As for the DCF release patterns and kinetic models at specificpH values, the release showed a diffusion- and swelling-controlledprofile, effortlessly fine-tuned by external environmental pH stimuli.Overall, we show that the modified beads exhibit excellent characteristicsfor transport across the gastrointestinal tract and enhance the bioavailabilityof the drug. Their therapeutic efficacy and biocompatibility are alsoevident from the studies on human fibroblast cells. We anticipatethat this platform could support and inspire the development of novelsustainable and effective polysaccharide-based delivery systems.The path to greatersustainability and the development of polymericdrug delivery systems requires innovative approaches. The adaptationand use of biobased materials for applications such as targeted therapeuticdelivery is, therefore, in high demand. A crucial part of this relatesto the development of porous and hollow structures that are biocompatible,pH-responsive, deliver active substances, and contribute to pain relief,wound healing, tissue regeneration, and so forth. In this study, wedeveloped a facile single-step and water-based method for the fabricationof hollow spherical cellulose beads for targeted drug release in responseto external pH stimuli. Through base-catalyzed deprotection, hydrophobicsolid and spherical cellulose acetate beads are transformed into hydrophiliccellulose structures with a hollow interior  by a stepwise increment of temperatureand treatment time. Besides the pH-responsive fluid uptake properties,the hollow cellulose structures exhibit a maximum encapsulation efficiencyof 20–85% diclofenac (DCF), a nonsteroidal anti-inflammatorydrug, used commonly to treat pain and inflammatory diseases. The maximumamount of DCF released  Stimuli triggers can be categorized into organ-leveltriggers, related to pathophysiological changes and cellular compartment-specifictriggers.1 Although the potential environmentaltriggers for medical applications are limited, variations in the pHof different cellular compartments, tissues, and organs offer pH asan accessible stimulus. The change in the external pH acts as a stimulus,to which the response is observed in the form of a change in the propertiesof the selected responsive material. pH-responsive materials undergophysical and chemical changes, resulting in swelling, shrinkage, dissociation,degradation, or membrane fusion and disruption. This is triggeredby either the protonation of ionizable groups or degradation of acid-cleavablebonds in the pH-responsive materials.1 Forinstance, carrier materials bearing −C(=O)OH functionalgroups demonstrate higher solubility at the basic pH range and canbe used to protect low pH-sensitive medications, for example, intestine-targeteddelivery.6 Every oral delivery must consider the changein pH along the gastrointestinal tract, including saliva (6.0–7.0),gastric fluid (1.0–3.5), bile (7.8), pancreatic fluid (8.0–8.3),small intestinal fluid (7.8–8.0), and large intestinal fluid(5.5–7.0).7 Additionally, the nominal pH rangeof chronic wounds and inflamed tissues has been reported between 7.4and 5.4, while cancerous tissues have a more acidic extracellularpH in the microenvironment of a tumor.11Stimuli-responsiverelease carriers exhibit altered propertiesfollowing changes in environmental variables, typically pH, temperature,salt concentration, enzymes, redox (oxidation–reduction), andlight.12 Hence, targeted drug delivery with simultaneousdrug protection is among the prime goals of drug delivery researchtargeting this organ. Among drug carriers, spherical beads (in mmor μm) with unique properties, such as high porosity, largeavailable specific surface area, low density, and adjustable chemistry,provide an exceptional template for drug delivery.13 These spherical beads also comply with durability and areeasily transported and stored. Various types of hollow beads havebeen fabricated from a plethora of materials for diverse applications, ranging from carbon 17 to inorganic oxides 22 to synthetic polymers 27 and natural polymeric materials .32 Polymers, as one of the most extensively utilized materials forthis purpose, have made remarkable advances in drug delivery and offerseveral advantages (pH-responsive). In addition to the stable physicochemicalproperties offered by an ideal polymeric delivery carrier, it shouldbe biocompatible, cost-effective, and protect the incorporated drug.Polymers applied in drug delivery primarily reduce degradation, immunogenicityand toxicity (burst release); improve circulation time; and serveas passive targeting agents. pH-responsive polymers used in medicinemust also respond to a range of biological conditions by a changein solubility, swelling, conformation, degree of ionization, and releaseof an active compound.However, many drugs cannotsustain the gastrointestinal environment.36 These polysaccharide-formulated beads have become attractive ascarriers in drug delivery, owing to the abundance of the raw material,relatively inexpensive production, and intrinsic properties: biocompatibility,swelling capacity, biodegradability, and positive environmental factors(sustainability and renewability).37 Furthermore,the functional groups carried by polysaccharides can be fine-tuned to shape and advance their properties as deliverycarriers.38 Cellulose beads are high-performancematerials used in a large range of applications.36 Cellulose is not only the most abundant renewable biopolymer39 but also a promising and comprehensively investigatednatural material, covering applications ranging from water treatmentto tissue engineering,40 packaging,41 biosensors,42 anddrug delivery.43 The high porosity withinternal surface area,45 gradual release,47 and porosity of the matrix48 make cellulosebeads an attractive absorbent and competitive granulate material forbiomedical applications . Importantly, cellulose enables the drug itselfto be stabilized in an amorphous state with high stability towardrecrystallization.49 Compared to the syntheticpolymer-derived beads, cellulose-based beads (size: mm or μm)show higher mechanical stiffness and are relatively more simple tomanufacture.52 In recent decades, various methods for the preparationof cellulose beads have been reported, including (1) dissolution ofcellulose, (2) spheronization, and (3) coagulation.53 In this study, we show for the first time the fabricationof polysaccharide cellulose beads with a hollow interior, which canbe an advantage to achieve high encapsulation and release efficiencyfor therapeutic drug molecules. Especially, a single-step base-catalyzeddeacetylation method to obtain hollow and porous cellulose beads fromthe commercial cellulose acetate (CA) spherical beads as drug carriershas never been reported. We chose diclofenac (DCF) as the model drugcomponent to demonstrate the encapsulation and pH-responsive releaseefficiency of the hollow cellulose beads. DCF is a nonsteroid anti-inflammatorydrug that is widely used in relieving pain, fighting fever, and decreasinginflammation.54 Although modification ofcellulose beads has been around for decades,53 it commonly adds to the economic and environmental expenditures.55Polysaccharide-based beads are a newgeneration of porous polymericmaterials that exhibit outstanding properties compared to synthetic-basedbeads, while being regarded as the most sustainable.in vitro release, concerning their morphology, structure, swelling capacity,and release kinetics using different kinetic models. To show the potentialof the prepared beads for biomedical applications, their biocompatibilitywas further tested against human fibroblast cells.Inspired by all the abovementioned methods,we present a straightforwardand scalable method to design cellulose beads and improve their functionality.In this method, the commercially available solid CA spherical beadswere transformed into cellulose beads with a porous surface and hollowinterior through a simple and environmentally friendly base-catalyzeddeprotection method by the fine-tuning of treatment time and temperature.Consequently, due to the increased swelling capacity of cellulosebeads in water, increased active substances like DCF can be encapsulatedinto the beads, and, correspondingly, a sustained and longer-releaseprofile can be achieved. To investigate the applicability, the drugof choice, DCF, was used in a controlled release triggered by thepH of the environment (pH 1.2–7.4). The beads were characterizedbefore and after encapsulation, as well as after 22.13) were purchased fromCospheric, USA. DCF sodium salt, 5-fluorescein hydrochloride (DTAF), sodium bicarbonate, potassium hydroxide(KOH) pellets, disodium phosphate heptahydrate (Na2HPO4·7 H2O), and sodium dihydrogen phosphate monohydrate(NaH2PO4·H2O) were purchasedfrom Sigma-Aldrich, Austria, and used as received. Advanced Dulbecco’smodified Eagle’s medium (DMEM) and fetal bovine serum (FBS)were purchased from Thermo Fisher, Germany. Human-derived skin fibroblasts were purchased from ATCC,UK. Ultrapure water (18.2 MΩ cm) from a Milli-Q water purificationsystem  was used for deacetylation, bufferpreparation, and drug release experiments.White, spherical CA beads for 24 h at room temperature andstirred continuously (300 rpm) with a mechanical stirrer. Later, theywere immersed in 100 mL of water for 2 h, rinsed extensively withwater, and dried at room temperature for 12 h. Method II: 20 CA beadswere immersed in 100 mL of 5 M KOH for 3 h at different temperatures and stirred continuously with a mechanicalstirrer (300 rpm). Subsequently, the beads were immersed in 100 mLof water for 2 h and then rinsed extensively with water and driedfor 12 h at room temperature. The completely deacetylated celluloseacetate (DCA) beads at 90 °C .The chemical structure/compositionof the CA and deacetylated beads (before and after drug encapsulation)were analyzed using a Bruker Alpha attenuated total reflection Fouriertransform infrared (ATR–FTIR) spectrometer at a scan rangeof 4000–650 cmin et al.12where R2.4Themicroscopic morphology of all bead samples was analyzed using a CarlZeiss FE-SEM SUPRA 35 VP scanning electron microscope. The scanningelectron microscopy (SEM) images were recorded with an accelerationvoltage of 1 kV. The samples were mounted on aluminum sample holders,and no sputtering was performed on the samples’ surfaces.2.5c = 0.1 mg/mL, dissolvedin 100 mM bicarbonate buffer, pH 9). 10 CA and DCA beads were leftto react for 30 min in a dark place under the exclusion of light.Afterward, the beads were immersed in water for 60 min and then rinsedextensively with water, dried, and stored at room temperature underthe exclusion of light. A confocal laser scanning microscope  was used to observethe surface morphology of the stained samples. The DTAF dye was excitedat 495 nm, and the emission was recorded at 516 nm. The image sizewas 512 × 512 pixels, and the images were scanned at a scan speedof 290 frames s–1.TheCA and deacetylated beads (surface and interior) were stainedwith 100 mL of DTAF solution (2.6via methodII at 90 °C) in three different aqueous media: simulated gastricfluid ,phosphate-buffered saline (PBS) with pH 5.5, and PBS with pH 7.4.Accurately weighed amounts of beads (2.5 g) were put in a sphere lattice(sieve pouch) and immersed in 30 mL of swelling media at 37 °C.At fixed time intervals, the beads were taken out from the mediumand wiped gently with Whatman (115A) filter paper and weighed. Thedegree of swelling of the beads (%) with respect to time was calculatedaccording to eq Ws is theweight ofthe beads in the swollen state, and Wi is the initial weight of the beads.Swelling studieswere performed for the DCA beads (prepared 2.7via method II at 90 °C)were immersed in 5 mL of DCF solution at different concentrations for 3 h, ensuring 2 beads/cm3 of thesolution. Following this, the beads were taken out from the DCF solution,kept on a glass plate, and dried at room temperature for 24 h.DCA beads  in 500mL of SBF (pH 1.2) and PBS buffer (5.5 and 7.4) at 37 °C. Thereleased DCF amounts were measured with a UV/Vis spectrometer  at a wavelength of 276 nm, and the concentrationswere calculated using calibration curves. The release experimentswere carried out in triplicate.The DCF release from the encapsulated beads was determined accordingto the USP paddle method .The drug loaded in the DCA beadswas determined by the following eq To evaluate the release mechanismsof DCF from the DCA-encapsulatedbeads, the obtained release data at different pH values and differentdrug concentrations were fitted with four kinetic models, namely,zero-order, first-order, Korsmeyer–Peppas, and Higuchi models,respectively.mt is the amount of drug released in time t, mb is the initial concentration of the drug inthe solution before release, and k0 isthe zero-order release rate constant.The zero-order kinetic model can be describedas shown in mt is the amount of the released drug in time t, mb is the initial concentrationofthe drug and Kt is the first-order rateconstant.First-order kinetics 6 is usedm∞ is the amount of drugreleased after an infinitive time , kK–P isthe Korsmeyer–Peppas rate constant, and n isthe diffusional exponent used to describe the drug release mechanism.In general, the value of n varies, depending on theshape of the drug carrier system. In spherically shaped systems, forwhich an n value of 0.45 is determined, the releasemechanism is Fickian diffusion. Whereas, if 0.45 < n < 0.89, then a non-Fickian diffusion release, or an anomaloustransport mechanism, is implied. If n > 0.89,thenthe drug release is dominated by the case II release mechanism, thatis, a combination of diffusion and the swelling-controlled process.The Korsmeyer–Peppas kinetic model is as follows77where mmt is the amount of drug released at time t and K is the Higuchi constant.The Higuchi kinetic model is described by 2.82 for 24 h . The fibroblasts were seeded in a concentrationof 10,000 cells/well in a 96-well microtiter plate. After 24 h ofcell incubation, the samples were added to the cells in four repetitions.For this purpose, we used the as-prepared samples and their dilutionsof 1:2, 1:4, 1:8, and 1:16 in advanced DMEM with 5% FBS. DCA beadsencapsulated with different amounts of DCF (0–20 mg/mL) werealso tested. After 24 h of incubation at 37 °C and 5% CO2, the samples’ biocompatibility/viability were assessedusing the 3--2,5-diphenyltetrazolium bromide(MTT) assay . The purple formazan crystalsformed by active cells were quantified by measuring the sample absorbancespectrophotometrically  at 570 nm. The overall testing was performedfollowing the ISO 10993-5 standard.Allbead samples were transferred into Eppendorf tubes (2 mL) andwere sterilized under ultraviolet light for 30 min. After sterilization,they were transferred into the cell culture medium–advancedDMEM with 5% FBS. Before the bead samples were added to the cells,the beads were acclimatized by being incubated at 37 °C and 5%CO33.159 Although differentstarting cellulose sources, solvents, and regeneration methods havebeen used for the fabrication of cellulose beads, all of them resultin the formation of porous beads, which is achieved by either thedropping or dispersion techniques.53 Thesetechniques involve a multistep and tedious process, as reported inthe literature.53 In the current study,we aimed to develop a simple, single-step, and water-based procedureto achieve spherical hollow cellulose as one of the most attractivealternative pH-responsive delivery systems. Cellulose beads with amicrometer-sized hollow interior, which are sufficiently porous, areparticularly advantageous, as they allow a higher drug encapsulationand enable a controlled drug release. For this purpose, for the firsttime, we combined the use of commercially available spherical beadsof solid CA and a well-known base-catalyzed deacetylation procedureto regenerate the cellulose structure from electrospun nanofibers43 and the spin-coated thin films of CA.61 CA beads are beneficial for drug delivery applications in many ways,as CA itself is inexpensive, biodegradable, and biocompatible, amongothers.62 Base-catalyzed deacetylationis a simple and straightforward method to convert CA directly intoDCA and control its properties .60 Therefore, in the current study,the spherical solid CA beads (diameter: 2 mm and density: 1.3 g/cm3) were transformed into hollow spherical DCA or cellulosebeads, as shown in e.g., targeted,sustained drug delivery).Cellulose beads with micrometer (μm) size havebeen used in various advanced applications ranging from chromatography,solid-support synthesis, and protein immobilization to the sustainedrelease of targeted drugs.3.1.1–1 (−C=O), 1458 cm–1 (O=C–OR), 1365 cm–1 (−CH2), 1030 cm–1 (C–O–C), and902 cm–1 (−C–H), with a minor contributionof an −OH peak at 3328 cm–1.63 At 1 M KOH , the carbonyl peak disappeared,indicating that the surface of the beads was completely deacetylated.However, in the interior of the beads (Supporting Information) was estimated using the ratio between the acetylC=O stretching of the ester peak at 1730 cm–1 and the C–O stretching of the cellulose backbone at 1030cm–1, as described by El Nemr et al. are shownin  1 M KOH 2a, the ehe beads 2b, neithmr et al. A DS of –1 was detected at the surface of the beads,regardless of the temperature (–1 with increasing temperature wasobserved for the bead’s interior (–1 and other characteristicpeaks of cellulose in the fingerprint region (between 800 and 1400cm–1)65 was also observed. This effect was even more pronounced at 80 °C,where DS decreased from 2.85 to 0.94 . No carbonyl peak wasobserved at 90 °C, and the spectra resembled the structure ofneat cellulose,61 indicating that the CA beads had been completely transformed intocellulose beads. Overall, compared to method I, method II allows thecontrol of the hydrophilic/hydrophobic character and DS of the entirebead structure by simply varying the conditions of the base catalysis. This type of character is desiredif beads are to act as carriers in targeted drug delivery. They canbe used selectively to encapsulate hydrophobic or hydrophilic drugs,depending on the application.66To achieve complete deacetylationof the CA beads, the latter weretreated with 5 M KOH at increasing temperatures, from 25 to 90 °C. Although the direct measurement of roughness on thesurface of the beads is not possible, it was evident that the surfacebecame rougher with incremental temperature. It is suggested thatsome parts of the base material and/or the acetyl (−COCH3) groups are removed from the surface as the result of deacetylationat a high temperature and exposure to a strong base (5 M KOH). Thisled to changes in the surface morphology and porous structure. Thisis in good agreement with the ATR–FTIR spectroscopy results(The SEM morphology(a: surface and b: interior) of CA beads before and after treatmentwith 5 M KOH at different temperatures is depicted in  results2a, whichSupporting Information) and an inner diameterof 650 μm was formed, implying that the combination of highertemperature and strong base caused extensive deacetylation, leadingto the formation of the hollow structure. We can show that a single-stepdeacetylation treatment at high temperature is useful to transformthe spherical solid systems of acetylated polymers into deacetylatedspherical systems with partially or completely hollow interiors. Suchsystems with hollow interiors and porous surface structures may beadvantageous as drug carriers in medicine. For example, they can beused to enhance the encapsulation capacity of drugs such as DCF and/orto enable a continuous and simultaneous fluid exchange with the environment,as in the case of drug release.9As can be seen in 61 This is also in accordance withthe ATR–FTIR results, where the emergence of reactive −OHgroups at 3328 cm–1 was more pronounced with theincreasing temperature. At 90 °C, complete deacetylation of thesurface and interior of the beads was achieved, which was evidencedby their strong and high fluorescence intensity. This confirms thatthe unreactive hydrophobic CA beads were completely transformed intoreactive and hydrophilic cellulose.61 The ATR–FTIRspectroscopy results support this finding further, where all acetylgroups disappeared and only peaks related to the neat cellulose structurewere observed.To confirm the completion of deacetylation of the entire structureof CA beads further, we used a combination of staining and confocallaser scanning microscopy (CLSM)-based methods. 3.1.3–1  resulted in considerable mass loss and shape changes. This canbe correlated with the ATR–FTIR data, where the actual structuraltransformation of the base CA beads was observed. Interestingly, althoughthe beads shrank in size, they did not lose their spherical shape.The 800 μm size of the fully deacetylated CA beads remains comparableto the bead size of other drug delivery systems such as chitosan,34 carboxymethyl cellulose/chitosan,67 alginate/chitosan,68 alginate/carboxymethyl cellulose,69 pectin,70 TEMPO-oxidized cellulose,35 and so forth.Theinfluence of temperature (25–90 °C) on the mass loss  and size (in mm) of CA beads after treatment with 5 MKOH is shown in –1 2 in the –1 4. At hig71 To determine the swelling/fluiduptake capacity, hollow DCA beads prepared at 90 °C were immersedfor an extended period (24 h) in swelling media prepared at threedifferent pH values , corresponding to the gastric35 and cancer72 environments,as well as the values during wound healing or tissue regeneration.34 The results of the swelling measurements areshown in Swelling is one of the most importantparameters that determinethe diffusion and release rate of encapsulated drugs in differentfluid environments and, as such, affects the overall drug deliverymechanism importantly. The extent of swelling is used to determinethe ability of drug carriers to absorb water, which can be analyzedquantitatively by the weight gain of the beads. Among others, theswelling degree can be influenced by two main factors: (i) pH and(ii) time.3.2e.g., to make use of pH changes to controldrug release in different pathophysiological conditions). Therefore,we investigated the release properties of DCF-encapsulated DCA beadsin different pH environments. This was done to simulate the situationand to understand the release kinetics of DCF/DCA beads in variouspH environments, including the gastrointestinal tract (pH 1.2),35 tumor microenvironment (pH 5.5),72 and (soft and hard) tissue engineering (pH 7.4).34 The nonsteroidal anti-inflammatory DCF was chosenas a model drug, for its pH-dependent solubility, to aid investigationsin the encapsulation and release efficiency studies of the hollowDCA beads. The release profiles of DCF at different pH values  and at different drug loading concentrations(DCF: 1–20 mg/mL) at increasing durations of time are shownin ca. 20–85% (1–25mg/mL). In particular, the DCF loading content in the bead was inthe following order: 19.8% (1 mg) < 28.3% (5 mg) < 35% (10 mg)< 45.6% (15 mg) < 60.2% (15 mg) < 71% (20 mg) < 85.3%(25 mg). This is comparable to the values obtained with other drugloading systems, such as cellulose nanocrystals/polyvinyl alcohol(PVA),35 CA/PVA, carbon dot/alginate, ironoxide/PVA,73 polyethylene glycol-basedpolyprodrug amphiphiles,74 and so forth.Although DCF is highly water-soluble (21.3 g/L), the release profilesof DCF from all three media studied are nevertheless very different  was observedfor up to 300 min following the burst effect. The second release phasewas the so-called “plateau,” which is due to the diffusion-controlledrelease of the remaining drug in the bead. In the other two cases,a rapid release of the drug continued after the burst release, increasingmaximally for all DCF concentrations up to 300 min, followed by aslower and prolonged release for the duration of the next 19 h.At pH 1.2, the release curvesat all concentrations showed a biphasicbehavior, meaning that an initial fast release  is accompanied by a prolonged/sustained release (second part).These biphasic release patterns are also seen at pH 5.5 and 7.4. Atall pH ranges tested, the burst release was noticed up to 30 min andat all concentrations of DCF. At pH 1.2, 3–11% of DCF was releasedduring the first part when the DCF concentration increased from 1to 20 mg/mL. At pH 5.5 and pH 7.4, the maximum observed release ofDCF was 10–30 and 10–60% during the initial burst release.An explanation for the observed burst effect is the release of drugsinto the surrounding media, which were adsorbed heterogeneously onthe surfaces of the beads during encapsulation, keeping the surfaceof the pores open for subsequent diffusion of the drugs.ca. 30% at the highestconcentration (20 mg) of the drug after 24 h, whereas at pH 5.5 andpH 7.4, the release of the drug was increased to 80 and 100% at thesame concentration. The release of the drug is due to the diffusionof water into the porous structure of the beads, resulting in swelling.This may cause the incorporated drug to dissolve, diffuse out of thebeads, and, subsequently, be released into the surrounding medium.It is expected that the swollen DCA beads will release higher amountsof the drugs to the medium. From the results of the swelling experiments,it can be stated that the less swollen DCA beads at pH 1.2 releasedlower amounts of DCF than the other two pH values at which maximumswelling of the beads was observed. DCF is a weak acid with a pKa value of 4.0, is weakly soluble in acidicsolutions, and dissolves readily in intestinal fluid and water.76 Therefore, the lower solubility of DCF at pH1.2 limits the initial release of drug from the surface and hollowinterior of the beads and, hence, the overall release of DCF. Increasingthe pH to 5.5 and pH 7.4 caused DCF to dissolve readily due to ionization,ultimately resulting in greater solubility and a higher release profile couldnot be fitted satisfactorily with any of the models used in this study.Therefore, we divided the release curves for all samples and all pHvalues into two parts (part I and part II). Part I refers to the initialfast release of 0–300 min, while the prolonged release, thatis, 300–1500 min, was set as the second part. The divisionof each release curve into two parts can be verified further by calculatingthe first derivative (Supporting Information), it can be seen that, at each pH,despite the different models employed for the best fit, only one modelcan describe the release mechanism fully and satisfactorily. Consideringthe best fit , the best fit with maximum 3.2.1in vitro release studies by infrared spectroscopy(in vitro release are shown in –1 (−NH stretchingof the secondary amine), 1571 cm–1 (−C=Ostretching of the carboxyl ion), 1500 cm–1 (−C=Cring stretching), 1450 cm–1 (−CH2 bending), 945 cm–1 (−C–O–Cstretching), and at 746 cm–1 (−C–Clstretching), both on the surface and interior of the bead. After in vitro release at pH 1.2, 7.4 ,no such characteristic peaks were observed in the interior at pH 7.4.These results agree well with the release studies in which fewer amountsof the drug are released at acidic pH than at neutral pH. This maybe due to the low solubility of DCF at acidic pH and increased solubilityat pH 7.4, as mentioned above. It is clear that all the encapsulateddrug molecules in the interior diffused successfully through the poresof the beads and were released into the surrounding medium at pH 7.4,whereas at pH 1.2 and pH 5.5, the unreleased drug molecules remainedin the interior of the structure of the beads during release. Overall,ATR–FTIR spectroscopy is a useful tool and sensitive enoughto detect the successfully encapsulated and the unreleased part ofDCF from the carrier matrices.We also verifiedthe encapsulation of DCF in the whole bead structure before and afterthe troscopy7. The sp3.381Biocompatibilityis a prerequisite for any new drug delivery system to be used foreither type of application in humans. This is also true for the targetedapplications in the case of the DCA beads developed herein, for example,cancer therapy, in wound healing, and/or in boosting tissue regeneration,where pH-responsive drug delivery might improve the treatment outcomes.This study aimed to evaluate the biocompatibility of the untreatedCA, DCA, and DCF-encapsulated DCA beads with human fibroblast cells,which are commonly applied for such purposes.4Invitro release studies showed a pH-dependent release behavior,where the amount of drug released was lower at acidic pH and increasedwith increasing pH of the release medium. The kinetics of drug releaseat different DCF concentrations followed the same pattern, with aburst release during the 30 min release period, followed by prolongedrelease. Among the four kinetic models chosen for data analysis, thezero-order model showed the best r2 valuesat pH 1.2, while at the values of pH 5.5 and pH 7.4, the best r2 values are obtained for the Higuchi and Korsmeyer–Peppasmodels, indicating that the release of the drug is controlled predominantlyby swelling and diffusion. The results of biocompatibility with humanfibroblasts proved that not all the investigated beads encapsulatedwith and without DCF at different concentrations exhibited cytotoxicityand were, therefore, biocompatible. Considering the one-step aqueousmethod to prepare hollow cellulose beads and their potential use forhigh encapsulation efficiency and pH-responsive release behavior,it can be concluded that the biocompatible hollow cellulose beadshave great potential for various biomedical applications .In this work, cellulose beads with a porous surface and hollowinterior were fabricated from solid spherical CA beads for the firsttime. This was achieved by single-step deacetylation treatment atdifferent temperatures and varying concentrations of base (KOH). Thissimple aqueous and temperature-dependent treatment resulted in deacetylatedcellulose beads (DCA) with adjustable DS (acetylation), wall thickness,mass, and hydrophobic/hydrophilic properties, as proven by scanningelectron microscopy, infrared spectroscopy, and thickness and massloss measurements. The fluid uptake properties of the fully deacetylatedCA hollow beads (size: 800 μm) increased with the increasingpH (1.2–7.4) of the solutions, as demonstrated by the swellingstudies. Encapsulation efficiency of 20–85% was achieved withincreasing the DCF concentrations (1–20 mg/mL).


Hyalomma asiaticum ticks in northwest China and serologic evidence of human Tamdy virus infection in the same region. These findings highlight the need to further investigate a potential causal relationship between Tamdy virus and febrile illnesses of unknown etiology in that region.We report the isolation of Tamdy virus from  Tamdy orthonairovirus  includes 5 viruses: Tamdy virus (TAMV), Burana virus (BURV), Tǎchéng tick virus 1 (TcTV-1), Huángpí tick virus 1 (HpTV-1), and Wēnzhōu tick virus (WzTV)  from Xinjiang in northwest China and divided the ticks into 55 groups according to the sampling location (n = 50–100 ticks/group) , confirming findings using real-time reverse transcription PCR (rRT-PCR) (data not shown). We used homogenates of 2 TAMV RNA-positive brain samples from the A-M6 pool (Appendix Table 2) to isolate viruses in Vero E6 cells. As indicated by immunofluorescence assays (IFA) (Appendix), we observed increasing TAMV infection from first to fourth passages in Vero E6 cells, suggesting successful isolation .Negative-stain electron microscopy revealed an enveloped spherical viral morphology with a diameter of ≈90–110 nm , panel AT. orthonairovirus species and 34%–49% identity with Crimean-Congo hemorrhagic fever virus (CCHFV), another Orthonairovirus species. Protein sequences shared 44%–62% identity with other members of the T. orthonairovirus species and 33%–40% with CCHFV .The 2 TAMV isolates shared very high sequence similarities . We named the 2 TAMV strains YL16082 and YL16083, including an abbreviation (YL) for the geographic location (Yuli County) where the original tick samples were collected. TAMV genome sequences (YL16082) shared 37%–59% identity with other members of the th CCHFV . PhylogeHy. asiaticum ticks in northwest China as early as 2007, which warranted more in-depth investigation to establish the potential causal relationship between TAMV and febrile illnesses of unknown etiology in regions where TAMV is present.To investigate potential human infection by TAMV in northwest China, we conducted a seroprevalence study using archived serum samples from 725 healthy persons  and 87 febrile patients  (Appendix). Of the 87 febrile patients, 21 (24.14%) were TAMV IgG positive and 17 (19.54%) IgM positive . MoreoveT. orthonairovirus, TcTV-1, had been identified in a febrile patient in northwest China (Finally, because another st China , suggestTAMV was initially found in ticks in countries in central Asia, including Kazakhstan, Kyrgyzstan, Uzbekistan, and Turkmenistan (Findings of a potential role of TcTV-1 in causing human febrile disease (Among study limitations, the nature of using archived samples limited our ability to provide direct evidence of a causal relationship between TAMV and human febrile illnesses. Also, it is possible that the high TAMV antibody-positive ratio might have resulted not from the recent cases but from a small outbreak of human TAMV infection in northwest China in 2007.In summary, our study strongly suggests the potential of TAMV as a human pathogen and supports an urgent need to conduct more in-depth epidemiologic and pathogenesis investigations into this group of viruses in China, central Asia, and beyond. While the world’s attention is currently on coronavirus disease and batborne viruses, our study highlights the need to pay attention at the same time to emerging zoonoses of tick origin to prevent future outbreaks.Additional information on human exposure to Tamdy virus in China.


Little is known about the burden that overweight and obesity impose on Dutch society. The aim of this study is to examine this burden in terms of cost-of-illness and health-related quality of life.A bottom-up, prevalence-based burden of disease study from a societal perspective was performed. Cost-of-illness information including healthcare costs, patient and family costs, and other costs was obtained via the Treatment Inventory of Costs in Patients with psychiatric disorders (TiC-P) questionnaire. Health-related quality of life was assessed through the EuroQol (EQ-5D-5L) and the BODY-Q instruments. Non-parametric bootstrapping was applied to test for significant differences in costs. Subgroup analyses were performed on all outcomes.A total of 97 people with overweight and obesity completed the survey. Per respondent, mean healthcare costs were €2907, patient and family costs were €4037, and other costs were €4519, leading to a total societal cost of €11,463 per respondent per year. Total costs were significantly higher for respondents with obesity versus overweight and between low & intermediate versus highly educated respondents. The mean utility score of our population was 0.81. A significantly lower utility score was found for respondents with obesity in comparison with respondents with overweight. BODY-Q results show that respondents with obesity scored a significantly lower Rasch-score than did respondents with overweight in three scales. Respondents with a high education level and having paid work scored significantly higher Rasch-scores in two scales than did those with a low education level and without having paid work. The age group 19–29 have significantly higher Rasch-scores in three scales than respondents in the other two age categories.Overweight and obesity have a considerable impact on the societal costs and on health-related quality of life. The results show that the impact of overweight and obesity go beyond the healthcare sector, as the other costs have the biggest share of the total costs. Another interesting finding of this study is that obesity leads to significant higher costs and lower health-related quality of life than overweight. These findings draw attention to policy making, as collective prevention and effective treatment are needed to reduce this burden.The online version contains supplementary material available at 10.1186/s12889-021-12449-2. Furthermore, research from the Organization for Economic Cooperation and Development (OECD) shows an estimated cost of 172 Euros per capita for treating high BMI (≥ 25 kg/m2) and associated conditions in the OECD countries [Globally, the prevalence of overweight (defined as body mass index (BMI) ≥ 25 kg/m decades . Obesity class 3 . Secondl class 3 , 8. In a class 3 –12. Accoountries . In addiountries . Due to ountries . Other sountries –18.Despite the international studies showing that overweight and obesity have significant impact on the individual, the healthcare system and the society, there is no study in the Netherlands that reflects the total burden, including costs and HRQoL, that obesity and overweight have on the society as a whole. Furthermore, there is no study in the Netherlands that makes a comparison between overweight and obesity. Knowledge about these actual costs and the associated burden is needed to highlight the importance of the problem for policy and research agendas, and thereby stimulate collective prevention and treatment programs . The aimThis is a prevalence based, bottom-up, prospective study focusing on the burden of disease expressed in COI (Euros) and HRQoL (utilities and Rasch-scores) from a societal perspective, overall based on the Dutch guidelines for costing studies in the healthcare sector . The socWhen information was not present in the Dutch guidelines, such as cost information, other sources were used. A numerical code was assigned to each participant as identification, to ensure anonymity of the questionnaire. The results obtained were available only to the researcher and the supervisors.2. Weight and length were asked to respondents, based on this information researchers calculated respondent’s BMI. Participants were recruited in cooperation with Partnerschap Overgewicht Nederland (PON) and the use social media, such as Facebook and overweight/obesity platforms. An informative text was used to inform possible respondents about the background and usefulness of the study and requirements for participation, such as the inclusion criteria. All questionnaires that were finished completely were included in the present study. The volume of the study depended on the willingness of people to participate in the study and fill in the questionnaire. This study is a non-WMO research and is therefore reviewed by the Ethics Review Committee for Health, Medicine and Life Sciences (FHML-REC) of Maastricht University. The FHML-REC has approved the protocol of the study . All methods were carried out in accordance with relevant guidelines and regulations. An informed consent was obtained from the adult individuals with overweight and obesity who wanted to participate in this study before they filled in the questionnaire.Participants in this study were individuals with overweight or obesity. Inclusion criteria were met when individuals were at least 18 years old and when the respondent’s BMI was equal to or higher than 25 kg/mThe study adopted a societal perspective, which incorporates all costs, regardless who incurs them . The COIAll costs related to obesity and overweight were included. To calculate the COI different costing categories were identified. The first category is healthcare costs, defined as medical care expenditures for diagnosis, treatment, rehabilitation, and costs related to the purchase of supporting devices. The second category included the patient and family costs, i.e. transportation costs, household expenditures, clothing and informal cares of any kind . The thiOverweight and obesity have a strong mental component; therefore the Treatment Inventory of Costs in Patients with psychiatric disorders (TIC-P) was used to measure costs. To keep the focus of the questionnaire on overweight and obesity and to make it complete, questions about patient and family costs were incorporated into the questionnaire. These questions elicit information about the expenditures related to the respondent’s weight, such as adapted clothing, gym subscription, diet books, parking permit, food, etc.. The TIC-P gave insight into general information, such as BMI, age, gender, and socio-economic status, and the different types of costs, such as healthcare costs, and costs in other sectors, related to obesity and overweight . In shorThe costs were gathered and calculated in Euros. The valuation of the costs was based on existing costs and cost information derived from the questionnaire. Existing costs, such as costs of medication and outpatient visits, were taken from the Dutch guidelines for costing studies in the healthcare sector . In caseThe HRQoL was measured by means of the standard Dutch version of the five-dimensional, five-level EuroQol (EQ-5D-5L). This method is recommended by the Dutch guidelines . The EQ-Disease-specific quality of life was measured using scales of the BODY-Q, which are related to overweight and obesity. A health-specific questionnaire gives more depth and insight regarding to the quality of life . The BODThe five dimensions of the EQ-5D-5L were summed up into a health state. Utility values can be calculated for these health states. The utility score can be valued between 0 and 1, where 0 indicated death and 1 full health. Utilities corresponding with the measured health states were derived from the Dutch tariffs .The BODY-Q scales can be scored if at least half of the statements are completed; the mean is imputed when there are missing data. For each scale, a raw score was calculated; this is the sum of the levels ranging from 1 to 4. This raw score was computed and converted to a Rasch transformed score, ranging from 0 (lowest) to 100 (highest). Low Rasch scores indicate a low satisfaction with the outcome, whereas higher scores indicate a better outcome. For example, the scale psychological well-being contains 10 statements. When respondents answer all these statements with “totally agree” (score 4), the raw score will be 40, and the Rasch-score will be 100, meaning the best outcome possible.2) and obesity (BMI ≥ 30 kg/m2). In the subgroup “Living situation” a distinction was made between living together (meaning married or living with partner), or living alone, meaning respondents were not living with a partner (were not married nor living together), but were single . Furthermore, there are 2 groups in the subgroup “Level of education”, where respondents could have a low level of education  and or intermediate level of education  – these two levels were in one group - or a high level of education  – the second group. Last, there is a subgroup “Paid work” where respondents either have paid work or do not. The reasons for not having paid work could be that they are unemployed, retired, incapacitated, or other.Subgroup analyses were performed. These subgroups were based on gender, age, BMI, living situation, level of education and work status. The “Age” subgroup is split into 3 groups, namely 19–29, 30–49, and 50+. In the subgroup “BMI” a distinction was made between overweight (BMI ≥ 25 kg/mt-test) was computed. When data was not normally distributed, a nonparametric test (Mann-Whitney U) was computed. A value of p < 0.05 was considered to be a statistically significant difference. Analyses were conducted using SPSS Statistics version 24, except for bootstrapping, which was conducted with Microsoft Office Excel 2016.As cost data are usually skewed and not normally distributed, we had to take into account nonparametric bootstrapping (1000 replications) for all costs categories. The alpha level was set at 0.05 for all cost analyses. The Shapiro-Wilk test was used to test the normality of the HRQoL outcomes. When data was normally distributed, a parametric test (independent Over a time period of 6 months (June – December 2020), 97 individuals filled in the questionnaire. The average age of the population was 43.31 years (SD 13.54) and 79 were female. The average BMI was 33.31 (SD 6.71). Most respondents  were sufTable The average patient and family costs per 6 months were €2018.34 (SD 2538.53). Attempts at weight loss included several methods, including gym subscription, diet books, personal training, adapted diet et cetera. Thirty-four per cent of the respondents indicated that they were absent from work in the past 6 months due to sickness, and 37.1% of respondents indicated that they had physical and/or mental complaints while being present at work. This caused presenteeism. This leads to an average of total other costs of €2259.37 (SD 6141.23). The total societal costs per individual suffering from overweight or obesity in this study are €5731.33 (SD 8238.70) per 6 months, corresponding to €11,462.66 per year.The mean utility of the study population was 0.81 (SD 0.18). The dimension pain/discomfort is slightly affected, with 40.2% having minor problems, 16.5% having moderate problems, and a median of score 2 (minor problems). The majority of the respondents indicated that there were no or minor problems for the other dimensions, shown in Table Table A subgroup analysis was performed for all relevant subgroups, based on gender, age, BMI, living situation, level of education and work status. As our costs were highly skewed, the normality assumption was violated. Therefore, bootstrapping was performed on all subgroups Fig. . AdditioU test was performed to test for significant differences (p < 0.05). Significant lower utility scores were found for respondents with obesity (0.77) in comparison to respondents with overweight (0.86). In addition, respondents in the age group of 19–29 had a significantly higher utility score (0.87) than did respondents in the other age categories (0.79). Furthermore, respondents who worked in paid employment indicated the lowest mean utility score of 0.71. The results are shown in Fig. A subgroup analysis for the utility scores, derived from the EQ-5D-5L, was performed. The utility scores were not normally distributed. The Mann-Whitney t-test was used to test for significant differences. The scales of body image and physical well-being were not normally distributed. For these scales the Mann-Whitney U test was performed to test for significant differences (p < 0.05). The results show that respondents with overweight have a significantly higher Rasch-score than do respondents with obesity in the scales for body image, physical well-being, and sexual well-being. In addition, there is a significant difference in the subgroup “Level of education”; respondents with a low or intermediate level of education have a significantly lower Rasch-score than respondents with higher education in the scales for psychological well-being and social well-being. Furthermore, in the scales for social well-being and sexual well-being, a significant difference is found between respondents who have paid work and those who do not, with a significantly lower Rasch-score in the latter. Last, there are significant differences in the subgroup “Age”. In the scales for body image and sexual well-being, a significantly higher Rasch-score is found for respondents aged 19–29 and 30–49. Also, in the scale for physical well-being, respondents aged 19–29 have a significantly higher Rasch-score in comparison with respondents aged 50 + .Results for the subgroup analysis for the BODY-Q are shown in Fig. This study examined the societal burden of overweight and obesity on the Dutch population in terms of COI and HRQoL. Our COI results show that the average societal costs of people with obesity and overweight are €5731.34 per person over the last 6 months, corresponding to €11,462.66 per year. Of these yearly costs, productivity losses make up the biggest share, namely €4518.7, and the healthcare costs have the lowest share, of €2907.24 per person per year, illustrating that the impact of overweight and obesity is significant beyond the healthcare sector. Our HRQoL results show a mean utility score of 0.81 for our population, derived from the EQ-5D-5L. BODY-Q results show the lowest Rasch-score of 36.37 in the scale for “Body image”. The remaining BODY-Q scales have a Rasch-score between 58.69 and 72.93.2 [In the Netherlands, 50% of the population has a BMI of ≥25 kg/m2 , 33. Our2 . The higResults from analysing the results from the subgroups show that in our study obesity was significantly associated with higher costs; respondents with obesity reported higher healthcare costs, patient and family costs, and significantly higher other costs and total societal costs in comparison with respondents with overweight. There are several causes for these higher costs. There are studies that indicate that obesity leads to higher healthcare costs in comparison with overweight, including costs related to diabetes and heart disease –36. AnalIn addition, level of education was also a significantly associated with higher costs; other costs and total societal costs were significantly higher for respondents with a low or intermediate level of education in comparison with highly educated respondents. Findings of the OECD and the Dutch Central Bureau of Statistics (CBS) indicate that a lower level of education is associated with a higher BMI , 40. In The burden of obesity and overweight on the HRQoL is large if you compare the utility of our population (0.81) with the utility of the Dutch population in general (0.91–0.96), indicating that obesity and overweight carry a high burden for the respondents . In our This study has several strengths. To the best of our knowledge, this is the first study investigating the societal burden of obesity and overweight in the Netherlands, including costs and quality of life. This study distinguishes itself from other studies by including, next to a general quality of life questionnaire, a disease-specific quality of life questionnaire, namely the BODY-Q. Furthermore this study incorporates several costs categories, such as healthcare costs, patient and family costs, and other costs (such as productivity losses). Combining this information leads to a full overview of the total burden overweight and obesity have on the society. This was a prevalence-based, bottom-up study, which means that cost units were collected on an individual level within a specific time period. In comparison to the top-down approach, the bottom-up approach has more informative power . The preThere are some limitations in this study that need to be considered. First, we used retrospective questioning, which can lead to recall bias . To miniLast, in 2019–2020 we lived in the COVID-19 pandemic, which could have an influence on the total costs respondents incurred. In 2020 some non-essential healthcare services were not available. It could be possible that there is an underestimation of the healthcare services used, because individuals simply could not use some of these services. Furthermore, all restaurants were closed during some months in 2020–2021. This could lead to an underestimation of the patient and family costs. Regarding the other costs, it could be possible that respondents were more absent from work due to sickness, because it was not allowed to go to work with a minor cold. However, respondents filled in the questionnaire during June, July and August in 2020, which means that those questionnaires are only slightly affected regarding healthcare costs since only non-essential healthcare services were scaled down for a very short time in May 2020. Regarding patient and family costs, respondents were asked what their average monthly expenses were for dining out and food delivery. With this way of asking, we believe that most respondents did not keep the pandemic in mind and that the outcomes for patient and family costs are not or only slightly affected.There are some implication for policy and clinical practice. First, more attention should be paid to education about the causes and influencing factors on the existence of overweight and obesity, such as a healthy lifestyle, use of medication with a side effect of increasing weight, social economic, hormonal and/or genetic factors etc. at an early age and on all levels of education . The exiSince little research has been done on the burden of overweight and obesity, further research is recommended to increase knowledge on all aspects of this burden. This research should be performed when there is no pandemic. It would be interesting to make a direct comparison with the healthy population and/or other countries in future research. In addition, further exploration of the BODY-Q is needed to make a direct utility comparison with the EQ-5D-5L. Last, the use of the data gathered from this study could be important for economic evaluations in overweight and obesity.This study indicates that overweight and obesity have a considerable impact on the societal costs and HRQoL in the Netherlands. The results show that the impact of overweight and obesity go well beyond the healthcare sector, as the costs of productivity losses have the biggest share of the total societal costs of this disease. Another interesting finding of this study is that obesity leads to significant higher costs and lower HRQoL than overweight. This impact draws attention to policy making, as collective prevention and effective personalized treatment are needed to reduce this burden.Additional file 1. Questionnaire (in Dutch).Additional file 2. Costing prices.Additional file 3. Prescribed medication list.Additional file 4. Over the counter medication list.Additional file 5. Subgroup analysis of healthcare costs.Additional file 6. Subgroup analysis of patient and family costs.Additional file 7. Subgroup analysis of other costs.Additional file 8. Subgroup analysis of total societal costs.Additional file 9. Subgroup analysis utility score derived from the five-dimensional, five-level EuroQol.Additional file 10. Subgroup analysis Rasch-score derived from BODY-Q, scale of psychological well-being. Subgroup analysis Rasch-score derived from BODY-Q, scale of social well-being. Subgroup analysis Rasch-score derived from BODY-Q, scale of body image. Subgroup analysis Rasch-score derived from BODY-Q, scale of physical well-being. Subgroup analysis Rasch-score derived from BODY-Q, scale of sexual well-being.


The global damage that a widespread viral infection can cause is evident from the ongoing COVID-19 pandemic. The importance of virus detection to prevent the spread of viruses has been reaffirmed by the pandemic and the associated social and economic damage. Surface plasmon resonance (SPR) in microscale and localized SPR (LSPR) in nanoscale virus sensing systems are thought to be useful as next-generation detection methods. Many studies have been conducted on ultra-sensitive technologies, especially those based on signal amplification. In some cases, it has been reported that even a low viral load can be measured, indicating that the virus can be detected in patients even in the early stages of the viral infection. These findings corroborate that SPR and LSPR are effective in minimizing false-positives and false-negatives that are prevalent in the existing virus detection techniques. In this review, the methods and signal responses of SPR and LSPR-based virus detection technologies are summarized. Furthermore, this review surveys some of the recent developments reported and discusses the limitations of SPR and LSPR-based virus detection as the next-generation detection technologies. The current COVID-19 pandemic caused by SARS-CoV-2 has revealed how highly infectious emerging viruses spread in modern society and the gravity of social consequences such infections can cause . Viral iPolymerase chain reaction (PCR)-based detection methods form the gold standard methods for virus detection . HoweverOn the surface of a solid material such as a metal with a free charge, the surface charge (mostly electrons) oscillates collectively due to light irradiation. This phenomenon is called surface plasmon resonance (SPR). . SPR-indIn this review, virus detection techniques and their performances based on signal amplification characteristics assessed by SPR and LSPR on a micro/nanoscale basis are explained. SPR uses as a signal the optical changes that occur when a virus is coupled to a sensor that has a prism bonded to a metal film to couple the light. For LSPR, the strong plasmons generated locally on the nanoparticles are used as a signal or as an enhancer for intensity of fluorescence material . In SPR-Metal substrates show strong SPP when irradiated by an appropriate laser . The mosSPR imaging (SPRi) has also been actively studied as a method to apply SPR in biosensing. SPRi is broadly classified into spectral, intensity, phase, and polarization contrast depending on the signal used . The varIn order to effectively utilize the properties of SPR, a method to specifically bind the virus to the metal film is necessary. Antibodies are suitable as trapping materials because they can be modified based on the substrate using chemical cross-linking, and by selecting a structure with high specificity, a highly selective sensor can be easily constructed . Table 13 plaque forming unit (PFU)/mL, 10⁴ copies/mL, and 100 pg/mL 104]. TheMany studies have reported the application of the fluorescence enhancement effect of LSPR for virus detection. Nasrin et al. recently reported a nanocomposite in which the plasmon particles and QDs were combined by chemical cross-linking and modified with antibodies for supplementation, thereby providing reactivity against viruses . The disThe LSPR-based fluorescence virus sensor should have a controlled nanoscale distance between the plasmon particles and fluorescence material. In the case of antigen–antibody reaction, forming a thin film on the plasmon particles beforehand to control the nanometer distance is effective . A typicSPR and LSPR-based virus sensors amplify the signal response of samples at extremely low concentrations, which is otherwise difficult to read and analyze using conventional methods. Commercially available virus detection systems, except PCR, are easy to use and can process multiple samples but have considerable sensitivity limitations, resulting in false negatives. The detection methods based on SPR and LSPR require only virus capturing at the sensor site, rendering the methods simple and highly sensitive. To achieve high sensitivity, the structure and thickness of the thin metal film must be optimized for the SPR-based sensor, and the size and shape of the plasmon particles must be controlled for the LSPR-based sensor. In addition, the SPR-based sensors are witnessing advancements with respect to reusability and miniaturization. As for LSPR, it is easy to construct a simple system to observe the optical response effectively. Collectively, studies on SPR and LSPR-based virus sensing research have already demonstrated sufficient sensitivity.


During the last year, science has been focusing on the research of antivirally active compounds overall after the SARS-CoV-2 pandemic, which caused a great amount of deaths and the downfall of the economy in 2020. Photosynthetic organisms such as microalgae are known to be a reservoir of bioactive secondary metabolites; this feature, coupled with the possibility of achieving very high biomass levels without excessive energetic expenses, make microalgae worthy of attention in the search for new molecules with antiviral effects. In this work, the antiviral effects of microalgae against some common human or animal viruses were considered, focusing our attention on some possible effects against SARS-CoV-2. We summed up the data from the literature on microalgae antiviral compounds, from the most common ones, such as lectins, polysaccharides and photosynthetic pigments, to the less known ones, such as unidentified proteins. We have discussed the effects of a microalgae-based genetic engineering approach against some viral diseases. We have illustrated the potential antiviral benefits of a diet enriched in microalgae. Viruses are the smallest and the most common entities on Earth. Their uniqueness lies in the fact that they are able to multiply only inside the cells of other living things. Viruses are generally considered non-living, since they are made up of a core of genetic material, either DNA or RNA, surrounded by a protective protein coat called a capsid .i) the virus binds to cell receptors on the cell membrane, transferring its genome into the cytoplasm—this process allows the virus to integrate into the host’s genome; (ii) the cells are forced to reproduce the virus genome; (iii) the protein and virion progeny are produced.Viruses have had evolutionary success for four main reasons: a massive genetic variation, a variety in means of transmission, a good mechanism to replicate in the host and the ability to survive in the host  A viral Viruses cause several human infections ; the so-Coronaviridae family. The coronavirus spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) receptors, located on the surfaces of many human cells, allowing virus entry and replication, and consequently the development of the disease [SARS-CoV-2 is a spherical, enveloped single-stranded RNA virus of the  disease . This viThe main weapons to prevent viral diseases are vaccines; for some viruses, such as human immunodeficiency virus (HIV), Zaire ebolavirus (ZEBOV), and Dengue virus (DENV), vaccines are still not available ,8. MoreoSome antiviral compounds have natural origins, and microalgae are among the most interesting candidates for their production. Among marine biomedical compounds, 9% are isolated from algae , and somChlorella pyrenoidosa was shown to have an inhibitory effect in mice against vesicular stomatitis virus (VSV). However, the greatest interest in the antiviral compounds within microalgae spread only in the mid-1990s, when some cyanobacteria, such as Nostoc and Spirulina, showed interesting results against HIV, which was considered a plague in the 1980s [One of the first studies on the antiviral activity of microalgae was carried out by Umezawa et al. : an extrhe 1980s ,19,20,21Recently, the interest in microalgae with antiviral effect has also developed in one of the biggest economic sectors, that of aquaculture and its derivatives . Indeed,In this review, we analyzed the antiviral effects of microalgae against some common human and animal viruses. Moreover, we examined antiviral compounds produced by microalgae, the effect of genetic engineering in this field, and perspectives on a diet enriched in microalgae. We focused our attention on some possible effects against SARS-CoV-2.Among the different strategies to fight viruses, one involves the prevention of the entry of the microbes into the cells by blocking their interaction with the cell membrane. The most famous preventive agents are lectins, proteins of a non-immunoglobulin nature capable of binding carbohydrates without altering the structures of specific glycosyl ligands; for this reason, they are called carbohydrate-binding agents (CBAs) . LectinsThe orientation of CRDs determines the affinity of the carbohydrate structure. Therefore, some lectins only interact with high-mannose polysaccharides, some with glycan branches, and others with the galactose core . LectinsMicroalgae lectins with antiviral activity all originate from cyanobacteria, and all of them have anti-HIV activity .Lentivirus of the Retroviridae family that infects CD4+ cells. This virus induces the lowering of CD4+ cell levels, making the body more vulnerable to all types of infections and causing acquired immunodeficiency syndrome (AIDS). There are two main types of this virus: HIV1, diffused around the world, and HIV2, diffused only in West Africa because of its low transmissibility [HIV is a positive single-stranded enveloped RNA virus of the genus sibility ,27.+-like receptor and the CCR5- or CXCR4-like coreceptor. After this interaction, the gp120 and gp41 peptides (glycosylated trimeric assemblies interacting with lectins) fuse inside the membrane [In more detail, HIV enters the cells thanks to the envelope protein gp120 that engages the CD4membrane ,29 , Microcystis Viridis Lectin (MVL), Microvirin, and Scytovirin . Oscillatoria agardhii strain NIES-204 produces a lot of lectins to support blooms on the surfaces of lakes and ponds; among these, there is Agglutinin OAA (molecular weight 13.9 kDa). This lectin shows anti-HIV1 and anti-HIV 2 activity in vitro, and high mitogenic activity [activity ,31,32,33Nostoc ellipsosporum [CV-N was extracted from sosporum ,35 and csosporum ,38,39,40Different studies have shown that CV-N inhibits the entry into ocular human cells of three viruses: hepatitis C virus (HCV), ZEBOV  and herpHCV is an enveloped positive RNA virus from the Flaviviridae family that replicates on intracellular lipid membranes in hepatocytes, but also in peripheral blood mononuclear cells . ZEBOV is a negative-stranded virus from the Filoviridae family causing a hemorrhagic fever with a high mortality rate . Herpesviridae family causing lithic infections in human mucoepithelial cells, such as fibroblasts and epithelial cells. After the infection, the virus moves to sensory nerve axons and causes latent infection in trigeminal ganglia, thanks to some genes that control the latency-associated transcript. Therefore, it can show up again periodically. The two main forms, HSV1 and HSV2, respectively cause oral and genital herpes [HSV is a double-stranded DNA enveloped virus of the l herpes . the Orthomyxo-viridae family. IAV is the only species of the genus Alphainfluenzavirus, and is categorized into subtypes based on the type of each of the two proteins present on the viral envelope, hemagglutinin (H) and neuramidase (N) (HxNy). IBV is the only species of the genus Betainfluenzavirus [CV-N also contains anti-influenza A-B viruses . They are negative stranded RNA viruses belonging to nzavirus . Virusesnzavirus .Escherichia coli, Streptococcus gordonii, Lactobacillus Jensenii [CV-N binds neuraminidase, opening a gap in the cell membrane . GeneralJensenii ,52,53,54Microcystis aeruginosa PCC7806 in higher quantities in the presence of iron and light stress conditions [Escherichia coli, and has a good anti-HIV activity, helping to avoid syncytium formation. It has a higher safety profile, but it is not possible to achieve a high productivity level of CV-N [Microvirin is an alpha--mannose-specific lectin; it is a protein of 14.7 kDa and shares 33% amino acid identity with CV-N [nditions . General of CV-N ,57.Microcystis viridis [Scytonema varium, and it has a weight of 9.71 kDA and a high affinity with tetrammonose. In vitro, its N-terminal residue of the SD1 domain links to HIV1 [MVL is a protein of 13 kDa containing two carbohydrate-binding sites per monomer. It is produced by the PCC7806 strain of  viridis . As for  viridis ,59,60. K viridis  showed,  to HIV1 ,62. West to HIV1 . In more to HIV1  showed tPolysaccharides originate in a great variety of organisms. Among these substances, sulphate polysaccharides and acidic polysaccharides showed the highest antiviral activities.Sulphate polysaccharides have a high antiviral activity against enveloped viruses. They interact with the positively charged domains of the virus glycoprotein envelope and create a non-reversible complex; as such, they occupy the sites for virus attachment .The activity of sulphate polysaccharides is linked to different factors: the degree of sulphation, molecular weight, the distribution of sulphate in the structure and stereochemistry, the effect of counter cations, and hydrophobic and hydrogen bonding interaction ,66,67,68Spirulina and Porphyridium , which was isolated by Hayashi and Hayashi in 1996 from a hot water extract via cellulose chromatography. Ca-SP showed its activity against HIV1 by interacting with the V3 loop region of the viral envelope, containing gp120, and stopping the syncytium formation between infected cells and non-infected cells [The most famous sulphate polysaccharide from ed cells ,69,70. TLastly, this compound also acts against other enveloped viruses, such as human cytomegalovirus (HCMV), and Mumps virus (MuV), IAV .Herpesviridae family, showing its action against people with compromised immune systems and causing retinitis, pneumonitis, and possible liver failure [Paramyxoviridae family causing pain in the salivary glands [HCMV is a double-stranded DNA virus of the  failure . MuV is y glands ,72.O-hexuronosyl-rhamnose). It has a high antiviral activity against all enveloped viruses thanks to the chelation of calcium ions with a sulphate group [Mass spectrometry showed that Ca-SP is composed of two types of repeating units of disaccharides  and Ectromelia virus (ECTV), but it also has some effect against HSV1. VV is a double-stranded DNA-enveloped virus of the Poxviridae family, and is a constituent of the vaccine that eradicated smallpox and caused smallpox in cattle [Radonic et al.  showed tn cattle . ECTV isn cattle .Arthsospira fusiforme produces a sulphate polysaccharide with interesting anti-HSV1 and HSV2 activity, and is thought to be linked to the presence of phycobiliproteins [proteins .Porphyridium is a red microalga of the Porphyridiophyceae family that, in recent years, has received great attention for its possible biotechnological uses in the biomedical field. This microalga is encapsulated by an envelope of sulphate polysaccharides with antitumor, antibacterial and antiviral activity [activity ,76,77,78Porphyridium sp has high activity against murine leukemia virus (MuLV), a single-stranded RNA-enveloped virus from the Retroviridae family causing cancer in mice [It has been seen that  in mice ,79, and  in mice ,71.Porphyridium cruentum produce some sulphate polysaccharides with high antiviral activity. They have some effects against Varicella zoster (HH3), a double-stranded DNA virus of the Herpesvirus family, and VV [VHS) and death in salmonid fishes [Asfarviridae family with a high mortality rate and causing a hemorrhagic fever, African swine fever (ASF), in domestic pigs [Among the different species, , and VV ,72. In a, and VV , methanod fishes . ASFV istic pigs .Gyrodinium impudium and Cochlodinium polykrikoides.Two dinoflagellates produce some sulphate polysaccharides with antiviral activity: Gyrodinium impudium produces a sulphate polysaccharide rich in galactose and glucose called p-KGO3, and this inhibits IAV until six hours after the viral infection. It has some effects on Encephalomyocarditis virus (EMCV) [Piconaviridae family, which is the causative agent of myocarditis and encephalitis, neurological diseases, reproductive disorders and diabetes in many mammalian species [s (EMCV) , a non-e species .Cochlodinium polykrikoides produces a sulphate polysaccharide composed predominantly of mannose, galactose, glucose and uronic acid, with antiviral effects and no cytotoxicity effect against HIV1, IBV, HSV, MuV and parainfluenza viruses (HPIVs) [Paramyxoviridae family causing severe respiratory disease in children under 5 years of age [ (HPIVs) , which as of age .Navicula directa, produces a sulphate polysaccharide called Naviculan, which is rich in fucose, xylose, galactose, mannose, rhamnose, and has other trace amounts of sugar moieties. It showed its activity in vitro against HSV, IAV and HIV [One type of diatom collected in deep seawater,  and HIV .Chlorella autotrophica have shown some antiviral effects against animal viruses such as VHSV and ASFV [Lastly, sulphate polysaccharides of green microalga and ASFV ,82,83,84Not all the polysaccharides of microalgae showing anti-HSV activity are sulphated, but some of them are acid polysaccharides with carboxyl group, phosphate group or ester group.Nostoc flagelliforme. It shows a high activity against HSV: in the presence of this substance, there is a reduction in gD, a major component of the virion envelope glycoprotein thought to be required for the fusion of the virus [Nostoflan is one of the most famous acidic polysaccharides. It is extracted from the cyanobacterium he virus ,86. Nosthe virus . These aChlorella pyrenoidosa produced an acid polysaccharide with some effects in mice against vesicular stomatitis virus (VSV) [Rhabdoviridae family causing mucosal vesicles and ulcers in the mouths of cattle, horses and pigs [us (VSV) ,86,87,88Microalgae pigments are extensively used in the biomedical field, and several studies show they could be used similarly to antivirals. Among these, the main substances that showed the most interesting results are pheophorbide a, carotenoids, astaxanthin, and phycobiliproteins  .Lingbya and green microalga Dunaliella, in particular the species Dunaliella primolecta, are active against HSV1, and inhibit the cytopathic effect of the virus [Pheoporbide a (PPba) is formed after the dephytylation and demetallation of chlorophyll a . This suhe virus . The preDunaliella salina directly inhibit the plaque formation of Suid herpesvirus (su-HV1), an enveloped DNA virus of the Herpesviridae family, causing fever and high mortality in piglets [Carotenoids are used in different biotechnology fields, and their antiviral action could be both direct and indirect. It has been seen that extracts of  piglets .However, it is also known that carotenoids decrease the harmful effects of some viruses indirectly. Indeed, viruses increase the level of reactive oxygen species (ROS) and reactive nitrogen oxygen (RNS), which inhibit virus replication. However, an excessive production of these substances activates transcription nuclear factor-KB (NF-KB), inducing the jak/stat pathway. Consequently, the cytokine is produced at higher levels, and this could cause a fatal attack by the immune system, called a cytokine storm. This hyperinflammation of the cytokine storm is also the origin of two severe syndromes: acute respiratory distress syndrome (ARDS), causing breathlessness and rapid heart rate, and acute lung injury (ALI), involved in the damage of different tissues. One is the consequence of the other: firstly, the alveolar walls are damaged, and consequently, there is a scarcity of oxygen in other organs .Dunaliella salina inhibits the level of nitric oxide and cytokine; moreover, it downregulates the gene and protein expression of the jak/stat pathway in the presence of pseudorabies virus (PRV), an Alphaherpes virus creating neurological problems in mice.Li et al.  showed tHematococcus pluvialis and some diatoms [Whispovirus , a double-stranded enveloped DNA virus of the Nimaviridae family that causes white spot disease (WSD), a lethal syndrome in penaeid shrimps, and against infectious hematopoietic necrosis virus (IHNV), a single-stranded DNA virus of the Rhabdoviridae family [Furthermore, astaxanthin, a xanthophyll produced at high quantities by  diatoms , showed e family ,73,98Phycobiliproteins are other substances with antiviral activity.Spirulina platenis extracts containing allophycocyanin (APC) were able to delay the RNA synthesis in vitro of Enterovirus 71 (EV71), a single-stranded RNA virus of the Piconaviridae family, causing neurological and cardiovascular disorders [isorders .Spirulina platensis showed anti-IAV activity. Its action is probably linked to the presence of phycocyanin (CPC), which generally downregulates the expression of inflammatory factors caused by viruses [A cold-water extract of  viruses ,101,102.Spirulina or Ankistrodesmus convolotus showed an anti-Epstein Barr virus (EBV) effect, inhibiting some proteins, such as LMP1, EBNA and ZEBRA, linked to the lytic cycle of the virus. This activity is probably linked to unidentified pigments of the phycobiliproteins family [herpesvirus family that is generally asymptomatic, but, in some cases, it leads to lymphoproliferative disorders, such as Burkitt’s lymphoma (BL) and Hodgkin’s lymphoma (HL) [Some methanolic extracts of s family . EBV is oma (HL) .antibioSpirulina maxima produced a peptide called SM that showed antiviral activity against HIV. This peptide inhibits the reverse transcriptase of the virus and p24 antigen production [oduction .Other peptides with antiviral activity are cyclic depsipeptides (CDPs), which are particular peptides wherein one or more amino acids are replaced with a hydroxylated carboxylic acid .Microcystis ichthyoblabe, had a high IAV activity inhibiting the proteins of the virus cycle.These peptides are generally synthesized by non-ribosomal peptides in combination with fatty acids in algae, sponges, and other marine organisms . ZainuddDifferent works in the field of aquaculture showed that proteins can also act against viruses.Baculoviridae family killing the silkworm Bombyx mori, is linked to the presence of some proteins produced by Spirulina platensis. However, the action is not known [Studies using HPLC showed that the antiviral effect against nuclear polyhedrosis virus (NPV), a double-stranded DNA-enveloped virus from the ot known .Nannochloropsis oculata increase α actin activity and immunity system defense, decreasing the mortality linked to Betanodavirus , an RNA non-enveloped virus from the Nodaviridae family causing the nerval necrosis of the humpback grouper [Proteins of  grouper .Geitlerinema sp. strain containing a substance of the flavonoids group showed high anti-HCV activity, reducing the ATPase activity and consequently RNA helicase and virus replication [Flavonoids are potent antivirals  and somelication ,111,112.Haslea ostrearia during blooms. The pigment’s name is derived from the French region Marennes-Olèron, a place rich in this diatom [A pigment with a probable polyphenolic nature that shows interesting antiviral activity is marennine . Water-ss diatom ,116,117.Marennine is produced in high quantities in a photobioreactor in the presence of shear stress , and it Two glycolipids, monogalactosyldiacilglyceride and sulfoquinovosyldiacyglycerol, showed an antiviral effect .Coccomyxa sp., a green microalga containing more than 30% lipids by dry weight [Monogalactosyldiacilglyceride is produced by y weight . This suy weight .Phormidium sp. and Lyngbya sp. It is a glycolipid that is rich in sulfur, is associated with photosynthetic membranes, and has high antiviral activity, in particular against HIV and HSV [Sulfoquinovosyldiacyglycerol is produced by two cyanobacteria,  and HSV ,123. ThiConsequently, this enzyme cannot interact with the negative charge of phosphodiester bonds ,123,124.Significant assistance in the fight against viruses is offered by the genetic engineering techniques widely used in microalgae, considered as model organisms ,126,127 One of these is the recombinant antibodies technique that allows the production of cloning antibodies in a vector, which are then subsequently expressed in a host.Phaeodactylum tricornutum to treat hepatitis B virus (HBV). In the experiment, monoclonal recombinant human antibody CL.4mAb, which acts against the HBV surface proteins, was expressed and assembled in the endoplasmic reticulum of this microorganism, showing a direct effect against the virus in vitro in human cells.Hempel et al. ,129 usedGenetic engineering is used to create vaccines, which are fundamental weapons against viruses.The main strategy used to create vaccines, in particular against Sars-CoV 2, is based on a viral vector replicating or not replicating DNA and RNA .An example is the technique of RNA interference (RNAi), wherein a double-stranded RNA interferes with virus mRNAs.Chlamydomonas reinhardtii, a green microalga, is used as a vector to express double-stranded RNA in yellow head virus (YHV), a single-stranded RNA virus of the Roniviridae family infecting shrimps and prawns. It is given to animals by oral introduction. The organisms treated with these bioengineered microalgae survive the infection of this virus. We have observed that this virus is related to the family of coronaviruses [aviruses .Other interesting strategies derived from microalgae could be used to create vaccines.Chlorella pyrenoidosa via Agrobacterium tumefaciens; serotype-specific antigenic determinants, located on this protein, induce neutralizing antibodies, and confer protection in young chicken against the action of the virus  to contrast IAV, but also as a food additive (approved by GRAS) [It has been shown that the Carrageenan and Chitosan polysaccharides produced by two different macroalgae have some anti SARS-CoV-2 effects ,160,161.by GRAS) ,160,161.Macrocystis Pyrifera, and it is also used as a food additive for cholesterol regulation [Chitosan is produced by gulation ,162,163.Immurella, a mix of extracted polysaccharides from Chlorella, had interesting effects when supplemented into the diets of broilers ,152,164.Alloherpesviridae family causing a high death rate in koi carps, decreases in the presence of a diet rich in Spirulina platensis exopolysaccharides.Reichert  showed tThe SARS-CoV-2 pandemic has demonstrated the necessity of investing in research into new antiviral compounds. Microalgae are considered good candidates to this end. In line with European objectives, they display eco-friendly and eco-sustainable characteristics, produce a high variety of antiviral compounds, and can be used as a supplement in diets without collateral effects. Moreover, these organisms are considered very good candidates for the genetic engineering approach, giving rise to interesting results in recombinant nucleic acid fields that are the basis for new types of vaccines, such as the SARS-CoV-2 vaccines.