To describe a guide for the construction of a laparoscopic trainingsimulator.Step-by-step description of an inexpensive and easy to assemble homemadelaparoscopic training box, capable of simulating the laparoscopicenvironment in its peculiarities to enable technical skills training.The total cost of the materials for the construction of the simulator was US$75.00 (about R$ 250.00 “reais”) and it can be reduced to US$ 60.00 if thebuilder judges that there is no need for internal lighting. The use of realtrocars imposes the same challenges as real surgeries regarding positioning,visibility and limitation of movements.The proposed economical and efficient alternative can contribute to theteaching and practice of laparoscopic surgical technique worldwide,benefiting surgeons and patients. The popularization of laparoscopic surgery since its introduction in the 1980s hasbrought with it the need for surgeons to adapt to this new surgical modality and toacquire the necessary skills to master this technique.–Anatomical knowledge as well as manual dexterity are essentials for laparoscopy,whose main challenges lay on ambidextrous, loss of tactile sensitivity andbi-dimensional images with loss of depth. The fact is that only performing actuallaparoscopic operations the surgeon can become truly competent; however, patientsafety issues, costs, time constraints and logistics, inevitably limit the trainingopportunities for beginners in the operating room,For decades, surgical training was based on the famous “see, do, teach” modelcredited to William Halsted in 1904. Today, with the evolution of minimally invasivesurgeries, basic laparoscopic educators know that surgical skills need to be taughtoutside the operating theater and most training uses a variety of models, such asblack boxes, virtual simulators, animals, and corpses,It is known that standardization, easy reproducibility of exercises, immediatefeedback after each training, with acceptable cost provides benefits on coaching inlaparoscopy, leading to the easy transference of techniques acquired to realsurgeries, culminating in a shorter learning curve and surgeons and patientsbenefitsIn the pursuit of this objective, we have developed at UroScience lab a step-by-stepguide for the construction of a laparoscopic training box, which is inexpensive,easy to assemble and capable of simulating the laparoscopic environment in itspeculiarities to enable the training of novice surgeons and experiencedlaparoscopists.®, bel line). It is important to choose a box that has someresistance to making holes without cracking its structure. Three holes were madewith a 1.1 / 4’ (32 mm) hole saw at the bottom of the box, due to the higherstrength of the bottom compared to the cap. It is important that an expansion ismade to adapt the drill bit of the hole saw avoiding cracks in the boxstructure. We used sequentially drill bits of 3 mm, 5 mm and 6 mm and only thenthe holes were made with the hole saw. These holes will be used for theinstallation of the trocars. Around each of these holes another 8 holes  of 3 mm were made for later fixing, withcotton threads, the EVA (Ethylene Vinyl Acetate) sheets. Two more holes (32 mmwith hole saw) were made in one of the smaller sides of the box, following thesame principles as the others; these holes will be used to install the cameraand the light source. On each of the larger sides a 3 mm hole was made for laterfixing the camera holder. The locations and distances of each of the orificesare shown in We use a transparent plastic box of 15 liters (29.7 cm × 44.0 cm × 19.0 cm), wereglued, with plastic glue, in the side of the trocar's holes, one external andtwo internal. The box itself was used to demarcate the shape and size of thepieces of the original sheet. The first (outer) must be glued in isolation fromthe others to allow the fixation through the 3 mm holes, using 2-0 cottonthreads, by a 40.0 × 1.2 mm (18 G × 1 1/2″) needle . The othIn the inside of the lid a corkboard of 24.0 × 28.5 cm was glued together with asmall piece of EVA to finish the lid, as shown in ® camera, model c270 (HD 720p), attached to the cameraholder with cable tie, was used. The chosen light source was a NSBAO®, flexible table lamp, with 14 LEDs (Light EmittingDiode), 3 levels of intensity and maximum brightness of 90 lumens. The base ofthe luminaire was glued with double-face adhesive tape, reinforced withcyanoacrylate glue (Super Bonder®), on the small side of the box. Theflexible stem was passed behind the camera holder with the LEDs pointing towardthe center of the box. The stem was attached to the top of the box with arubberized cable organizer. The camera and luminaire wires were passedseparately through the previously made holes in the smaller side.A LogitechThree trocars were installed through linear cuts with scalpel blade, in the EVAsheets, in the topography of each of the holes of the box, to facilitate theentry of the trocars. Four rubber organizers were glued on the outside of thesmaller face of the box to organize the camera and luminaire wires. The finalappearance is shown in The total cost of the materials for the construction of the box was US$ 75.00 (aboutR$ 250.00 “reais”) and it can be reduced to US$ 60.00 if the builder judges thatthere is no need for internal lighting.The advantages of our box, besides its low cost, lightness and versatility, are thepossibility to work only with the ambient light which further decreases the cost ofthe box, possibility of recording for analysis of movements after training andpossibility to observe the movements performed by the surgeon, from an outside,three dimension, perspective.The use of real trocars imposes the same challenges as real surgeries regardingpositioning, visibility and limitation of movements.We believe that the possibility of access to more economical and equally efficientalternatives to the current “black boxes”, available in the market, can contributeto the teaching and practice of laparoscopic surgical technique worldwide.–From the 80's, the art of surgery began an unprecedented process of transformation,with minimally invasive surgery assuming an important role in this scenario. Amongits advantages we could mention less postoperative pain, shorter hospital stay,faster return to usual activities, better aesthetic result, significant reduction ofoverall costs, especially considering the period of retirement from work activities,and a lower rate of infections,In 2006, in the United States, only 55% of residency programs had facilities forlaparoscopic training. In 2014, only 73% of the programs in all North America hadteaching in laparoscopic skillsIn Latin America, including Brazil, there is no established teaching and practicingmodel for training laparoscopic skills to be followed, nor validated tools for itsevaluation. Nevertheless, studies have shown that those who practice laparoscopicskills in a simulated environment verify good acceptance and also improvement oftheir technical skillsThe present article presents the instructions to build a homemade laparoscopictraining simulator, with low cost and easy assembly, with potential to improvetechnical skills in the environment of laparoscopy surgery, benefiting surgeons andpatients.


The effect of dual modification of corn starch, including hydrolysis and succinylation, were evaluated through peak viscosity (PV) analysis, differential scanning calorimetry (DSC), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. This dual modification was shown to increase the reaction efficiency (RE) and degree substitution (DS) compared with starches that were not subjected to acid hydrolysis pretreatment with a 44% and 45% increase respectively. After acid hydrolysis pretreatment, the surface of the corn starch granules exhibited exo-erosion and whitish points due to the accumulation of succinyl groups. The peak viscosity was reduced significantly with the acid hydrolysis pretreatment (between 3 and 3.5-fold decrease), which decreased the pasting temperature and peak time to 20 °C and 100 s respectively. In addition, the dual modification of corn starch altered certain thermal properties, including a reduction in the enthalpy of gelatinization (ΔH) and a higher range of gelatinization (around 6 °C), which may effectively improve industrial applications. Modifications on the FTIR spectra indicated that the dual modification affected the starch crystallinity, while the Raman spectra revealed that the dual modification disrupted the short-range molecular order in the starch. Rearrangement and molecular destabilization of the starch components promoted their granular amphiphilic properties. Starch is a polymer that is synthesized in the form of a granule and used as a gelling agent, stabilizer, thickener or emulsifier in the pharmaceutical, food, textile and packaging industries and for bioethanol production ,2,3. CorAcid hydrolysis is one of the commonly used methods for the depolymerization of starch granules to improve certain physicochemical properties, modifying the cooking and gelatinization temperatures that permit obtaining gel of higher strength ; on the For this reason, dual modified starches are used for the encapsulation of food ingredients, however, such modifications has been evaluated by means of chromatographic methods ; but, thSeveral studies have been reported that show that the succinylation process improves starch characteristics for food applications, nevertheless, for improvement of the starch characteristics, several changes on the modification conditions have been tested e.g., temperature, pH, time and starch concentration, with a combined method used in all cases ,18,19. HFew studies have reported the use of FT-Raman and FTIR spectroscopy as tools in the study of the structural changes of the polymeric components that take place in starch granules as a function of hydrolysis and esterification. The goal of this study was to evaluate if the dual modification of corn starch generated the highest difference in their physical and chemical characteristics than a single modification, for future applications as wall material for food encapsulation. For this reason, the present study employed both Raman and FTIR spectroscopy in addition to microscopy and analyses of peak viscosity, thermal properties, and hydrophobicity to investigate the changes occurring in the starch structure and the characteristics resulting from a dual modification (acid hydrolysis and succinylation).Native corn starch (CS) was purchased from the industrial corn company . Hydrochloric acid  and 2-octen-1-yl succinic anhydride  were used for the dual starch modification as is shown in A total of 100 g of corn starch was suspended in a solution of hydrochloric acid at 5% (300 mL) for approximately 5 h at room temperature. Subsequently, the solution was neutralized with NaOH solution 20%). The neutralized solution was washed three times with distilled water, centrifuged at 6000 rpm for 12 min , the supernatant was discarded and the pellet was dried in a forced convection oven  at 40 °C. The dried starch was ground and sieved using a 250 μm mesh. The dried samples were stored for further analysis ). Spectral data were obtained at a wavenumber resolution of 4 cmp ≤ 0.05). Using Origin software v. 8.0  was employed for the data analysis and all assays were performed in triplicate.Quantitative data were expressed as the mean ± standard deviation. Analysis of variance (ANOVA) was performed followed by Tukey’s test with a confidence interval of 95%  and consequently, a higher DS. The acid hydrolysis pretreatment likely generated more available hydroxyl groups in the starch, which enhanced the succinylation process. However, it was previously observed that the substitution by succinyl groups is not uniform in all starch granules due to the compact structure of the starch, for this reason, the OSA groups appear to primarily esterify the glucose at the starch surface ,24.The DS in the starches with dual modification was higher compared with the starches without acid hydrolysis pretreatment . The conditions used in the acid hydrolysis pretreatment resulted in the corn starch granule destabilization and exhibited increased depolymerization increasing polymerization, due to the primary attack of the acid on the amorphous region of the starch granule, where hydroxonium ions hydrolised the glycosidic linkages, consequently a decline in molecular weight of chains was observed . These mAccording to an FDA report, for the modification of starch with OSA, the highest level permitted is around 3%. Considering that the DS of modified starch with OSA, is influenced by the temperature, pH and reaction time, the results obtained in this study (0.0208) were consistent with those reported by other authors who indicated that the DS in modified starch with 3% of OSA, was between 0.0034 and 0.022 ,19,25,26SEM images of corn starches with or without acid hydrolysis or succinylation are shown in When the native corn starch was subjected to the dual modification, the number of whitish points was lower compared with the starch that was subjected to octenyl succinate without acid hydrolysis pretreatment. This finding indicated a higher degree of incorporation of the succinyl groups into the starch, which was a consequence of the surface erosion, fracture of the granule and polymeric decompaction in the starch that resulted from the acid hydrolysis pretreatment . In the The peak viscosity profiles are presented in The effect of acid hydrolysis on decreasing the PV in starch has been reported by other authors who indicated that the dual modification of acid hydrolysis and citric acid substitution generates a lower PV of the starch extracted from yam cultivars .Acid hydrolysis pretreatment improved the succinylation process, increasing the interaction between the succinyl groups and molecular components of the starch. Previous studies have reported that the pasting properties of starches are modified not only by acid hydrolysis but also by the molecular weight and type of the incorporated ester groups, which potentially promotes cross-linking or molecular networks with amylose or amylopectin . Evidentp < 0.05) with respect to native corn starch and octenyl succinate-treated starch without hydrolysis pretreatment. The acid hydrolysis pretreatment of the corn starch influenced the onset temperature (To), peak temperature (Tp), concluding temperature (Tc) and the enthalpy for gelatinization (ΔH).The DSC thermogram is showed in As shown in Tc–To) was observed in the octenyl succinate-treated starches; this increase indicated that the succinyl groups modified the crystalline structure, interfering with the reassociation of the amylose and amylopectin during the gelatinization process. It has been reported that succinyl groups weaken the hydrogen bonding of the amylopectin, leading to an increase in linear segments that ease the absorption of water into the granule, thereby changing the thermal properties [A higher range of gelatinization temperatures  is indicative that the succinyl group was successfully esterified with the starch. According to Ye et al. [−1 and 1730 cm−1) is enhanced when the DS is increased. For this reason, in this study, the signal of these bands in the FTIR spectra of the OSA-starches with an esterification level of 3% was lower than that of OSA-starches with an esterification level of 15%, as reported by Wang et al. [The octenyl succinate-treated starches exhibited one new band in the FTIR spectra at around 1573 cmg et al.  have inde et al. , Miao ete et al.  and Zhane et al. , the intg et al. .−1, which corresponds to the scissor vibrations of –OH from hydration water on the amorphous regions of the starch [−1  for the starch without modification (CS) and the starch with the dual modification (HOSCS), respectively; this change indicated a decrease in the crystallinity [The alteration of the crystallinity was confirmed based on the slight change in the ratio of the band intensities of 1047/1022 cmallinity , apparen−1 and 770 cm−1, which were related to the stretching vibration of C–O–C and C–O–H from glycosidic bonds. This finding confirmed the α–configuration of the glycosidic linkage in the starch [−1 indicated the skeletal modes of the pyranose ring.All starches showed bands around 864 cme starch . A well-The Raman spectra of the starch samples are presented in −1, and a change in the intensity of this band was observed in the starch with dual modification  of corn starch generated alterations on the surface of the corn starch granule, facilitating the succinylation process and thereby increasing the RE above 50%. The succinylation of the corn starch altered its pasting properties, thereby lowering the PV and Δ


The in vitro anticancer activity of SFNs-PTX was tested against CFPAC-1 cancer cells; results demonstrated a very high cytotoxic activity of SFNs-PTX, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL.The aim of this work was to load an anticancer drug, paclitaxel (PTX), on Silk Fibroin Nanoparticles (SFNs) by using an exogenous approach. SFNs were produced, freeze-dried and then loaded with PTX. An exogenous method allowed us to reduce both drug loss and environmental impact. In order to quantify PTX loaded in SFNs, a simple and reliable method using reversed phase liquid chromatography coupled to tandem mass spectrometry (rp-UHPLC-MS/MS) was developed. This methodology was validated by the determination of spiked QC samples in three consecutive days. Good accuracy and precision of the method were obtained, while the intra-day and inter-day precisions were less than 10.3%. For PTX, the limit of quantitation (LOQ) was 5.0 ng/mL. Recovery from the matrix (SFNs-PTX pellets) was calculated  as PTX was entrapped in a new matrix like the polymer silk fibroin-based. This method was successfully applied to determine the encapsulation efficiency (1.00 ± 0.19%) and the nanoparticle loading (0.12 ± 0.02%  Nowadays nanotechnology represents one of the most promising approaches to improve the solubility and the bioavailability of poor soluble drugs . In partThe same approaches could be applied to the traditional nanosystems; actually, most of published papers proposed the addiction of the selected drug during the nanoparticle’ preparation, unfortunately this approach causes the leak of drugs. In case of highly cytotoxic anti-cancer drugs, the active pharmaceutical ingredient (API) must be manipulated in adequate facilities, making the nanosystem preparation complicated. Furthermore, a loss of cytotoxic compounds must be reduced to prevent the dispersion in the environment and the possible consequent operator exposure. Based on this evidence, the exogenous loading of anti-cancer drug on nanoparticles could be advantageous; in fact, the high surface area of nanoparticles could increase the potential loading surface. Moreover, related to the cytotoxic drug manipulation problem, the exogenous approach could provide the preparation of the naked nanoparticles and then to load the API in a controlled environment .For these reasons, we proposed an exogenous approach to load an anti-cancer drug, paclitaxel (PTX), on silk fibroin-based nanoparticles (SFNs).Bombyx mori cocoons, which has been used for many years in biomedical fields thanks to its biocompatible and biodegradable properties [Silk fibroin (SF) is a natural protein, derived from operties ,17,18,19operties ,21,22,23operties ,25. Silkoperties . Overalloperties ,28,29. BOver the past three decades, several analytical methods based on Ultra High Performance Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS/MS) systems were developed for PTX ,33,34,35®), tax-11-en-9-one,5β, 20-epoxy-1,2α, 4,7β, 10β, 13α-hexahydroxy-4,10-diacetate-2-benzoate-13-(α-phenylhippurate) and the internal standard, trofosfamide , were supplied by Nova Chimica  as 40 and 250 mg powder, respectively. Acetonitrile, methanol, and formic acid (HCOOH), all of LC gradient grade were purchased from VWR International Ltd. . Hydrochloric acid 37%  and phosphate buffered saline (PBS) were obtained from VWR International Ltd. . Deionized water was generated from Milli-Q Plus water-purifying system purchased from Millipore . Sodium carbonate, lithium bromide and acetone were purchased from Sigma-Aldrich . Dialysis tubes were obtained from Visking . Eppendorf tubes (15.0 mL), as well as pipette models (from P200 to P5000) were purchased from Eppendorf . Disposable pipette tips were obtained from Rainin Instruments, Woburn, MA, USA. The centrifuge  was used throughout the study to separate impurities from nanoformulation.Reference standard Paclitaxel, PTX,  and then rinsed four times in water. After the drying of the fiber at room temperature, we dissolved the fibroin in 9.3 M LiBr solution  [w/v) was preserved at +4 °C until the use.To obtain the silk fibroin water solution, we started cutting the °C, 4 h) . Fibroinw/v) was added dropwise into an acetone bath, maintained under magnetic stirring, considering a volume ratio silk: acetone of 1:5. After 1 min of magnetic stirring, nanoparticle suspension was dialyzed against distilled water, using cellulose tubes (Cut-off 3–5 kDa), removing all acetone. Obtained SFNs were lyophilized . Three SFNs batches were prepared to determine the mean production process yield .SFNs were prepared as previously reported . BrieflySFNs were characterized in terms of particle size distribution, morphology by scanning electron microscopy (SEM), and physico chemical properties by Fourier Transfrm Infrared (FT-IR).Nanoparticle Tracking Analysis (NTA) was used to evaluate the SFNs size distribution . Each measurement was repeated 6 times (60 s each) . DynamicFor both NTA and DLS analyses, SFNs were suspended in aqueous solution (0.5 mg/mL), sonicated and filtered (0.45 µm) before the analyses in order to eliminate nanoparticle aggregates. Three batches were analyzed to determine the reproducibility of the production method.Nanoparticle morphology was evaluated by SEM analysis . Freeze-dried SFNs were gold-sputter coated under argon and then analyzed. SEM analyses were performed at Arvedi Laboratory, CISRiC , University of Pavia .−1 (resolution 4 cm−1); the IR spectra were recorded in transmittance mode. Each experiment was performed in triplicate.FT-IR spectroscopy was selected to evaluate the secondary structure of silk fibroin in nanoparticles. SFNs spectra were obtained with Spectrum One Perkin-Elmer spectrophotometer  coupled with a MIRacle™ ATR device . We considered the spectral region between 650 and 4000 cmg, 10 min). This washing procedure was repeated twice to eliminate the unbounded PTX and to obtain the final pellet of SFNs-PTX. SFNs subjected to the same procedure, in the absence of PTX, were considered as controls.5 mg of SFNs were rehydrated with 1 mL of PBS for 2 h at 37 °C. After this time, SFNs were co-incubated with PTX , at 37 °C for 2 h under magnetic stirring. SFNs-PTX were diluted in 50 mL PBS and collected by centrifugation . Hydrochloric acid (HCl 37%) was diluted to the working standard conditions. The matrix solution composed of PBS-suspended SFNs, 0.12 M hydrochloric acid and mobile phase (0.1% formic acid in acetonitrile) was used to prepare daily standard calibration curves and quality control samples (QCs). The samples were centrifuged and then filtered through syringe filter devices .v/v) (solvent A) and acetonitrile (solvent B). The starting mobile phase conditions were 95% A and 5% B. Then we applied a linear gradient of mobile phase B that was held for 3 min and then was increased to 60% in 4.0 min. This gradient was held for 2.0 min, followed by a reconstitution of starting conditions. The equilibration process was obtained by using 5% of B for 3.0 min, resulting in a total analysis time of 11 min. UHPLC flow rate was set at 0.4 mL/min and the column temperature to 40°C. The retention times of paclitaxel and IS were 4.5 and 3.5 min, respectively. The concentrations of paclitaxel was expressed as ng/mL of PTX, for the validation procedure, and ng of PTX, for the real samples of SFNs-PTX.A UHPLC system is composed of an Agilent Technologies 1200 series system operating with a degasser, binary pump, and high-performance autosampler (HiP ALS SL+) with a thermostatic column compartment . The chromatographic column was a Zorbax Eclipse plus C8 2.1 × 50 mm, 1.8 m . The mobile phase consisted of 0.1% formic acid in water ; accelerator voltage 7. We used a retention time window of between 2.0 and 6.0 min to adjust the scheduled SRM measurement segment.The analysis was performed with the UHPLC system coupled with a 6460 quadrupole mass spectrometer . Equipment control, data acquisition and analysis were carried out by using an Agilent Mass Hunter workstation. 6460 Agilent and electrospray ionization (ESI) interface was used for MS/MS analysis. To enhance the sensitivity, we selected the Jet Stream technology with a super-heated sheath gas. Ions source parameters for the positive mode were as follows: vaporizer temperature, 300 °C; sheath gas, 11 mL/min with a temperature of 300 °C; nozzle voltage, 500 V and capillary voltage, 3500 V. Nitrogen was selected as nebulizer gas . The PTX working standard solution, at the concentration of 1 µg/mL, was used to obtain the selected reaction monitoring (SRM) transitions of paclitaxel. PTX was quantified using the following transitions: The calibration solutions were prepared starting from the PTX standard stock solutions. The molecule was diluted in methanol (10 µg/mL). Calibration curve and quality control solutions were prepared by a serial dilution of PTX. For the calibration curve, we considered the following working standard solution concentrations: 0.10, 0.25, 0.50, 1.50, 2.50 and 5.00 µg/mL. Quality control solutions (QCs) were 0.20, 1.00 and 2.00 µg/mL. Internal standard was solubilized in methanol (0.2 µg/mL) to obtain the working solution. Solutions were stored at +4 °C in the darkness.For PTX recovery, the stock standard solutions were prepared in mobile phase at the same concentration levels of those prepared in matrix. A mixture, composed of 100 µL of each point of these working standard solutions and 100 µL of internal standard, was vortexed and directly injected in the UHPLC-MS/MS system. During the validation study, we freshly prepared every day seven-point matrix calibration curves. Each calibration standard was obtained adding 100 µL of each working stock solution in methanol to 1 mL of matrix solution to obtain the final calibration curve for PTX with the following concentrations: 5.0, 12.5, 25.0, 75.0, 12.5, 125.0 and 250 ng/mL.The validation study was performed according to “Guidance for industry: bioanalytical method validation, FDA, US Department of Health and Human Services, 2001” .N = 3). The least-squares linear regression equation y = a + bx  was used for the plotting of calibration curves. In order to minimize the deviation between back-calculated values and theoretical concentrations, we chosen the weighting factor. Then, QCs were prepared in a similar way at concentrations within the range of the calibration standards and were analyzed in quadruplicate (n = 4) on each day of the validation study. Three QCs were, therefore, at the concentrations of 10.0, 50.0 and 100.0 ng/mL. The lowest level of quantification (LLOQ) was determined by analyzing spiked samples (n = 4) at the concentration of 5.0 ng/mL. Moreover, each calibration curve included a blank sample (matrix processed without IS) and a zero blank sample (matrx processed with the IS).The linearity of calibration curves was validated for different working days  as measure of precision. The intra- and inter-assay accuracies should respect the recommendation, reported in the international Guidelines (2001) . At eachThe stability of PTX in matrix was assessed by analyzing QC samples during storage and handling. QC samples were frozen overnight at the normal storage temperature (−20 °C) and thawed unassisted at room temperature. Long-term stability was not assessed because the complex SFNs-PTX were revealed to be unstable after the first cycle of the freeze-thaw procedure.w/w) of SFNs-PTX was calculated from the ratio between the total drug content  was determined as percentage ratio between the actual loaded drug and the drug dissolved during SFNs-PTX preparation .50 was determined according to the Reed and Muench formula [The anticancer activity of SFNs-PTX was in vitro tested on a human pancreatic carcinoma cell line (CFPAC-1) according to a previously described procedure ,42. The  formula  as the cn = 3).Desolvation method allowed us to minimize the loss of fibroin during the production process, as demonstrated by a high process yield of 88.56 ± 6.32% , with narrow size distribution (PDI = 0.14), as confirmed by the Zetasizer analyzer. The effect of lyophilization on the SFNs size distribution was evaluated comparing the mean diameter before and after the freeze-drying procedure . The lyophilization causes the nanoparticle aggregation and, to overcome this inconvenience, we sonicated and filtered the nanoparticle suspension before the use.NTA analyses showed a SFNs mean diameter of 115.23 ± 5.11 nm .FT-IR analyses were performed on lyophilized SFNs to evaluate the secondary structure of silk fibroin. Typical bands of crystalline β-sheet domains were observed in the spectra of SFNs, demonstrated that silk fibroin was in its stable conformation. In particular, we identified the bands related to amide I, amide II and amide III at about 1620, 1520 and 1230 cmThe UHPLC-MS/MS method was validated for PTX loaded on silk fibroin-based nanoparticles over a concentration range of 5.0–250 ng/Ml considering six successive linear calibration curves (average correlation coefficients >0.998 in matrix). The average calibration curve equations obtained in mobile phase and in SFN matrix are reported in y mean) that was prepared in SFNs matrix over the three days of the validation procedure.Provided for a good ratio between the slopes, the average recovery percentage of PTX was 76.5% under the optimized experimental conditions (SFNs matrix). As PTX recovery value has been considered satisfactory to release paclitaxel from the silk fibroin nanoparticles, PTX drug free was quantified in real samples (SFNs-PTX) by means of the calibration curve  and over the three days of the validation study. The measured concentrations had precisions included between 3.21 and 8.84. Accuracy was also less than 108.2%. A complete overview of the validation parameters is detailed in The recovery percentages, measured at the three QCs and LOQ levels, were obtained by comparing the integration ratio values for PTX spiked in mobile phase and the same concentrations spiked in SFNs matrix. The integration values and standard deviation associated to the obtained results at the QCs and LOQ levels are summarized in SFNs per HCl 0.12 M were stable at least for 8 h at room temperature in autosampler. SFNs-PTX treated and hydrolyzed with HCl were unstable at 4 °C in dark conditions overnight. Freeze and thaw cycles indicated a loss of more than 25% of the theoretical concentration at QCs levels. These results demonstrated that this type of matrix if used, it is necessary to prepare fresh samples for each analysis.m/z 854.1→286.2 and 854.1→509.3 were chosen for the quantitative and qualitative assessment of PTX, respectively. With the chromatography conditions described, the retention times of PTX and IS were 4.5 and 3.4 min, respectively. Thus, a total run time of 6.0 min was allowed for each injection.The transitions of the mass-to-charge ratio at The representative SRM chromatograms for PTX drug free released by silk fibroin-based nanoparticles are reported in The concentration assessed for PTX drug free in matrix solution was set at 2832.67 ± 580.001 ng/mL  over the concentration range of the calibration curves obtained through the validation process. All samples were diluted before carrying out chromatographic analyses in order to obtain concentration values within the calibration curve; then, these results were properly converted by using the applied dilution factor.w/w  while the loading efficiency reached 1.00 ± 0.19% .Results obtained from validated analytical quantification were used to determine SFNs-PTX drug loading expressed as percentage weight/weight and the loading efficiency percentage. Considering exogenous loading approach allowed us to obtain a drug loading of 0.11 ± 0.023% The in vitro anticancer activity of free PTX, SFNs and SFNs-PTX, tested against CFPAC-1 cancer cells, was expressed as proliferation percentage referred to untreated cells considered 100%. As expected, the unloaded silk fibroin nanoparticles did not produce any pharmacological effects on CFPAC-1 proliferation and the IC50 value was estimated by extrapolation to be higher than 750,000 ± 1500 ng/mL. Free cytotoxic PTX evidenced a dramatic dose dependent inhibition of cancer cell proliferation, confirmed by the IC50 value that of 1.55 ± 1.2 ng/mL. Also, SFNs-PTX expressed a very high activity, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL.10(PTX concentration)+1 was calculated  from the same amount of free PTX  as biocompatible drug delivery systems that are able to load hydrophobic compounds . Here, wSFNs size distribution, morphology and physico-chemical properties confirmed the data previously obtained by other researchers ,25,44,45w/w . Other authors used silk fibroin nanoparticles to encapsulate PTX [w/w. Wu and colleagues [Our exogenous approach for PTX loading on SFNs allowed us to obtain a drug loading of 0.11 ± 0.023% late PTX ,46,47; tlleagues  supposedlleagues . In thisAccording to other authors , we demoFurther investigations will be performed to use a carrier-in-carrier drug delivery system based on the combination of SFNs-PTX and MSC-EVs. The combined use of fibroin nanocarrier and the biological vehicle (EVs) could improve the in vivo efficacy of the final product thanks to both the homing ability of the EVs and the PTX bioavailability and solubility improvement related to the fibroin nanoparticle use.


Human norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis. Vaccine design has been confounded by the antigenic diversity of these viruses and a limited understanding of protective immunity. We reviewed 77 articles published since 1988 describing the isolation, function, and mapping of 307 unique monoclonal antibodies directed against B cell epitopes of human and murine noroviruses representing diverse Genogroups (G). Of these antibodies, 91, 153, 21, and 42 were reported as GI-specific, GII-specific, MNV GV-specific, and G cross-reactive, respectively. Our goal was to reconstruct the antigenic topology of noroviruses in relationship to mapped epitopes with potential for therapeutic use or inclusion in universal vaccines. Furthermore, we reviewed seven published studies of norovirus T cell epitopes that identified 18 unique peptide sequences with CD4- or CD8-stimulating activity. Both the protruding (P) and shell (S) domains of the major capsid protein VP1 contained B and T cell epitopes, with the majority of neutralizing and HBGA-blocking B cell epitopes mapping in or proximal to the surface-exposed P2 region of the P domain. The majority of broadly reactive B and T cell epitopes mapped to the S and P1 arm of the P domain. Taken together, this atlas of mapped B and T cell epitopes offers insight into the promises and challenges of designing universal vaccines and immunotherapy for the noroviruses. Human norovirus (HuNoV) is a major cause of acute gastroenteritis and has emerged as the leading cause of severe childhood diarrhea in populations vaccinated against rotavirus ,2. NorovNorovirus disease is associated with a higher risk of life-threatening dehydration in the young and old, immunocompromised individuals, and those with limited access to health care ,9,10,11.Several factors have hindered the development of broadly protective HuNoV vaccines and treatment, including the marked antigenic diversity of norovirus strains and an incomplete understanding of protective immunity . This reCaliciviridae. They are small (30–40 nm), nonenveloped viruses with a single-stranded, positive sense RNA genome that is organized into three open reading frames (ORFs) (Noroviruses are classified in the family s (ORFs) A. ORF1 eThe major structural capsid protein, VP1, and the minor structural protein, VP2, are encoded by ORF2 and ORF3, respectively . VP1 monNorovirus and has been assigned genotype GI.1. VP1 sequences can vary up to 60% between genogroups and up to 30% between genotypes, but this genetic diversity has not yet been correlated with a classical serotyping system based on neutralization. In addition to genotypic diversity, noroviruses, like other RNA viruses, undergo error-prone replication, which results in the generation of highly diverse viral RNA populations [Noroviruses are classified into seven genogroups (GI though GVII), which are further subdivided into more than 40 genotypes ,24,25 FC. Norwalulations ,27. ThisMost human norovirus infections are caused by GI or GII genotypes, with fewer cases of GIV. The majority of HuNoV outbreaks and sporadic illnesses over the past few decades have been caused by a single genotype, GII.4 ,29,30,31Difficulties in the efficiency of cell culture systems and animal models have historically limited an in-depth analysis of HuNoV immunity and viral escape . Murine Interferons (IFNs) are released by infected cells during the initial innate immune response and play a key role in limiting MNV replication ,52,53. HHumoral immunity supplements the primary Th1 response and plays a critical role in MNV clearance and protection from subsequent infection. Mice incapable of expressing MHC II could not mount a protective immune response, indicating that Th2 responses, which are responsible for full B cell activation and maturation, were also necessary for controlling MNV infection . MNV-speAlthough it has been reported that MNV and HuNoV share common features in structure and replication ,66, it hDuring human norovirus infection, monocyte recruitment and cytokine activation have been detected ,74,75,76Humoral immunity to HuNoV has been studied in far more depth, and is considered stronger and more long-lasting than T cell immunity ,80,81,82Virus-like particles (VLPs) have largely informed immunological studies of HuNoV. VLPs are replication-deficient, empty capsids that are self-assembling and antigenically indistinguishable from live virus ,92. NoroModified VLPs have been used extensively to map epitopes via site-directed mutagenesis, P2 domain swaps, and chimeric VP1 proteins ,105,106.Monoclonal antibodies (mAbs) have become essential tools for defining immunologically relevant norovirus epitopes. The majority of norovirus mAbs (228 mAbs) have been isolated from naïve animals initially hyperimmunized with a single VLP or live strain and followed with various boosting and adjuvant regimens. The antigens and strategies used for screening or panning antibodies play important roles in the selection process. Immunizations and screens with GI VLPs, primarily NV, yielded mAbs that predominantly bound homotypic VLPs . LimitedNatural HuNoV infections stimulate polyclonal IgA, IgG, and IgM responses . Though The tissue distribution of an antibody in vivo is arguably the most important attribute in determining protective potency . TherefoNewer antibody technologies have also proven useful in defining norovirus epitopes. Nanobodies (VHHs) are ~15 kDa, single-polypeptide chain antibodies derived from the unique variable heavy chain domains of camelid antibodies A. ImmuniNorovirus mAbs are subjected to a number of standard assays in their initial characterization such as ELISA and western blot analysis on denatured antigen. These assays help identify reactive regions and their respective conformations. Of the 307 mAbs reviewed here, 77 recognize linear epitopes that map to the S and P1 domains of both GI and GII genotypes . Though fut2, is responsible for HBGA expression on cell surfaces and the secretion of soluble HBGAs in humans [fut2 are designated as “secretor-positive” and tend to be more susceptible to HuNoV infection [The majority of monoclonal antibodies have been selected to define HuNoV epitopes that are likely immunodominant, conformational, and neutralizing. In the absence of a cell-based neutralization assay for many years, surrogate neutralization assays were developed. While a proteinaceous receptor for HuNoV has not been identified, as it has for MNV , HuNoV Vn humans ,122,138.nfection ,140,141.nfection ,143,144.nfection ,22,141. One of the first HuNoV surrogate neutralization assays measured hemagglutination inhibition (HAI)  Figure B. HAI anAntibody neutralization is traditionally defined with in vitro or in vivo studies and inhibition of live virus infections. Neutralization activity of NV mAbs in the chimpanzee model was shown by pre-incubation of infectious virus with antibodies prior to challenge, but this animal model is no longer available ,88. For Mapping of conformational mAb binding sites has been conducted using various mutagenized or chimeric HuNoV VLPs in HBGA blockade assays. By monitoring VP1 residue changes that result in the loss of blockade activity, a minimal binding epitope for an mAb can be assigned. GII.4 epitopes have been mapped with VLPs that represent naturally-circulating pandemic strains, as well as those from immunocompromised patients with chronic norovirus infection ,10. ChroStructural studies have modeled the precise interactions between mAbs and their epitopes in VP1 ,156,157.Blockade mAbs have been correlated with reduction in the severity of gastroenteritis in challenged individuals and therefore likely play a critical role in HuNoV immunity . The bloThe majority of HBGA blockade epitopes are conformational and include residues that are within or proximal to HBGA binding sites. Intuitively, direct binding of a mAb to the HBGA binding pocket would prevent viral interaction with HBGAs. Monoclonal antibodies that bind residues immediately adjacent to an HBGA binding site could prevent ligand interaction via steric hindrance by the bulky antibody itself. Antigenic drift in the P domain can overcome both blockade mechanisms by change in the mAb epitope sequence. Over years of selective pressure, amino acids surrounding an HBGA binding domain tend to undergo more antigenic drift than the binding pockets themselves . This reThe flexibility of the norovirus capsid likely complements antigenic drift in the evasion of humoral immunity. Conserved epitopes found buried within the VLP structure are often occluded at room temperature and become exposed at 37 °C  Figure C. OcclusParticle flexibility can be both useful and detrimental to mAb HBGA blockade. mAbs can confer blockade indirectly by binding residues distant from the HBGA binding pocket that result in allosteric changes to the P domain ,161. HowOf the 91 mAbs that demonstrate GI-specific reactivity, 62 have been tested for HBGA blockade . Most GIGenogroup I nanobodies have recently been generated and mapped to their corresponding epitopes. Nano-7 and Nano-64 bound the GI.1 dimer interface, while Nano-94 bound an epitope shared with mAb 54.6 A. Nano-7Several blockade epitopes have been successfully characterized for genotypes GII.2, GII.4, and GII.10 . A majorThe hypervariable Epitope A  has demonstrated significant immunodominance compared to other GII.4 blockade epitopes. Although a GII.4 virion is predicted to have the same number of Epitopes A, E, F, and G, ~40% of serum blockade responses target Epitope A . SimilarEpitope D  mediates the binding of both mAbs and HBGAs directly. Like Epitope A, Epitope D faces the exterior of the VLP in the P2 domain and is unaffected by particle conformation caused by changes to the NERK domain . ResidueEpitope E  maps to loops T and U in the P domain and is lateral to Epitopes A and D on the outermost surface of the VLP. However, Epitope E is less surface exposed than either Epitope A or D . ResidueSimilarly, access to Epitope F (residues 327 and 404) is temperature-dependent and can be influenced by mutations in the NERK domain . EpitopeThe remaining lettered GII.4 epitopes have been mapped with a single mAb, yet their exact epitope residues are uncharacterized. Epitope G is hypothesized to be nearby, if not overlapping with, Epitope F . mAb GIISeveral mAbs block GII.4 HBGA binding at epitopes not included in this lettered system. VHH M6, for example, exhibited cross-reactivity to 7 different GII genotypes as well as blockade against GII.4 MD145 VLPs. Unlike other HBGA blockade mAbs, M6 mapped to a linear epitope located on the C-terminal P1 domain . The mecMurine norovirus replicates efficiently in cell culture and classical viral neutralization assays such as plaque reduction can be applied to the analysis of antibodies. The MNV receptor CD300lf  is a celA repertoire of mAbs has been generated against MNV 21 mAbs, . The earWith the very recent establishment of the intestinal enteroid system, the majority of HuNoV blockade mAbs have not yet been validated for neutralization in vitro. However, a study in 2018 generated and characterized 25 IgG and IgA mAbs from naturally-primed human PBMCs . Twenty Antigenic diversity is the hallmark of the noroviruses. However, the maintenance of structural integrity and key capsid functions such as receptor binding undoubtedly require certain sequences to remain constant over time. For human noroviruses, the ability to target conserved epitopes buried within the VLP structure offers insight into mechanisms of antibody-mediated neutralization involving such conserved sequences. Nanobodies in particular have begun to define these mechanisms. Depending on the VHH treatment, nanobody binding has resulted in the aggregation, disassembly, and structural rearrangement of VLPs from several HuNoV genotypes ,130. ThuSeveral mechanisms of particle reorganization have been characterized using VHHs. Treatment with GII.10-specific Nano-32 produced large aggregates of apparently intact VLPs when viewed under EM. Nano-32 binding induced a conformational rearrangement of several P domain loops, thereby altering the hydrophobic landscape of the P domain surface . Nano-94Antigenic variation of HuNoV has clearly challenged humoral immunity in the establishment of long-lasting cross-protection. Similarly, HuNoV diversity may also confound T cell-mediated immunity. Human challenge studies found evidence of cross-reactive T cell responses to heterologous VLPs in the form of an increase in IFN-γ ,78. HoweT cells interact with MHC proteins on infected cells or professional antigen-presenting cells (APCs), which present short peptides from digested intracellular proteins. While a single antigen-presenting cell may present many peptides simultaneously on various MHCs, T cell recognition is restricted to a single peptide. The antigenic diversity of norovirus and the relative infrequency of conserved amino acid sequences may directly limit the ability of T cells to establish a cross-reactive memory response. Additionally, it has been suggested that MHCs may preferentially present peptides that are not conserved between different genotypes . This, iThere have been only a few T cell epitopes identified for norovirus . Most T Human T cell epitopes have recently been identified . A totalToday, most norovirus diagnostic assays are nucleic acid-based with multiplex panels and genome sequencing . HoweverSeveral IgGs and scFvs have been generated that may expand the possible genotypes that can be detected in immunoassays, including NS14, NS22, and HJT-R3-A9 . In factNorovirus mAbs have promise for use in immunoprophylaxis and treatment. Passive antibody therapy with HuNoV mAbs could provide immediate protection for individuals at risk for infection, such as transplant recipients and those exposed during outbreaks ,197. TheThe therapeutic potential of the current repertoire of HuNoV mAbs is variable, and some would require humanization by genetic engineering. Protective HuNoV mAbs produced in non-primate animals, including mice and chickens, cannot be directly administered to humans due to allergy or hypersensitivity concerns ,199,200.Therapeutics based on scFv and VHH circumvent the need for humanization of Fc domains. Moreover, VHH coding sequences can be engineered into any human immunoglobulin scaffold ,204, andAdministration of pooled immunoglobulins from healthy donors has been shown to clear chronic infections in some immunocompromised patients ,211,212.Early adult challenge studies reported that norovirus immunity was predominantly homologous and lasted from six months to two years post-infection ,217,218.An effective HuNoV vaccine would ideally induce long-lasting protection against a wide range of genotypes. Polyclonal cross-blockade antibody responses can be observed following natural infection and artificial challenge, even against highly variant GII.4 strains ,180,181.Currently, numerous monovalent and multivalent vaccines against HuNoV have been developed, with several progressing into phase I/II clinical trials ,229,230.Primary infections with HuNoV were initially thought to cause “original antigenic sin” (OAS). This theory asserts that an individual’s first HuNoV strain exposure would predetermine dominant immune responses to subsequent HuNoV infections, and evidence for such an effect has been observed in a young child . HoweverAlternatively, this memory response can work with vaccination to induce cross-protective responses against highly variable, immunodominant epitopes. Though the antigenic variation of immunodominant epitopes is high, a vaccination study noted evidence of polyclonal serum antibodies possibly containing a rare class of cross-reactive Epitope A-binding antibodies . By repeCurrent vaccination efforts report a reduction in disease severity, but varied degrees of protection from reinfection ,147,227.Several vaccination studies have measured evidence of T cell stimulation through the increased levels of IFN- γ, IL-4, and activated CD4+ and CD8+ T cells ,231,238.Decades of humoral immune studies have addressed the complex antigenic diversity of the noroviruses. Monoclonal antibodies have played a key role in defining regions of interest in the major capsid protein that may be therapeutically targetable. It is now confirmed that certain HuNoV mAbs mapping to the P domain of VP1 have both HBGA blockade and neutralization activity, an important validation of the surrogate carbohydrate blocking assays. These functional epitopes are consistently conformation-dependent, an important structural feature to consider in protein engineering or reverse engineering of vaccine antigens. Insight has been gained into mechanisms by which antibodies neutralize beyond blocking attachment to cells, including particle aggregation or disassembly. Additionally, the discovery of temperature-sensitive epitopes has provided insight into the dynamic nature of norovirus capsids, a structural feature that could potentially be harnessed to gain access to hidden functional epitopes. Such knowledge will be helpful in the design of antibodies to treat or prevent norovirus infection, and in profiling the humoral immune response to candidate vaccines. Importantly, an understanding of how antibodies function can inform the selective immune pressures that drive antigenic diversity and escape from herd immunity.Epitope mapping has now established that there are conserved B and T cell epitopes among noroviruses, despite the genetic diversity. The role of these cross-reactive epitopes in protective immunity is not yet known. Interestingly, both conserved B and T cell epitopes map to the S and C-terminal P1 domain. While B cells and the antibodies they secrete contribute to protection and control of norovirus infection, the role of T cells should also be considered in achieving long-lasting and cross-genotypic protection. Given the relative abundance of antibody mapping studies, further mapping of functional T cell epitopes is needed. The stimulation of lasting immunity at the intestinal mucosal surfaces may prove challenging for the noroviruses. Knowledge of norovirus B and T cell epitopes will undoubtedly continue to play an important role in meeting this challenge.