epithelial-mesenchymal transition. In addition, cancer-associated fibroblasts (CAFs) can promote the secretion of inflammatory factors and growth factors, and then promote the growth of the tumor. TDEVs are key mediators regulating cellular communication between CAFs and cancer cells. TDEVs can convert normal fibroblasts into CAFs, and cause fibroblasts to differentiate into myofibroblasts, releasing matrix metalloproteinases (MMP) and causing extracellular matrix (ECM) remodeling. ECM decomposes, causes the growth factor release, promotes the invasion ability of parenchymal cells, and promotes the tumor cell adhesion TDEVs promote tumor angiogenesis. When new capillaries are formed in the tumor environment, tumor cells can enter the bloodstream and get more nutrients, oxygen, and growth factors, so new advances in cancer occur. The process of angiogenesis includes the stimulation of angiogenic factors to endothelial cells, the degradation of the vascular basement membrane, the proliferation, germination, migration of endothelial cells, the formation of the lumen, and the maturation of blood vessels. A variety of growth factors and signal pathways are involved in regulating this process. Therefore, TDEVs are indirectly involved in the regulation of angiogenesis by influencing these factors and pathways. For example, TDEVs can control the angiogenesis process by altering the net balance between angiogenic and antiangiogenic factors at the tumor site. Many kinds of nucleic acids and proteins in TDEVs can affect angiogenesis through many pathways, such as miR210, miR9, miR135b, and long non-coding RNAs such as LincPou3F3, LincRNAH19, LincCCAT2, which are transferred to endothelial cells (ECs) and activate the angiogenesis signal pathway Figure ). Drug resistance is often associated with ATP binding cassette (ABC) transporter proteins family. Some studies have shown that miRNAs regulate the expression of some drug transporters in the ABC family after transcription, including P-glycoprotein (P-gp or MDR1), multidrug resistance-associated protein (MRP), and breast cancer resistant protein (BCRP) TDEVs promote drug resistance in tumors. The mechanisms of drug resistance include drug efflux, changes in drug metabolism, and changes in energy programming, DNA damage repair, cancer stem cells, and epigenetics + T cell-mediated tumor-targeting immunity. The apoptosis of CD8+ T cells was directly induced by the expression of FasL and TNF-related apoptosis-inducing ligand (TRAIL). TDEVs increases neutrophil promotes tumor progression and activates or suppresses natural killer (NK) cells, which play an important role in tumor immunity. The antigen delivered by TDEVs can activate dendritic cells (DC) and participate in CD8+-mediated anti-tumor response Figure ). During immune editing, TDEVs may act as an immunostimulator for cancer cells to germinate and then transform into an immunosuppressive factor during the progression of cancer. In the clinical application of cancer vaccines based on TDEVs, it is necessary to pay attention to its function of inhibiting tumor immunity TDEVs can affect immune system homeostasis. TDEVs transport nucleic acids, lipids, and proteins mediate immune regulation in the microenvironment, and protect tumors through immunosuppression. For example, TDEVs can activate and increase Treg and myeloid-derived suppressor cells (MDSCs) and inhibit CD8in situ can induce vascular leakage and interact with resident cells of distal organs to promote the proliferation of TDEVs in vivoIn the tumor distant microenvironment, TDEVs mainly showed the promotion of pre-metastatic niche (PMN) for tumor formation. In the process of tumor metastasis, TDEVs released by tumor cells in vivo, and compared with other tumors, TDEVs are more likely to be ingested by the same type of tumor cells. In zebrafish embryo models, TDEVs were absorbed by endothelial cells and macrophages, stored in degradation chambers, activated macrophages, and ultimately promoted tumor metastasis Notably, TDEVs tend to be ingested by tumor cells In summary, TDEVs play an important role in the progression of cancer. Tumor cells can both directly or indirectly affect tumor cells and tumor microenvironment through TDEVs-mediated signaling pathways. Therefore, the TDEVs-mediated tumor cell-to-cell pathway is not only a target for cancer therapy in the strategy of tumor therapy. At the same time, when TDEVs is used as a drug carrier to treat cancer, the TDEVs cargo can be changed or surface modified, so that the therapeutic effect of TDEVs drug carrier system cannot be counteracted by its promotion of tumor development Figure ).The construction of the TDEVs as DDS starts from the isolation and extraction of EVs. Several methods can be used for the purification of EVs. The usual methods mainly utilize the physical and biological characteristics of EVs. Based on the physical characteristics of separation methods include the use of EVs density or size and impurities for separation, such as ultracentrifugation, size exclusion chromatography, ultrafiltration, and flow field-flow fractionation. The method based on biological characteristic separation mainly utilizes the specific molecules on the surface of EVs, including polymer precipitation and the immunoaffinity method. There are also integrated approaches such as microfluidic technology Ultracentrifugation is a kind of separation method based on the difference in particle density and size. There are two types of ultracentrifugation: differential ultracentrifugation (DU) and density gradient flotation (DGF). Ultracentrifugation is the most common technique for the separation of EVs from cell culture supernatants or other biological fluids. However, it has the disadvantages of time-consuming operation, limited production, low automation, and dependence on expensive instruments.In DU, the usual procedure for separation is the removal of large cell fragments and cells at a low speed , followed by further removal of proteins by precipitation of EVs at a high speed . However, the disadvantage of DU is the operation time is long, which may damage the integrity of EVs, resulting in protein contamination and yield reduction ™ for isolating EVs secreted by leech microglia, the results showed that DGF was superior in removing protein impurities and other membrane particles DGF is another kind of ultracentrifugation. The first step is to construct a density gradient. Common solvents include sucrose and iohexanol, both of which are viscous solutions, with the latter having a lower viscosity. In the solution with an increasing concentration gradient, EVs can migrate to their equilibrium density after centrifugation, and the migration velocity depends on its size, shape, and density. By using this technique, the protein contamination in the sample can be eliminated and therefore, be more effective than DU. One study compared the difference between DU and DGF with OptiprepSize exclusion chromatography (SEC) is a method of separation using particle size and shape. In this method, small porous polymer beads were used as stationary phphaseor separation of EVs Compared with the DGF method, the SEC method is less time-consuming, more convenient, and more automatic Ultrafiltration is also a method of separation based on particle size. The principle of separation is that the sample passes through a semi-permeable membrane with certain pore size. The particles with smaller pore sizes pass through the membrane, while the particles with larger molecular weight are retained. The extraction of EVs by ultrafiltration has the characteristics of high efficiency, not easy to destroy the structure of EVs, and high yield and stable EVs can be obtained.The separation efficiency of EVs by ultrafiltration is similar to that of SEC. The results of electron microscopy and nano-tracking analysis showed that the number of exosomes extracted from human melanoma cells by ultrafiltration and SEC was 58 times as much as that by ultracentrifugation. It shows the efficiency advantage of EV extraction by ultrafiltration Flow field-flow fractionation (FFFF) is also a method of separation based on particle size. In this method, particles of different sizes are loaded into a small channel made up of two semi-permeable membranes. The liquid then flows because it interacts differently with particles of different sizes, and particle mobility is also affected by the size, which can distinguish different particles. Asymmetric flow field-flow fractionation (AF4) is an advanced FFFF technology. AF4 has high reproducibility and purity in the process of removing high-density lipoprotein and low-density lipoprotein from plasma to obtain EVs The method of separating EVs based on physical properties is to purify EVs by taking advantage of the difference between EVs and impurities in size, shape, or density. The advantages of the physical separation of EVs are high yield and efficiency, and the structure of EVs is not easy to be destroyed Polymer precipitation separation is a method of separation by using the biological properties of EVs. In this method, a special polymer is dissolved in the liquid, which reduces the solubility of the EVs and precipitates out. For example, polyethylene glycol (PEG) can precipitate EVs. Low-speed centrifugation can accelerate the separation of EVs when using PEG to precipitate EVs There are some commercial EVs extraction kits, such as Exo-Quick, ExoSpin, and so on, which use the principle of polymer precipitation to extract EVs Immunoaffinity chromatography is a separation method based on the specific affinity of EV surface molecules. Some membrane surface proteins or lipids, such as tetraspanins, glycosylphosphatidy-linositol-anchored protein (GPI-Aps), and tumor necrosis factor receptor 1 (TNF-R1), are known as specific binding sites. There are some extraction kits based on the immunoaffinity method, such as PAP/ME kits. Compared with the methods of ultracentrifugation and SEC, the yield of EVs obtained by the immunoaffinity method is lower, but the protein contamination is less Because immunoaffinity assay is a method of using special proteins on EVs, different EVs membranes from different sources may contain different proteins. Therefore, on the one hand, immunoaffinity may not be able to extract all the EVs, resulting in reduced extraction efficiency. On the other hand, this property can be used to extract specific sources of EVs. For example, monoclonal antibody 763.74 against a uniquely expressed chondroitin sulfate proteoglycan 4 (CSPG4) epitope on melanoma cells. Using immunoaffinity, the exosomes derived from melanoma cells were isolated from the plasma of patients with melanoma, and the exosomes from other sources were removed In recent years, separation methods based on microfluidic technology have been paid more and more attention. Microfluidic technology is to uses a continuous flow process to distinguish different sizes of nanoparticles, and then according to the surface markers, ligands, charges, particle size, and so on to identify the required EVs. The method has the advantages of the short operation time, less consumption of reagent, and sample volume newExoChip, was developed to isolate cancer-related exosomes from plasma. The capture efficiency of TDEVs and healthy exosomes was 90% and 38% respectively Microfluidic technology can be combined with the immunoaffinity method to design a variety of microfluidic devices. This kind of device can be combined with a diverse of physical or chemistry principles EVs can be separated in a number of ways. Therefore, in the preparation of DDS, the first step is to select the appropriate separation method according to the need and the actual situation. The separation method based on EVs' physical properties has the advantages of high yield, stable properties, but low purity. The separation method based on the biological characteristics of EVs has high purity and specificity, but the yield may be damaged Figure ). This section introduces the related research progress (Table 1).After extracting enough EVs to meet the requirements, the drug can be loaded. Drug loading methods can be divided into endogenous drug loading and exogenous drug loading. The exogenous drug loading methods include incubation, electroporation, sonication, extrusion, and freeze-thaw Endogenous drug loading means that the drug is first added to the medium and cultured with the cell. In the physiological activities of the cell, the drug is taken up automatically. As the cell secretes EVs, the drug enters EVs along with the cytoplasm, so the cell can secrete drug-loaded EVs. The successful preparation of drug-loaded EVs involves the successful uptake of the drug by the cell and its excretion by the cell via EVs. Therefore, if choose the endogenous drug-loading method to prepare DDS, the first step is to prove that the drug can be successfully transported through the biogenetic pathway of EVs. The release of drug-loaded EVs may involve autophagy. Autophagy is a highly regulated homeostatic process that prevents cell damage by removing, degrading, or removing damaged cell components. The mechanism involves the biogenesis of EVs. During the formation of ESCRT, there is a protein Ndfip1 with L-domain, which helps drugs enter EVs In addition to the endogenous drug loading method, an exogenous drug loading method can also be used. The EVs were first isolated from the cells and then loaded drug by a series of drug-loading methods. The commonly used drug loading methods include incubation, electroporation, sonication, extrusion, and freeze-thaw. Different types of drugs should be loaded in different ways. For example, some hydrophobic or small-molecule drugs can enter EVs passively, so they can be loaded by simple incubation. On the contrary, hydrophilic compounds cannot passively pass through lipid vesicles. So a proactive approach is needed to enable drugs to penetrate the pores of the EVs membrane. The principles of electroporation, sonication, extrusion, and freeze-thaw are to create pores on the surface of EVs with sufficient pore size to allow drugs to enter Some hydrophobic molecules, such as paclitaxel (PTX), doxorubicin (DOX), and curcumin, can be passively loaded into EVs by co-incubation with EVs. EVs were first isolated from the culture supernatant of Raw264.7 cells in the construction of EVs vector for the treatment of methicillin-resistant staphylococcus aureus infection. After the incubation with linezolid antibiotics at 37ºC for 1 h, the unbound drugs were removed by 10,000 × g low-speed centrifugation for 10 min and then sterilized by 0.22 μm microporous membranes, to get an EVs loaded with antibiotics. The drug-loaded EVs were 5.06% ± 0.45% by HPLC analysis 3 times Electroporation is an extrinsic drug loading method in which a change in voltage caused by an electrical signal leads to the formation of temporary pores in the EVs membrane to allow drugs to enter. Electroporation is often used for loading nucleic acid drugs, but it may cause nucleic acid precipitation or vesicle fusion. Therefore, in recent years, some new drug loading methods and facilities have been developed based on the principle of electroporation The sonication method is a kind of exogenic drug loading method, which can form pores on the membrane by vibrating EVs with periodic ultrasonic waves. In a study of DOX loading for tumor targeting, DOX-loaded EVs were prepared using a sophisticated ultrasound device with 20% amplitude and /150 sec. 6 cycles for 30 sec. The setting of sonication parameters directly affects the result of drug loading. The disadvantage of the sonication method is that it may change some physical properties of EVs, such as size, electric potential, and so on. Therefore, the optimization method based on traditional ultrasound improves the drug loading effect. One study combined microfluidics and sonication to prepare poly (lactic-co-glycolic acid) (PLGA) NPs coated with the exosomal membrane (EM) or cancer cell membrane (CCM) to reduce drug clearance and improve tumor-specific targeting Other methods include extrusion and freeze-thaw, but these two methods are not often used for TDEVs drug loading. According to the nature of drugs, different methods should be chosen for drug loading Drug-loaded EVs can be modified on the surface of the membrane, which is helpful to improve the targeting, change the biological distribution and improve the curative effect N-terminal of the mature peptide by gene engineering technology. Therefore, it can express the target homing peptide on the EV membrane Figure ).Endogenous engineering is a kind of genetic engineering technology that can change the gene sequence of donor cells to change the structure of secreted EVs. The spatial conformation of transmembrane proteins that are selectively enriched in EVs is the same as the donor cells. As a result, some transmembrane proteins can be used to express ligands or homing peptides on the surface of EVs, such as tetraspanins , CD protein (LA), and lysosome-related membrane protein-2b (Lamp-2b) Figure ). In addition, there are also studies on the surface of EVs to prepare a film, EVs included in the internal, forming a protective barrier. For example, the nano-film prepared by Fe3+ supramolecular complex and tannic acid can encapsulate exosomes, protect the exosomes from ultraviolet radiation or heat damage, and control the drug release. Gold nanoparticles are also attached to the nano-film to serve as visual markers Physical modification is a kind of method which directly affects the surface molecule of EVs membrane. Specifically, lipo-solubility molecules were anchored into the bilayer membrane of EVs, and the targeting of EVs was improved by rearrangement of the bilayer membrane Figure ).Chemical modification is a method to modify the molecular structure of EVs film by chemical reaction. It is a kind of chemical reaction with high efficiency and strong anti-interference. This method is based on the synthesis of small units, with the click of a chemical reaction to obtain a diversity of molecules in vivo may be affected by surface glycosylation modification In addition to the addition of specific ligands to the surface of EVs, it is also possible to remove the endogenous substances on the surface of EVs, such as stripped surface glycans. As mentioned above, glycometabolism not only regulates the biogenesis of EVs, but also the distribution of EVs Figure ). Studies show that a microfluidic ultrasonic method can fuse two kinds of membranes and simultaneously load drugs in the membrane The membrane fusion technology is to mix EVs membrane with another lipid membrane from different sources to get a hybrid membrane. The hybrid membrane has the same characteristics as the original lipid membrane. It can also be used for the combination of bio-membrane and liposome Table 2).The prepared drug-loaded EVs can realize the effect of targeted drug delivery. In particular, TDEVs can be used to target tumor cells to achieve information transfer, so the use of TDEVs based DDS has become a promising tumor treatment method. Among them, chemical drugs and biological drugs, especially natural products, can be delivered to the administration site through TDEVs, thereby enhancing the therapeutic effect of or reducing toxicity. Moreover, based on the direct encapsulation of simple compounds or biomolecules, some engineered drugs have also been designed to further optimize the construction of DDS Figure 6).Since drug-loaded TDEVs carry tumor-specific antigens and other substances that can be efficiently taken up by tumor cells, they can improve the targeting effect of drugs and effectively reduce the viability of cancer cells. The traditional DDS construction is mainly through the packaging of simple chemical drugs into TDEVs. Among them, natural products are popular drugs of choice. In particular, many monomeric active ingredients are extracted from traditional Chinese medicines, such as paclitaxel (PTX), curcumin, camptothecin, vincristine, β-elemene, catalpol, tanshinone, and triptolide, etc. Most of these active ingredients of traditional Chinese medicine have the characteristics of high hydrophobicity, low solubility, poor stability, and short half-life. As a result, its bioavailability is low and it is difficult to be widely used in clinical practice. With the help of the administration of TDEVs, the curative effect of these Chinese medicine monomer components can be improved Taxus chinensis. It has poor water solubility, poor efficacy in intravenous injection, and has dose-dependent toxicity. These problems limit the use of PTX. Packing PTX with TDEVs can significantly improve its therapeutic effect. Saari et al. 8-5×109 /mL were incubated in 1 mL of 5 μM PTX-DPBS for 1 h, loaded with PTX in TDEVs, and then centrifuged into balls. The loading rate was 9.2 ± 4.5%. The efficacy of the drug-loaded TDEVs was tested by bioassay. The results showed that the viability of LNCaP and PC-3 cells decreased by 80% and 40% respectively after 24 h administration. The mechanism of drug release is known as endocytosis.PTX is a natural anti-tumor drug extracted from Curcuma wenyujin. It has the effects of anti-inflammatory, anti-oxidative stress, and inhibiting the proliferation of malignant tumor cells. Curcumin can regulate different cancer factors without being toxic to normal cells. The National Cancer Institute has listed it as a third-generation cancer chemopreventive drug. Exosomes loaded with curcumin can increase its anti-tumor effect by 3-5 times. Co-culturing curcumin and pancreatic cancer cells can encapsulate curcumin in exosomes secreted by pancreatic cancer cells. By targeting pancreatic cancer cells, it can increase cytotoxicity. Studies have shown that after curcumin treats tumor cells, the secreted exosomes can inhibit IL-2 stimulated NK cell toxicity Curcumin is one of the effective ingredients of the TCM Camptotheca acuminata. It has a good effect on gastrointestinal as well as head and neck cancers. The exosomes secreted by colon-26 cells were loaded with camptothecin, and it was found that the killing effect on colon cancer cells was significantly higher than that of the free camptothecin group, the camptothecin exosomal mixed group, and the control group. It shows that the exosomes encapsulating camptothecin enhance the anti-cancer effect of camptothecin. In addition, a variety of TCM monomer components have been shown to be loaded by EVs to improve efficacy, but there are few reports on the use of TDEVs as a delivery platform Camptothecin is a plant anti-cancer drug isolated from TDEVs delivery of active ingredients of traditional Chinese medicine is expected to play a role in the treatment of a variety of tumors. One of the classic cases is the treatment of glioma. Glioma is highly malignant, has a poor prognosis, and is easy to relapse, which seriously threatens the safety of human life. At this stage, the clinical treatment of glioma is based on surgical treatment, radiotherapy, and chemotherapy. However, the infiltrating growth of glioma cells can easily cause radiation damage to the surrounding normal tissues. Surgical treatment is difficult to remove completely, and the side effects of radiotherapy and chemotherapy on patients are more obvious. A variety of active ingredients of traditional Chinese medicine show good glioma inhibitory effects. Including polyphenols, flavonoids, saponins, terpenes, alkaloids, and some mineral medicines containing arsenic, mainly natural products such as curcumin, resveratrol, silymarin, and quercetin. However, most of the drugs have problems such as poor solubility and poor stability, resulting in low bioavailability in the body after administration. In addition, due to the existence of the blood-brain barrier, the traditional Chinese medicine treatment of glioma still has problems such as poor drug targeting and many adverse reactions, resulting in poor therapeutic effects. In response to the above problems, in recent years, researchers have developed various drug delivery systems to increase the delivery of drugs in the brain. The targeted delivery system of traditional Chinese medicine can reach deep in the brain through blood circulation, increase the concentration and retention time of central nervous system drugs, improve the targeting efficiency of traditional Chinese medicine for glioma and reduce adverse reactions, and improve the therapeutic effect of glioma.Traditional Chinese medicine has been increasingly used in the treatment of tumors due to its multi-targets and synergistic effects in recent years. The reports of EVs as emerging natural drug carriers have gradually increased, but there are still many biological problems need to be resolved. Compared with western medicine nano-preparations, the tumor therapeutic DDS prepared by encapsulating the effective ingredients of traditional Chinese medicine monomers does not show obvious advantages. The targeted delivery system of traditional Chinese medicine for the treatment of tumors still has some shortcomings. First, some drug delivery vehicles are easily recognized and eliminated by the immune system, which affects the targeting efficiency of drugs. Some carriers have a certain degree of biological toxicity, some carriers cannot be degraded in the organism, and there is a long-term hidden danger of toxicity to the organism. Moreover, the preparation methods of some vectors are complicated and cannot be mass-produced, and it is difficult for the Chinese medicine targeted delivery system to be transformed into clinical practice. In addition, there are few studies on the multi-component Chinese medicine compound targeted delivery system developed based on the theoretical characteristics of Chinese medicine to treat tumors. This kind of treatment method has not exerted the anti-tumor characteristics of Chinese medicine under the guidance of Chinese medicine theory. Therefore, the current research is mainly focused on the direction of TDEVs to deliver multiple components in the traditional Chinese medicine compound. Many of these problems have yet to be resolved, such as multi-component co-loading, coordinated delivery between components, and so on. At present, the reported preparations containing Chinese herbal compounds include Buyang Huanwu Decoction and Tongxinluo Capsules. However, DDS constructed by TDEVs containing Chinese herbal compounds has not been reported yet. However, with the continuous development of new materials, the continuous improvement of preparation processes, and the continuous development of carrier surface modifiers, the above-mentioned problems should be gradually solved. It is believed that the advantages of Chinese medicine targeted delivery system for the treatment of glioma will be fully reflected in the future.The active ingredients in traditional Chinese medicine not only play a significant role as therapeutic drugs, but some traditional Chinese medicine ingredients themselves can exist as drug delivery carriers. Not only that, but traditional Chinese medicine also can produce EVs. At present, most researches focus on EVs derived from animal cells. In recent years, researches on EVs derived from Chinese medicine plants as active ingredients are gradually being carried out. Plant-derived EVs are easier to obtain in large quantities and the yield and economic benefits are greatly improved. They not only participate in the regulation of plant innate immune function, but also can perform the regulation of transboundary gene expression, achieve anti-inflammatory, anti-viral, and anti-oxidant effects, and provide new ideas for the study of active ingredients in traditional Chinese medicine. The active ingredients of traditional Chinese medicine and chemical drugs are used in combination, and they are jointly loaded in EVs, which can act on multiple signal pathways and play a synergistic effect. Play a slow and controlled release effect, increase drug loading, and reduce drug leakage while reversing tumor multi-drug resistance.et al. In vivo studies show that DDS can target A33 positive colon cancer cells, inhibit tumor growth, prolong the survival of mice, and reduce cardiac toxicity. Ingato et al. In addition to TCM ingredients, some antibiotics can also be used as encapsulated chemical drugs. DOX is one of the most effective anti-cancer drugs in clinical use. It has a significant inhibitory effect on many kinds of cancer cells. Due to its cardiac toxicity and other side effects, the dosage is limited In recent years, biopharmaceutical preparations have developed rapidly. Biopharmaceuticals are various regulatory substances related to metabolism, such as proteins, enzymes, nucleic acids, hormones, antibodies, cytokines, etc., extracted from the body. After the proper treatment, these biological drugs can also be incorporated into TDEVs to exert special effects.et al. In vitro and in vivo experiments showed that EV-virus-PTX significantly inhibited tumor growth. Garofalo et al. et al. Chemotherapeutic drugs and other biological drugs can be loaded into TDEVs to improve efficacy. Garofalo in vivo, resulting in low clinical efficacy. Therefore, it may be considered to load the programmable RNA drug into the low immunogenicity TDEVS and deliver it directly to tumor cells to improve the therapeutic effect et al. ™. Expression of CRISPR/Cas9 plasmid in exosomes by electroporation. The expression of poly (ADP-ribose) polymerase-1(PARP-1) was inhibited by the exosomes loaded with CRISPR/Cas9, which activated the apoptotic pathway and inhibited the proliferation of ovarian cancer cells. Moreover, compared with the exosomes derived from epithelial cells, they are more targeted. Wang et al. Figure ).Gene therapy is an important method of cancer treatment. Gene therapy can directly regulate the growth of tumor cells, but also can activate the body's immune system to eliminate cancer cells. However, the low absorptivity and high cytotoxicity of gene drugs limit their delivery et al. in vivo. TDEVs loaded with some special kinds of miRNAs can modulate the immune response and tumor microenvironment. Then the DDS can initiate the anti-tumor response. Taghikhani et al. et al.Tumor immunity, as a common cancer treatment strategy, has received more and more attention in recent years. Cancer cells constantly express new tumor antigens that normal cells do not possess et al. Past studies usually directly load chemotherapeutic drugs or biomolecules in TDEVs. However, in recent years, more and more researchers tend to combine or modify drugs and then deliver them through TDEVs to improve the therapeutic effect. This process usually involves the recombination of multiple drugs. For example, simple active molecules can be first made into biomimetic nanoparticles et al. Figure ). In the above example, EVs are not isolated from the tumor, which opens a new way for TDEVs based DDS fabrication. Other studies have used DDS with a similar structure to EVs to improve efficacy.DDS based on apoptotic bodies is also an effective cancer therapy and can enhance combined chemical drugs penetration. Zhao  al. et al. Figure 8).There are also new drugs that can be used to make DDS. The emerging nano-catalytic drugs have become an alternative to chemotherapeutic drugs. Wu etet al. Figure ).Not only are the principles of passive and active targeting applied to the construction of DDS, but the strategy of physical chemistry can also realize the targeting effect of drug-loaded TDEVs. This principle can also achieve good results et al. ™ kit and then loaded with Olaparib (PARP inhibitor) by electroporation and labeled with superparamagnetic iron oxide (SPIO). Compared with other DDS, hypoxic cells preferentially absorb the exosomes released by hypoxic cells, increase cell apoptosis and slow down tumor growth in vivo. Zhu et al. et al. in vivo bioorthogonal click-response to azide on the surface of deep tumors was observed by intravenous injection of a water-soluble dibenzo cyclooctyne modified chloride e6 (DBCO-Ce6). Through the strategy of DDS construction, the accumulation and penetration of DBCO-Ce6 in the tumor were enhanced .Some studies have produced EVs that target hypoxic tumor cells, for example, to enhance tumor response The EVs-based cancer therapy drug delivery system has many advantages. Specifically, EVs can prolong the half-life of drugs to maintain stable drug properties. Moreover, EVs have good biocompatibility, which makes the drugs easier to be absorbed by the body and avoids suffering from drug resistance. In particular, TDEVs can better recognize tumors and contain tumor-associated antigens and immunosuppressive molecules, which can down-regulate the immune system's response and deliver certain inhibitory signals. Therefore, DDS based on TDEVs can bring better therapeutic effects to a certain extent. The above-mentioned advantages have made the research of EVs as drug carriers attract more and more attention in recent years in vivo function and safety of EVs. In the clinical use of EVs for drug delivery, potential risks need to be considered. Since TDEVs play a role in assisting cancer cells to build the local and distant microenvironment of the tumor during the tumorigenesis and development process, these DDS can not only deliver drugs but also increase the proliferation and spread of tumor cells in the body. In addition, because EVs can carry drugs across biological barriers, it may increase the dose of drugs in certain special tissues and organs, leading to increased toxicity. Moreover, the cell-to-cell communication of endogenous EVs may be interrupted due to the influence of a large number of exogenous EVs. The protein on the surface of the vesicle may cause biological toxic side effects. In in vivo and in vitro studies, how to determine the final active substance, inactive ingredients, and mode of action is also a challenge. Therefore, when applying EVs-based DDS, it is necessary to consider selecting EVs derived from appropriate cells and tissues, as well as appropriate drugs and modifying them with other compounds to avoid the occurrence of the above-mentioned situations (Table 3).However, there are also many noteworthy problems in the clinical application of the DDS. First, to improve the efficiency of EVs administration, appropriate cell sources should be selected, taking full account of tumor cells and cells in the tumor microenvironment. The selection of parental cells not only needs to consider factors such as the activity of EVs, tissue homing ability, immunogenicity, and carcinogenicity but also whether the parental cells are convenient for large-scale culture. In addition, the parental cells should have the characteristics of genetic stability and fewer pollutants. In the process of large-scale cultivation, genetic stability and the influence of pollutants should also be closely monitored. Second, appropriate extraction methods and drug loading methods should be selected. The EV concentration is low, the content is small, and there are complex environmental components. If there are more impurities in EVs, it may affect the quality of the preparation, so sufficient purification should be given to ensure the removal of impurities, such as cell debris. When selecting an extraction method, comprehensive consideration should be given to the yield, purity, integrity, and efficiency of EVs, and whether the method can be mass-produced. The drug delivery method should be cost-effective and efficient. The design of the EVs specific drug delivery program is a cautious process, and comprehensive experimental verification is required to determine a EVs preparation that can achieve large-scale clinical production. When performing operations such as destroying the membrane during the drug loading process, there is a risk of changing the direction of the membrane protein, which may cause recognition by the immune system and then cause adverse reactions. When constructing DDS, the targeting of TDEVs may be insufficient, and surface modification is needed to improve the effectiveness of targeted therapy. Third, the storage of EVs is also a problem that should be paid attention to. Higher temperatures may change the physical and chemical properties of the vesicles and reduce the effectiveness of the load. For example, storage of exosomes at 4°C will cause aggregation and structural destruction. The current better storage condition is less than -80°C. However, this storage condition also increases the cost of transportation and storage. It is worth noting that there are still many controversies about the TM has entered clinical phase I and can treat skin T-cell lymphoma, melanoma, and breast cancer. The clinical trials showed positive results and demonstrated good safety. exoSTINGTM can treat solid tumors such as the neck, head squamous cell carcinoma, and breast cancer, and has entered phase I/II trials. Other drugs such as exoASO-STAT6 and exoASO-STAT3 are in the preclinical stage. Evox Therapeutics, a British biotechnology company dedicated to engineering exosomes for delivery, raised US$95.4 million to advance its exosome therapy into clinical trials. MD Anderson also initiated the first clinical trial of therapeutic exosomes. At present, China's innovative "drug-loaded vesicle therapy technology" has completed the clinical transformation in the Chinese market and has been approved for use in 7 provinces and cities in Hubei, Hunan, Hebei, Shandong, Anhui, Shenzhen, and Tianjin, and has become EVs research and clinical transformation The most advanced new tumor treatment technology. In the process of clinical transformation of EVs, the biological research, technology, and application of EVs are closely related and complement each other. The development of production technology relies on a deeper understanding of the biological research of EVs, and clinical transformation is inseparable from the innovation of production technology Since more than 10 years ago, many pharmaceutical companies have announced the development of therapeutic EVs as drug delivery vehicles. The clinical research of EVs on cancer treatment began to rise, gradually transforming from scientific research results to applications, and the safety and effectiveness of the technology have been verified. Drug-loaded EVs have been applied to the treatment of a variety of cancers, such as prostate cancer, brain cancer, lung cancer, bowel cancer, melanoma, and so on. At present, the clinical transformation of most EVs on a global scale has progressed to preclinical or clinical phase I, and some have entered clinical phase II. Most clinical trials have shown good efficacy. In recent years, some biotech companies have listed several EVs drug delivery systems. Aegle Therapeutics is the first company approved by the FDA to enter human clinical trials. Founded in 2015, CODIAK is a biotechnology company focusing on cancer immunity. The candidate drug exoIL-12In summary, the application prospects of EVs are full of hope. Even if many unsolved problems require further research by experimenters, if DDS based on EVs can effectively achieve targeted delivery, a new era of tumor therapy will come. This technology will fill the gaps in multiple tumor treatment methods and bring the gospel to more cancer patients. It is believed that EVs will bring new disease treatment opportunities in the near future.


Nanomaterials possess valuable physical and chemical properties, which may make them excellent candidates for the development of new insecticides, acaricides, fungicides, drugs, catalysts, and sensors, to cite just some key categories. To avoid the utilization of toxic or high-energy inputs, which are routinely used in nano-synthesis, the “green synthesis” concept has been proposed, outlining the use of microbial-, animal-, and plant-borne compounds as reducing and stabilizing agents. Even though a large number of studies have been published on the topic, many potential applications of nanomaterials have scarcely been explored, and their real-world applications are poorly implemented. This is often due to a lack of prolonged efficacy of the bioactive compounds and challenging regulations. Nanotechnologies are currently considered a strategic option to improve the efficacy and stability of classic and green insecticides, acaricides, repellents and antiparasitic drugs, relying on various nanocarriers, including nanoencapsulation and nanoemulsions.Nanomaterials has already dedicated a successful Special Issue to the topic “Green Synthesis of Nanomaterials” in 2019 [In this framework, the journal Green Synthesis of Nanomaterials and Their Biological Applications” represents a continuation of the former, with another collection of top-quality articles in this research area. Particular attention has been devoted to entomological research, because the widespread overuse of synthetic insecticides leads to the rapid development of resistance in target species, and non-target effects on human health and the environment. A comparable scenario is well recognized in parasitology concerning the use of drugs to manage parasites of public health importance. The Special Issue “Herein, both original research and reviews have been considered for publication. The present Special Issue includes contributions on the following research topics:(i)Green-based processes improving the stability of nanomaterials ;(ii)The development of green synthesis protocols for the preparation of nanomaterials with antibacterial and anticancer activity, as well as acting as catalytic reductors of nitrophenols ;(iii)The development of novel insecticides against key insect pests, including highly stable insecticidal nanoemulsions toxic to moth larvae , as well(iv)The development of new terahertz metamaterials for biosensing applications ,7.The Special Issue ends with a review on cystic echinococcosis , a dangeOverall, I am grateful to all the authors for their fine contributions to the present Special Issue, and hope that the published studies will pave the way for novel real-world applications of green nanomaterials.