This research explains how attributes perceived during the festival celebration generate loyalty in terms of satisfaction. As regard, tangible aspects  and intangible aspects (escapism and education) shall be differentiated. A theoretical model is proposed, which explains the effects of festival attributes on satisfaction and loyalty through structural equation modelling. The model was estimated with a sample of 440 people attending Weekend Beach Festival in Spain. The research proves the relationship between attributes and loyalty through satisfaction as a moderating variable; likewise, tangible attributes are deemed to have a greater influence on loyalty, specifically, the aesthetic/environment experience. Nowadays, festivals are considered to be a fundamental pillar within the tourism industry. Festivals are valued, among other characteristics, for their ability to create an image in destinations and for being a tourist offering itself . AccordiAcademic interest on this topic is global, therefore studies can be found in any region around the world, whether in Europe , the MidIn recent years, research has focused on the analysis of visitors’ characteristics: the segmentation of attendees , their bMany authors define festival tourism as an experience product and a tourist offering made up of experiential aspects , 11. On Some studies which analyse festival-goers’ loyalty highlight that festival’s characteristics influence loyalty through festival experience , 15; otha future festival study could use structural equation modelling to test the hypothesis that tangible attributes influence loyalty through attendee satisfaction, whereas intangible attributes influence loyalty directly (p.216). Thus, we apply structural equation modelling to find out the impact of tangible and intangible attributes on satisfaction and loyalty based on experience.The aim of this study is to demonstrate the effect of the tangibility and intangibility of attributes on loyalty through satisfaction in festival tourism, bearing in mind the distinctive value of attributes . This reLikewise, this research is aimed at increasing knowledge on festival attendees’ perceptions and obtaining useful results for festival managers. We believe that a better analysis of the experiences and attributes of festivals clearly influence a more suitable management of these events. These two above-mentioned elements help to design the offer, distribution and sale of festivals, and allow a better understanding of the behaviour and evolution of the market, and explain the role of satisfaction and loyalty as essential elements in the competitiveness of festivals.To this end, a structural equation model shall be used in order to discover the specific weight of each of the attributes of a festival and their relation with the variables of satisfaction and loyalty. The research is conducted on the basis of a survey on 440 attendees of the music festival Weekend Beach Festival in 2018 (WBF-2018), located on the Costa del Sol, in Andalusia (Spain).Consumers have experienced a process that pursues the satisfaction of psychological needs, such as inspiration, authenticity and sense of belonging to a community . It is aMany studies have been conducted on festivals, with several of them focusing on the aspects and attributes that influence on these events.  analysedOne of the first definitions of experience was described as “a subjective mental state felt by visitors during service delivery” .  understa, 21 propHaving presented the classification of festival studies according to the type of attribute and their connection with satisfaction and loyalty, studies relating to the hypothesis proposed in this research are analysed, linking experiences with tangible and intangible attributes and thus establishing a connection between these elements. In the following two sections experiences and attributes are related in accordance with One of the key aspects to understand the role of experience in the analysis of festivals is entertainment, which is a crucial aspect for satisfaction and loyalty. The entertainment experience in the festival context occurs when people passively watch other peoples’ performances . The attAn example of entertainment analysis as an economic experience is the study conducted by . This stH1Entertainment will significantly predict satisfaction with the festival.servicescape and festivalescape. The first term is defined as the environment or influence on consumer’s feelings and behaviour  co coservicperceive  highlighquently,  verifiedRegarding aesthetics and environment,  find autSouvenir shops and food stalls have a relevant role at festivals, leading to various studies , 29. At The aesthetic experience attribute is found in the authenticity and properties that are discovered in the town in which the festival is held . At a cuH2The festival aesthetics will significantly predict satisfaction with the festival.The educational dimension is considered as the participants’ need to know, to experience new skills and abilities that may help intellectual and physical growth  12]. Ed. Ed12]. H3The educational experience will significantly predict satisfaction with the festival.Escapism is defined as the participants’ desire to get out of their routine where they are often stuck in, to escape reality, to live new experiences that change their routine  67]. In. In67]. Attendees’ pleasure or hedonism was evaluated using phrases such as: the customer was satisfied; they appreciated the feeling of escapism and enjoyed the experience . The folH4The escapism experience will significantly predict satisfaction with the festival.an evaluation based on the global purchase and consumption experience of a good or service in time”; subsequent studies broadened knowledge on satisfaction, concluding that two precedents existed: the satisfaction of properties and information. Satisfaction of properties is explained as “a subjective satisfaction judgement resulting from observations of the attribute performance” (p.17) and the satisfaction of information as “a subjective satisfaction criteria of the information used when choosing a product” (p.18). Satisfaction has not been included as an attribute in previous studies, although it has been mentioned as a key word along with festivals [According to (p.54) , satisfaestivals  or visitestivals .Regarding the experience,  researchdefined loyalty as a strong commitment to buy back or endorse a consistent product or service again in the future, leading to repeat purchases of the same brand, despite influences and marketing efforts that can potentially cause a disruption and behaviour change (p. 34). According to this author, loyalty is a multidimensional concept that develops in several stages of loyalty: affective loyalty, cognitive loyalty, action loyalty and conative loyalty [ defined  loyalty . Loyalty loyalty . Usually loyalty , 74. In  loyalty  scale, rOther authors consider loyalty in festival tourism as the predisposition of users to repeat certain behaviour , which iSome studies measure loyalty as future expenses; for example, purchasing wine after attending an Australian wine festival  or the iIn other research conducted on festivals, satisfaction is a positive precedent towards loyalty , 81; morH5The festival satisfaction will significantly predict loyalty with the festival.2 enclosure with three stages just metres from the beach, hosting current national and international artists. The festival was held for four days in July and reached a record figure of 140,000 attendees in 2008; the growth experienced over recent years has been a breathtaking 42% in comparison to 2015. It is currently number 11 in the official Spanish festival ranking according to the Social Network Festival Awards 2018. The tourists’ staying in the town has increased each year until it reached 100% capacity during its celebration over recent years. 20% of attendees are locals and 80% of them come from the rest of Spain and abroad.The study was carried out on the “Weekend Beach Festival (WBF)” held on the Costa del Sol, Torre del Mar, Spain. The festival was first held in 2014 and was one of the most cutting-edge tourism proposals on the coast of Malaga. The festival had a 67,000 mInformation gathering was carried out using a structured questionnaire. It was handed out personally in bars, establishments and streets in the town of Torre del Mar, Malaga, the city in which the festival was held. A two-stage method was used to choose the sample. In the first step, the method for selecting the sample was proportional and stratified according to the attendees’ origin. In the second stage, the respondents were selected in a non-probabilistic way for convenience. This method is suitable when it is difficult to find the participants and it is equally valid . Prior tFieldwork was carried out from June to July 2018, achieving 440 valid questionnaires, with a sampling error of 4.7% and a confidence level of 95%. The response rate was 89%. The research we have carried out has been based on anonymous surveys that respect ethical and analytical standards in the field of social sciences and do not require the prior approval of an ethical committee, in accordance with national and European legislation.The questionnaire does not ask about sensitive social aspects such as race, religion, sexual orientation, diseases, etc. The items are focused on the analysis of the analysis of the analysed phenomenon . The funding source has not requested the analysis of any particular group. All authors respect the rules of confidentiality and ethics regarding the analysis of data and results, in accordance with national, European standards and international agreements.In order to have a greater representation of data, a multi-stage sampling by quotas was carried out based on the gender . With reThe nature of the causal links is reflective , 86, becStatistical analysis of the data is based on a structural equation model (SEM) to identify the specific weight of each of the attributes and their relationship with the two variables: satisfaction and loyalty. For the analysis of the model being studied, the recommendations made by  were folIn the following tables, the psychometric properties are evaluated using the main measures of reliability, validity and goodness and the matrix of correlations between factors.First, in order to assess common method bias, we applied Harman’s single factor method –93. The In 2 (480.29). However, this is not considered to be a limitation, since it is very sensitive to the sample size [The amount of variance that each construct captures from its indicators (AVE) is higher than the variance that said construct shares with other constructs in the model; therefore, the discriminating validity of the measurement model is confirmed . Table 5ple size  and, freple size .2) has been calculated, and the results obtained are 0.39 for satisfaction and 0.14 for loyalty. It can be observed that all of the causal relationships are statistically significant (H1 and H5 are supported). In this sense, it can be stated that a positive causal relationship exists between entertainment experience and satisfaction (β = 0.2816), aesthetics experience and satisfaction (β = 0.3675), education experience and satisfaction (β = 0.1086), escapism experience and satisfaction (β = 0.1607) and, finally, between satisfaction and loyalty (β = 0.1832). Therefore, the results show a direct relationship between the different experiences and satisfaction, as well as satisfaction and loyalty. The most intense of the causal relationships between perceived destination attributes and satisfaction occurs between the aesthetics experience and satisfaction, followed by the entertainment experience, escapism and, finally, the education experience. This SEM analysis provides empirical evidence that tangible attributes, such as entertainment and aesthetics, have a greater influence on satisfaction and loyalty than intangible attributes (education and escapism). To analyse the predictive capacity of the model, the coefficient of determination ;;Fig 2); The practical implications of this research highlight that event managers should prioritise entertainment experience and aesthetics experience, because they are the attributes that have the highest influence on the satisfaction and loyalty of festival goers. Moreover, these managers should not forget tangible aspects such as education experience and escapism experience, because although they influence to a lesser extent, they also present a remarkable causal relationship with satisfaction and loyalty. This is in line with other authors who have studied the relationship and satisfaction through attributes , 74. TheWhen conducting this research, we came across some limitations which are discussed as follows. The festival participants studied are attendees to a current music festival  and, consequently, the data obtained may not be applicable to other festivals with different characteristics. With regards to creating the analysis scale, the results of the study indicate that tangible and intangible attributes only account for experiences. It would be appropriate for future studies to investigate other aspects such as emotions and experienced memories, which may be important for the analysis of affective variables. Another limitation of this study is the lack of attention paid to the interaction that visitors showed at the festival, emotional solidarity playing an important role in festival tourism, as described by the authors , 105.Likewise, no control effect has been included in the model for this study. Future research should include some control variables such as service quality, perceived value and any other variable that is considered crucial to explain the model. Lastly, research on festivals could focus on other aspects of interest such as the change in perception of festivals by new generations. In future research, emotional solidarity in festival tourism will be taken into account as a key element to better explain the behaviour of visitors and their interaction with the destination , 105.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Borreliaburgdorferi sensulato (s.I.) complex, the causative agent of Lyme borreliosis (LB), has been reported in tick-abundant areas worldwide, while no data have been reported in Greece. The aim of the study was to identify the hematological alterations in sheep with seropositivity against Borrelia burgdorferi (s.I.). Blood samples were obtained from 318 tick infested sheep for blood analysis and serological determination of IgG and IgM antibodies against B. burgdorferi by indirect immunofluorescence antibody (IFA) assay after exclusion of endo-ectoparasites and other tick-borne infections. A total number of 162 sheep met the inclusion criteria, allocated in four groups based on the presence or absence of IgG and/or IgM; sheep found negative for IgM and IgG (Group A), positive for IgM (Group B), positive for both IgM and IgG (Group C) and positive for IgG (Group D). Anemia, thrombocytopenia and normal or decreased leukocyte count, mainly due to lymphopenia were the main hematological features observed in seropositive sheep. The presence of these features raises the suspicion of Borrelia infection in tick infested sheep. The seropositivity of 23.58% in sheep raises concerns of Borrelia circulation, especially in rural areas and potential risk of transmission to humans.Exposure of sheep to  Borrelia burgdorferi sensu lato (s.I.) complex, helical-shaped bacteria belonging to the spirochaete phylum [Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii are of public health importance [Ixodes. In Europe the tick species of concern is Ixodes ricinus, while ticks of Ixodes scapularis and Ixodes pacificus species [Lyme disease or Lyme borreliosis (LB) is a tick-borne disease with zoonotic potential. The etiologic agent is e phylum . The come phylum , while mportance ,4. Trans species  are the  species .Ixodes , Rhipicephalus , Hyalomma (H. m. marginatum), Boophilus (B. annulatus) and Dermacentor (D. marginatus) [D. marginatus, Haemaphysalis parva, H. sulcata, H. punctata, I. gibbosus and R. sanguineus s.l. [I. gibbosus, R. sanguineus, D. marginatus, H. parva and H. sulcata [Borrelia burgdorferi s.l. (B.afzelii and B. garinii) has been reported in Ixodes spp. ticks collected only from birds in Greece [Ticks infesting animals have been reported in different studies in Greece. In a study, ticks collected from goats, sheep, cattle and dogs belonged to five genera: ginatus) . Moreoveeus s.l. . In anot sulcata . Howevern Greece , to the B. burgdorferi, whilst some of them, such as rodents, insectivores, hares and deer, act as an important reservoir. Additionally, migratory birds are major participants of the transmission cycle, spreading the infected ticks into new territories [Borrelia species, host and altered environment due to the climatic change. This reciprocal relationship between ecology and society has contributed to the emergence of new diseases as well the re-emerge of known ones to new geographical regions.Several mammalian species can be infected by ritories . Life anritories . Howeverritories . From thVector-borne diseases represent the 17% of the total human infectious diseases, leading to more than 700,000 deaths globally every year . LB is eB. burgdorferi in Greece, one was reported in 1985 concerning only 1 seropositive individual and in two years later, two seropositives in the island of Crete [B. burgdorferi antibodies was 1.1% in 1997 [In Greece, there are limited data on the prevalence of LB in humans . Among tof Crete . In the  in 1997  and 3.3% in 1997 . B. burgdorferi antibodies in dogs of different regions of Greece. In a population of 2620 dogs in Greece, the seroprevalence of antibodies against B. burgdorferi was 2.23% in 2019, while double or triple tick-borne pathogen seropositivity was detected [B. burgdorferi antibodies were detected in only 1 out of 1000 dogs, with triple tick-borne pathogen seropositivity [B. burgdorferi was not detected in a population of 1154 dogs living on Greek Ionian and Aegean islands including Crete in 2020 [B. burgdorferi DNA was detected, in a percentage of 6.2% of tick infested or not tick infested sheep in Tunisia [B. burgdorferi antibodies among sheep was 23.8% in Egypt [Moreover, several recent studies reported the seroprevalence of detected . The sam in 2020 . However Tunisia . The prein Egypt , 14.1% iin Egypt , 10% in in Egypt , while iLB is a multi-systemic disease affecting the nervous system, the heart and the joints . The mosBorrelia persica species were first reported in cats and dogs in Israel [B. burgdorferi, mild to moderate anemia, thrombocytopenia, leukocytosis, leukopenia, lymphopenia, neutrophilia and presence of band neutrophils were detected [B. burgdorferi in sheep remains unknown. Hematological alterations associated with Lyme disease in humans have been firstly described in 1994 in the United states  and in En Israel . Furtherdetected . The hemB. burgdorferi in sheep. The purpose of the study was to identify possible variation in hematological variables associated with the presence of antibodies against A total number of 318 animals was tested. Out of the 162 samples meeting the inclusion criteria, 87 were negative for both IgM and IgG (group A), 9 were positive for IgM (group B), 18 were positive for IgM and IgG (group C), while 48 were positive for IgG (group D). A positive reaction appeared as bright sharp stained spirochetes was observed. The size, appearance and density were compared with the positive and negative controls reactions. Patterns of reactivity different than that seen in the positive control was considered non-specific, which means negative, as per manufacturer’s instructions. Positive and negative antibody reactions are shown in p < 0.05). The lowest value was recorded in group C (IgM+/IgG+) and was significantly different from those in groups A (IgM−/IgG−) and D (IgM−/IgG+) (p < 0.05). No significant difference (p > 0.05) was recorded between groups B (IgM+/IgG−) and C (IgM+/IgG+) and between groups B (IgM+/IgG−) (IgM+/IgG−), and D (IgM−/IgG+).Average Packed Cell Volume (PCV)  was signp < 0.05); no significant difference was detected between the other three groups (p > 0.05). Band neutrophils were detected only in group C (IgM+/IgG+) and no basophil was detected in either sample. Monocyte counts were not significantly different among groups , B (IgM+/IgG−), C (IgM+/IgG+) and D (IgM−/IgG+), respectively; p < 0.05).As it is shown in p < 0.05). The mean platelet values recorded in group D (IgM−/IgG+) were significantly lower compared to those in group A (IgM−/IgG−) (p < 0.05) but not in group B (IgM+/IgG−) (p > 0.05). The highest average platelet value was detected in group A (IgM−/IgG−) and was significantly different than those in groups C (IgM+/IgG+) and D (IgM−/IgG+) (p < 0.05).Average platelet (PLT) count  was signHematological findings of seropositive sheep included anemia, thrombocytopenia, leukopenia, neutropenia and lymphopenia, as well as leukocytosis, neutrophilia, lymphocytosis and eosinophilia. Blood smear microscopical examination revealed presence of anisocytosis, polychromatophilia and nucleated erythrocytes in anemic sheep indicative of regenerative anemia (blood loss and/or hemolysis) ,41,42.p > 0.05). The percentage recorded in group A (IgM−/IgG−) was significantly lower (p < 0.05) compared to the other groups whereas no significant difference was detected between groups B (IgM+/IgG−) and D (IgM−/IgG+) (p > 0.05).Regarding PCV value , 2.3% ofp < 0.05). The percentages between groups B (IgM+/IgG−) and D (IgM−/IgG+) were not significantly different (p > 0.05).Thrombocytopenia was detected in 3.45% of the sheep in group A (IgM−/IgG−), 33.33% in group B (IgM+/IgG−), 100% in group C (IgM+/IgG+) and 50% in group D (IgM−/IgG+), as it is shown in p < 0.05) whereas the difference of the percentages of leukopenic sheep among groups A (IgM−/IgG−), B (IgM+/IgG−) and D (IgM−/IgG+) was not significant (p > 0.05).Out of the sheep of group A (IgM−/IgG−) and group C (IgM+/IgG+), 5.75% and 55.56% were leukopenic, respectively . No leukp < 0.05) whereas those in group B (IgM+/IgG−) were not significantly different than those in the other groups (p > 0.05).Neutropenia was detected in 33.33% of the sheep in group C (IgM+/IgG+) and 8.33% in group D (IgM−/IgG+) . The perp < 0.01).A percentage of 50% of sheep in the group C (IgM+/IgG+) presented lymphopenia but no lymphopenic animal was detected in other groups . The difp > 0.05). Thrombocytosis was detected only in group A (IgM−/IgG−) in 1.15% of the sheep and the differences among groups were not significant (p < 0.05) between group D (IgM−/IgG+) and groups A (IgM−/IgG−) and C (IgM+/IgG+), while no significant difference was detected among groups D (IgM−/IgG+) and B (IgM+/IgG−) (p > 0.05).A percentage of 22.92% of sheep in group D (IgM−/IgG+) presented leukocytosis whereas no leukocytosis was detected in the other groups. The difference was significant (p < 0.05) but not with groups B (IgM+/IgG−) and C (IgM+/IgG+) (p > 0.05).Neutrophilia was detected in 8.33% of the sheep in group D (IgM−/IgG+); this percentage was significantly higher than group A (IgM−/IgG−) (p > 0.05). Lymphocytosis and eosinophilia were detected in 2.08% of sheep only in group D (IgM−/IgG+) and were not significantly different from all other groups  are more likely to be in the early stage of infection, since IgM antibodies are usually present within short time after initial infection . TherefoB. burgdorferi [Depending on the limit of detection of each method, false-negative results for both antibodies can occur during the first weeks of infection. In the present study, IFA assay was performed as an accurate and highly sensitive method for detecting antibodies against gdorferi . Althouggdorferi .Borrelia is the main mechanism of host immune response. Despite the strong humoral response, the bacteria can survive for a long time in human [Borrelia persistence is challenging. This long term presence of Borrelia in tissues implies that immune system interacts with bacteria antigens, recursively [The production of specific antibodies against in human . Unravelursively  and thisBorrelia species may appear likely due to cross-reaction within the same bacterial genus (Borrelia complex known to cause Lyme disease and relapsing fever). As members of the Borrelia genus share common antigens with one another and with other spirochetes, cross-reactivity is expected [Ehrlichia and Babesia species as well as Helicobacter pylori [Rickettsia, Babesia, Leptospira also infect sheep triggering immune response. The first two hemoparasites were neither cytologically or serologically detected in sheep included in this study, while Leptospira is rather infrequent in Greece and mainly associated with abortions [False-positive antibodies against a certain expected . False-pr pylori . In the bortions , althougB. burgdorferi in patients with autoimmune disease such as systemic sclerosis and systemic lupus [Borrelia employs several mechanisms to escape immune system, provoke chronic inflammation and trigger autoimmunity. Certain Borrelia species are found to escape complement system [Borrelia and may favor chronic inflammatory processes [Borrelia and host tissue antigens may result to cross-activation of autoreactive lymphocytes by Borrelia antigens [False positive test results may be due to cross-reacting antibodies to ic lupus . Borrelit system  while ott system . Dissemit system . Extracerocesses . Finallyantigens . Borrelia induces gene expression of cytokines, chemokines, adhesion molecules and other immunomodulatory or immunosuppressive factors mediated by NF-kB and other transcriptional factors [B. burgdorferi was found to present cytopathic effects, killing B and T lymphocytes in cell cultures after active attachment, invasion through endocytic pits into vacuoles [B. burgdorferi to invade and kill lymphocytes may be a possible explanation of lymphopenia in the 50% of sheep in group C (IgM+/IgG+). Similarly, immune suppression was reflected and in other white blood cells with the presence of neutropenia in sheep of group C (IgM+/IgG+) and group D (IgM−/IgG+). Generally, host antigenic stimulation by  factors . Especia factors . Except vacuoles . This abB. burgdorferi. Likewise leukopenia was also detected in dogs and cats infected with B. hispanica [Borrelia by phagocytosis [Borrelia in sheep with a relatively past exposure. However, in the total population of sheep with antibodies against Borrelia the most prominent features in WBCs are lymphopenia and absence of neutrophilia in the vast majority of sheep. The presence of normal total WBC count is a typical finding in human LB and supports differentiation of Borrelia infection from other bacterial infections where leukocytosis and increased polymorphonuclear cells are characteristically observed [The lower mean count of WBC, as well as of neutrophils and lymphocytes, were decreased in sheep of group C (IgM+/IgG+), while the higher mean value of WBC and neutrophils, were detected in sheep of group D (IgM−/IgG+). The presence of normal or decreased WBC and mainly lymphopenia are in accordance with other studies in human ,63,64 anispanica ,66. On tocytosis . IncreasB. burgdorferi, as well as in all tick infested sheep sampled in the present study due to a certain extent to tick infestation itself [B. burgdorferi was found to induce hemagglutination [Borrelia hemolysin involvement. A hemolytic activity associated with B. burgdorferi has been detected, as certain B. burgdorferi strains produce oxygen-labile hemolysin enzymes with highest activity against horse erythrocytes, followed by bovine, sheep, and rabbit erythrocytes [B. burgdorferi infection [Anemia was observed in all groups with antibodies against n itself . Erythrotination . The deshrocytes . In the hrocytes ,41,42. Ahrocytes  and dogsnfection .B. burgdorferi. This decrease of the number of PLTs may be attributed to increased platelet consumption or destruction rather than diminished production. In previous studies in human [Finally, thrombocytopenia was detected in all sheep of group C (IgM+/IgG+) as well as in a considerable percentage of sheep belonging to other groups with antibodies against in human ,71,72 anin human , normal in human ,74,75. Ain human .Borrelia including the impact on the immune and hemopoietic systems and the possible reflection of this impact in blood variables. B. burgdorferi is a common pathogen in both human and animals. From a “One Health” perspective, the presence of antibodies against B. burgdorferi in the present study is indicative of the sheep exposure to the pathogen. This raises concern about the dynamics of the circulation of B. burgdorferi in the area as sheep act as maintenance hosts for tick populations and potential spreaders of infected ticks and, consequently, tick-borne pathogens among humans and animals.Further studies are required to elucidate the underlying pathogenetic mechanisms of http://glimmpse.samplesizeshop.org/) [Prior to the onset of the study the minimum required total sample size was calculated using General Linear Multivariate Model with Wilks Likelihood Ratio procedure at the GLIMMPSE software (op.org/) . The typAnaplasma phagocytophilum, Babesia sp., Theileria sp., Rickettsia sp.). If any animal was found positive to any of these tick-borne pathogens, it was excluded from the study.A total number of 318 sheep of Chios breed from 6 farms in Greece was enrolled for the study. Animals included for the study based on (a) presence of ticks (b) deworming treatment performed the last 2 months. Animals were excluded based on (a) presence of other ectoparasites such as fleas and lice (b) serological or cytological evidence of concurrent tick-borne infections  for serum retrieval and whole blood, respectively. All samples were transferred on ice, avoiding direct contact with the tubes, to the Diagnostic Laboratory, Faculty of Veterinary Medicine, School of Health Sciences, University of Thessaly, Greece, within 24 h.Packed cell volume (PCV) value was assessed by the microhematocrit method . The redBlood smear microscopy was performed in each sample. Blood smear preparations were fixed using methanol and stained with Giemsa. Quantitative assessment of white blood cells (WBCs) and platelets (PLTs) was performed as previously described . Brieflyg for 10 min. Serum supernatant was transferred into plastic vials  and stored at −20 °C pending analysis. IFA assay was performed for the detection of IgG and IgM antibodies against Borrelia burgdorferi in serum.Serum separation was performed after blood clotting by centrifugation of the tube at 300× Borrelia burgdorferi sensu lato antigens , a rabbit fluorescein isothiocyanate-conjugated anti-sheep IgG and a rabbit fluorescein isothiocyanate-conjugated anti-sheep IgM . Titers ≥1:64 were defined as positive [Commercially available slides coated with positive . Slides B. burgdorferi, (c) group C: sheep with IgM and IgG against B. burgdorferi, (d) group D: sheep with IgG against B. burgdorferi.Based on the results of the antibody detection assays sampled animals were allocated in four groups; (a) group A: sheep found negative for both IgM and IgG presence, (b) group B: sheep with IgM against ® statistical software. All comparisons were done at a significance level of p ≤ 0.05.Differences in the blood cell counts  and the PCV among the four groups were analyzed using the statistical software IBM SPSS 25. The normality of the data was evaluated with the Kolmogorov-Smirnov test and the homogeneity of variances with Levene’s test. One-way ANOVA was run to determine the significance of the differences among groups for each parameter that was normally distributed. Post hoc comparisons were done using Bonferroni test when equal variances were assumed and Tamhane T2 test when the variances were unequal. The results are expressed as means ± SE. Chi-square test was used to assess the significance of the differences among groups in the percentages of sheep of each group presenting each of hematological findings  d using in MedcalcB. burgdorferi causes significant alterations on the hematological profile of sheep. However, the stage of the disease cannot be assessed in accuracy, when complete blood count and serological tests are combined. From a clinical point of view, in cases with thrombocytopenia along with anemia, normal or decreased WBCs and mainly lymphopenia, the infection with B. burgdorferi should be included in the differential diagnosis especially when the sheep are parasitized with ticks. The relatively high seroprevalence of B. burgdorferi recorded at this study provides also evidence for potential increased risk for Lyme disease in humans in rural areas that are in contact with sheep.The results of the present study indicate that the infection with


The study aims to describe process evaluation measures of the three-year Health Promoting Schools’ obesity prevention program in Lima and Callao, Peru, and to assess factors that influenced the implementation of the intervention leading to the mentioned process outcomes results. The program was implemented in four public high schools located in low-income areas of Lima and Callao. Embedded in a Health Promoting School Intervention, the program consisted of two main components—an education program and several environmental activities. Quantitative data were collected and analyzed based on dose delivered and reach for each specific activity. Dose received was analyzed by satisfaction scores related to six specific activities. Furthermore, qualitative data including documentation of activities and transcriptions from individual in-depth interviews were qualitatively analyzed to identify factors influencing the implementation. The education component of the Health Promoting Schools’ program achieved a 78.4% average nutrition sessions delivery in 2015 and 88.0% in 2017; while for PA sessions, the average delivery in 2015 was of 79.7% and 93.8% in 2017. In the case of reach, at least 75% of total students participated in all sessions per year. Nevertheless, there were differences in terms of delivery and participation in a number of environmental activities within and between schools during the program period. Differences in delivery included education sessions for parents, teachers, Junior Health Promoters, and school food kiosk staff, besides execution of physical activity events every year. Impeding factors included the complexity of the overall intervention, limited strategies to facilitate program implementation and those to maintain the participation of subjects, and related contextual factors. While more emphasis has been placed on outcome evaluation to determine whether a health program has been successful or not, process evaluation, which allows us to understand and interpret such results, is equally important . ProcessOver the last twenty years, literature on how to measure program implementation has become widely available. The increase in the use of process evaluation is partly related to the inherent characteristics of public health interventions, the complexity that results when interventions are implemented at multiple levels and with multiple audiences, and the accountability demands of results as in the case of Official Development Assistance (ODA)-funded programs ,4. WhileA commonly used framework is the one outlined by Steckler and Linnan 2002), which includes seven recommended elements of a process evaluation plan: Context , reach (degree to which the intended target group participates in intervention components), dose delivered and received (amount of program delivered by implementers and extent to which participants responded to it), fidelity (extent to which an intervention is delivered as designed), implementation  and, recruitment  002, whic,8,9,10. However, reports of process evaluation for intervention studies in the area of childhood obesity are still not common. Childhood obesity has been recognized as a serious health challenge worldwide, for which effective and feasible preventive interventions are critically needed ,12. If pThe aim of the present study is to describe process evaluation measures of a school-based obesity prevention program in Lima and Callao, Peru, and to assess the factors that influenced the implementation process of the intervention leading to the mentioned process outcomes results. To achieve this, we have reported on two specific process outcomes for the various program activities (dose delivered and reach), the satisfaction of students and teachers to specific activities, and provided an overview of main factors affecting the implementation of the program.The school-based obesity prevention program, focus of the present study, is part of an overall health promotion study titled “Health Promoting Schools” (HPS), an Official Development Assistance (ODA)-funded project by the Korea International Cooperation Agency (KOICA). The HPS intervention was designed by KOICA and Yonsei University’s Global Health Center as a quasi-experimental study targeting adolescents of six public high schools located in Lima and Callao, Peru, among which four were allocated to receive the intervention. The overall aim of the HPS intervention was to improve the mental and physical health status of adolescents by decreasing the risk factors of suicide ideation and preventing obesity. For the first, risk factors comprised depression, substance use behavior, and abuse experience, and for the latter, the targeted risk factors included unhealthy dietary behaviors and physical inactivity among the participants. The HPS intervention was implemented throughout the coordination of three teams, a design team composed of two researchers, a monitoring team, and an implementation team. Both the design and monitoring teams were based in the KOICA Peru office; the design team  coordinated with the different collaborating organizations at all levels about program activities, while the monitoring staff received the self-administered forms and logbooks every week, and was in continuous contact with each school implementation team regarding execution of activities, reporting and local-level planning and coordination. Local implementation teams per school consisted of one nurse and a psychologist, who were based in health centers built by KOICA and located nearby each school premises.The overarching concept of the HPS obesity prevention program emphasizes that by providing health education and increasing opportunities to eat healthily and stay active, students can improve their health awareness, and thereby, engage in behaviors conducive to healthy nutrition and increase of physical activity (PA), preventing the development of obesity . The theThe HPS obesity prevention program consisted of two main components: an education program and several environmental activities targeting the entire school community each school year. A detailed description of the elements of each component and its intended delivery per year (as stated in program plans for intervention) are presented in The nutrition sessions of the education program for students were designed to be delivered sequentially. In addition, sessions were structured to have a media powerpoint presentation, a teamwork exercise, and a video presentation to portray a dynamic example of the subject taught during the session. Some elements of the education and environmental components varied between the first program year (2015) and consequent years (2016–2017). The Healthy Food Kiosk initiative included education sessions for kiosk staff, physical improvement by installing fruits exhibition stand and nutrition traffic lights, as well as 2-yearly evaluations.The HPS obesity prevention program process evaluation uses a modified model based on the work of Steckler and Linnan (2002) and Hasson (2010) . Based oA self-administered form developed by the design/monitoring teams and provided to each implementation team to fill-out and report weekly was used to collect information on the actual implementation of program activities (dose delivered and reach). For the dose received, we utilized question items included in follow-up questionnaires conducted at the end of each school year for students. Quantitative process-evaluation questions of dose delivered and reach for each program component were separately defined. Examples of questions were as follows: “To what extent were all nutrition and PA sessions for students implemented?”, “Was the education program delivered to at least 70% of all students?”, To what extent were all scheduled sessions provided to parents/teachers/JHP/school food kiosk staff?, and “How many parents/teachers/JHP/kiosk staff participated in respective education sessions?”.Dose delivered for education and environmental activities were measured using total counts of number of sessions/evaluations/materials/events carried out during the year in each recipient school, while reach was measured using total counts in terms of participants in each activity.Dose received was assessed using satisfaction scales included at the end of the year follow-up questionnaires for students. Satisfaction to education sessions on nutrition and PA, the overall Healthy Food Kiosk initiative, the minigym or provision of selected sports equipment, and PA events were measured using a 5-point scale ranging from ‘Very Unsatisfied’ coded as 1 to ‘Very Satisfied’ coded as 5. Overall scores and respective standard deviations were calculated and expressed on a yearly basis, and the higher the score, the higher the satisfaction. In addition, satisfaction of teachers who participated in capacity-building training in 2016 and 2017 was measured in the same way as aforementioned.Qualitative data was collected through program-related documents and individual interviews . DocumenIn addition, individual interviews with students, parents, teachers, implementers (nurses) and Peru officials were conducted using purposive sampling. The characteristics considered for each type of participant were as follow: students who had been enrolled during entire program duration; individual parent or both parents of a student enrolled during entire program duration who were aware of the program existence and/or participated in a program activity ; teachers who had worked in intervention schools during entire program duration and were aware of program and/or participated in any activity; nurses who were in charge of implementing the program activities between April 2015 and December 2017; and Peru officials who had been in charge of monitoring program activities as recipient country counterpart during its entire duration.The process to recruit the participants was established as follows: first, the Regional Health Direction in charge of supervising health-related programs in the four intervention schools was contacted via e-mail and phone by one of the authors (RCCH) to inform about the study and request permissions, given that some potential student respondents were still attending school. After receiving the respective approval, directors of the four intervention schools were separately contacted to request contact information of potential participants. Potential participants  were later contacted by phone by nurses who implemented the program in each school who provided a general explanation of the study and inquired their willingness and consent to participate in the interview. For students still attending school, parents were provided with respective information as well. The participants who agreed to participate were later contacted by the interviewer (RCCH) to schedule date and place of the interview. Interviews were conducted in Spanish using a semi-structured interview guide covering perceptions of the program activities and their implementation. Examples of evaluation questions were how much did students/parents/teachers engage in education sessions, and how did the program characteristics, nurses, and the school/home environment influenced the implementation of education sessions and environmental activities. Interviews were carried out using broad definitions or similar examples to avoid bias towards any particular type of answer.The Regional Health Direction recommended to include two participants per school in the case of the students, parents, and teachers’ group (24 respondents across three groups). In the case of two schools, one nurse who implemented activities during the entire program period in each was invited by e-mail and phone, while for two schools, two nurses in each were invited taking into account the resignation of the first implementer in each school (6 nurses across four schools). Finally, one Peru official who worked at the regional health direction and complied a monitoring role of the program activities during all intervention years was invited via e-mail and on-site to participate.The present study has a mixed methods design, where the quantitative and qualitative data were collected and analyzed in parallel but separately, and later brought together in the interpretation of the overall results  16]. Th. Th16]. Data on dose delivered, dose received and reach from self-reported forms were first entered into an Excel workbook that was uploaded into SPSS 24.0 Statistical software to obtain descriptive statistics  for each program activity per school. Dose delivered for education sessions, food kiosk evaluations and dissemination of information was expressed on a yearly basis as a percentage resulting by dividing the number of sessions/evaluations/information materials delivered in the year  by the total number of sessions/evaluations/information materials scheduled for the year and then multiplied by 100. In the case of activities concerning installation of equipment and PA events, the aforementioned formula was expressed as a percentage of activities carried out during total program duration (2015 to 2017). The reach formula established for education and environmental activities was expressed as a percentage resulting by dividing the number of participants in the activity by the total number of participants targeted and then multiplied by 100. In the case of parents’ sessions, the total number of parents per school was defined in terms of one parent per student for calculation. For walking events, information on total school administration staff and guests was not available, for which reach was expressed as a total count. Satisfaction mean scores ± standard deviations were calculated for the six selected activities as mentioned in the quantitative data section above.A total of 31 individuals (23 women and 8 men) 16–56 years old, participated in the individual interviews. Interviews were held for 35–70 min, with an average interview time of 46 min. Also, RCCH took daily field-notes of the interaction with respondents and problems encountered in data collection. All interviews were recorded and transcribed verbatim, some by the first author (RCCH), and some by one Graduate student whose mother tongue was also Spanish. Transcriptions were made in Spanish and English, and RCCH listened to all interview recordings to make sure transcriptions were correct.Qualitative data were analyzed using a deductive Qualitative Framework Analysis approach  using thEthical approval for the study was obtained from the Institutional Review Board of the Wonju Campus of Yonsei University (IRB 1041849-201909-SB-134-02). Regarding the in-depth interviews, informed consent was also obtained from each interviewee after providing information about the purpose of the study and use of the data. In the case of under-age participants, a parent present before the start of the interview signed the informed consent form instead.The results are presented regarding the modified process evaluation model . In orde“KOICA had a process already defined for education sessions which was the same every year”. Furthermore, school directors and teachers were mentioned to have had an overall key role in accommodating program sessions  in the school curriculum for their benefit. However, teachers reported about the difficulty of schools to adopt an external program and its different activities into individual year schedules, especially during the first years, which was described as “KOICA project activities in the first years were not easy to follow”. The mentioned difficulty was greater for schools with both, morning and afternoon shifts (School 1 to 3), which was expressed by teachers as “It was our first time having an external program in the school, so we needed time to adapt the KOICA activities to our everyday routine”.Between 2015–2017, the delivery of nutrition and PA sessions for students increased or was maintained across schools . With regards to environmental activities, with the exception of parents, delivery of teachers, JHP, and school kiosk staff sessions increased over time. The education component of the HPS program (sessions for students) was considered the main activity, while sessions carried out with other target groups  aimed to support the healthy dietary and PA behaviors promoted among the students. Implementers mentioned about the importance given to the education component of the program, as described in action plans every year, where detailed information was available on session process and standardized PPT tools to be used. This was expressed by the quote In the case of education sessions directed at parents, the complexity of being part of the overall HPS intervention influenced the delivery. Since 2016, mental health-related activities including parental sessions were increased based on the risk factors identified, thus, the obesity-related sessions targeting parents became sidelined or seen as a lower priority. As a result, only one of three scheduled sessions was implemented in 2016 (data not shown), and nutrition and PA sessions for parents were completely ruled out from the yearly plan in 2017.“in order to provide teachers a learning opportunity at graduate level in relation to core components of health promotion in the school setting not only for the intervention immediate impact but thinking of long-term effects for the individual, students and the school in general”. In this way, it was expected of teachers to enroll and actively participate in training out of school working hours.Education for teachers passed from non-delivery in 2015 to full delivery of training programs carried out in both years, 2016 (not shown) and 2017. The program design team determined to outsource capacity building programs for the years 2016 and 2017 with a local University. According to a meeting minute where design team and University representatives coordinated the training program in 2016, the decision was taken “I think coordination was not effective”. In spite of this, health center directors and new implementers were perceived to make efforts to accommodate the activities into daily work agendas, though there were delays in sessions implemented.Education sessions delivered to school kiosk staff averaged 67.9% in 2015, 81.3% in 2016 (data not shown) and 100% in 2017. While in 2015, program nurses carried out the education sessions, from 2016, staff from the KOICA-built health centers located nearby school premises were requested to develop this activity. The decision was made in order for program nurses to focus on sessions for students and provide support in planning, coordination, and implementation of duties for new mental health program activities. However, nurses considered there was a lack of timely coordination and monitoring for this specific activity between design team and health center directors on occasions, which affected the implementation of some sessions . This was described especially by nurses as “She (the school director) only focused on letting us carry out the students’ sessions”.In regards to JHP education sessions, 75% of sessions in average were carried out in 2015 and 2016 across four schools, while delivery was 100% in 2017. JHP sessions was another activity translated to health centers since 2016. In addition, in 2015, only one session was implemented in School 2, which was one of the schools that exhibited a greater difficulty to adapt some of the activities into their schedules. This was related to the fact that the school director during that year was perceived to give low importance to program activities other than school sessions. This was described by the assigned nurse as We had the intention to organize the walking event and sports Olympics like KOICA did but the many activities of the school didn’t leave us time to do it” represented the difficulty schools had to prepare and carry out both events by themselves within the school year. Thus, while both PA events were carried out in each school in 2015, there were no walking events done in 2016 across all schools, among which one (School 1) did not carry out any event until the last program year. School 1 and 2 were recognized by the nurse in charge of activities as giving a low importance to some of the program activities as compared to their own schedule, which was expressed by the quote “The school was very attached to its normative, own plans and activities, so it was difficult to insert some of the activities into such tight schedule”.The greater focus on the mental health program since 2016, made PA events another activity that was shifted directly to schools. The quote ““time availability was the biggest difficulty for parents’ sessions. We could not match the personal or working schedule of many”. In spite of sessions that were rescheduled to be carried out in the evenings and the use of individual attendance cards, parent participation did not improve across schools in 2016. Parents themselves described their difficulty to join sessions as “I couldn’t join sessions because of my work” or “I had to take care of house chores and my younger children”.All schools reached an important proportion of targeted students, with at least 75% of total students participating in all sessions per year. The average percentage of parents participating in all sessions provided across schools was 29% in both 2015 and 2016. While written notes on the school diary of students were the main method used to contact parents for activities, their participation into sessions was mainly affected by conflicts with working and personal schedules. As nurses expressed many worked both shifts in the intervention schools”, while others did the same even on weekends. The program tried to adjust this by offering two schedules of the yearly training programs (3 h each on Tuesdays and Thursday evenings or 6-h sessions on Saturdays); however, attendance did not improve considerably.Teachers attendance to capacity building training averaged 33.1% across schools in 2016 and 39.2% in 2017. The teachers themselves recognized extended working hours as the main barrier to their involvement in programs, since “few JHP students actually participated in the sessions, maybe because the schedule was out of class hours”. Given the aforementioned difficulty, during the second and third school year, incentives were provided for those participating in sessions, especially lunch boxes and presents like small stretching ropes and ecobags.Attendance of JHP to sessions increased from 47.2% in 2015, 54.7% in 2016 to 62.4% in 2017. Gathering specific students from different classrooms, school years, and shifts (morning and afternoon) for sessions during class hours was not an easy task. Thus, nurses reached an agreement with school directors to do sessions in between shifts from 2016 to avoid students losing respective class hours. However, this strategy placed a burden to JHP; those from the morning shift needed to stay after school hours while those from the afternoon shift needed to come to school early. Students described this as ““they said we can offer everything healthy, but it is actually a business, if students do not want it, outside the school there are street vendors”. To this, throughout program period, food options available outside each school were out of the influence of the program.In regards to the extent schools’ kiosk staff participated in sessions made available, attendance in 2015 was 65% across the four schools, 57% in 2016, and 47.4% in 2017. Food kiosks worked under a year renewable contract with the school and coordination was made through directors to ensure the participation of staff in sessions. However, kiosk staff were not always willing to receive sessions, given concerns about profit. Some staff believed changes introduced to products they offered and the physical presence of uncontrolled competing vendors could affect their “business” which was described by nurses as After the first year of implementation (2015), students rated the PA events as the activity they were most satisfied with (mean score across schools 3.88), followed by the nutrition sessions from the education component (mean score 3.72). PA events were developed in full in the mentioned year across schools, while in 2017 students from School 1 considered the activity as the one they were most dissatisfied with (1.35 score) given non-implementation. At the end of the last intervention year, students were mostly satisfied with the education component of the program , followed by the Healthy Food Kiosk Initiative with an average score of 3.63. In relation to the satisfaction of teachers who attended the capacity building training made available by the program in 2016–2017, average score across schools was above 3.9 in both years, indicating overall satisfaction with the courses .The aim of the study was to describe process measures of the HPS obesity prevention program in Peru, and to assess the factors that influenced the implementation process of the intervention leading to the mentioned process outcomes results. We found that the overall delivery and reach of education sessions for students between 2015–2017 increased or was maintained across schools, a decrease in delivery of parents’ sessions, an increase in delivery of sessions for teachers, JHP and school kiosk staff, and that the delivery of PA events differed across years and schools. About reach of environmental activities, less than 30% of parents in average participated in total sessions provided yearly, while less than 40% of teachers across schools attended both capacity building training provided. In regard to dose received, the activities students were most satisfied within 2015 were the PA events and nutrition sessions, while in 2017, it was the education sessions (nutrition and PA), followed by the Healthy Food Kiosk Initiative. In addition, it was found that the complexity of the intervention the program was embedded in, the extent strategies to facilitate implementation and maintain the involvement of participants were carried out, and contextual factors across and between recipient schools were the main factors that influenced the implementation process of program activities. The findings related to intervention complexity, and facilitation/recruitment strategies coincide with different studies which argued about their potential role as barriers to implementation ,9,10.In the present study, the complexity we refer to is the one that stems from being part of HPS, an ODA health-promoting school intervention that included separate mental- and obesity-related programs each with different goals and characteristics targeting the same audience and developed during the same period. In this sense, being part of a wider health promotion intervention considerably affected how the HPS obesity prevention program was planned, implemented, and evaluated. The HPS intervention worked under an agreement between the Peru Ministry of Health and KOICA Peru office, for which Peruvian stakeholders from different levels and hierarchical lines were involved from the start of the intervention. Previous studies have highlighted how the number of groups or organizational levels involved in an intervention constitute key dimensions affecting a program functioning ,19,20,21Year action plans constituted the main material provided by the design team to nurses for the implementation of activities. As the results section highlights, there was a detailed description concerning the education sessions process and tools to be used (mainly PPT slides). However, roles between design team and implementation nurses for coordination with related stakeholders  were not clearly defined, and such lack of task description became a barrier that affected the development of some activities such as PA events, JHP, and kiosk education sessions. This relates to a study were guideline implementers identified the use of multiple strategies to improve implementation success . AnotherContextual factors greatly affected how the implementation of the school-based intervention developed. This was represented by the characteristics of each intervention school and related stakeholders which acted as barriers to the implementation of overall activities. As the mentioned section highlighted, perceived importance by school directors and teachers differed between education sessions for students and environmental components. These results coincide with extensive research that identified context as a strong impeding factor for program planning, implementation, and achievement of outcomes results in health promotion and policy research, for which arguments of a thorough assessment of local circumstances at all stages of program planning and implementation duration have been found to increase program adaptability towards the achievement of outcomes ,14,25,26This study has different limitations. There was a lack of data collection methods for different elements that would have allowed a more comprehensive process evaluation. This included observations, questionnaires, and/or specific fidelity assessment tools. Furthermore, data availability only allowed us to describe total number of sessions/activities implemented within each year per school but not in regards to duration or other objective measures. Satisfaction scores were used to provide an overall idea of how participants responded to selected program activities. Thus, averages might represent a favoring but not strong process evaluation measure. Also, there is the possibly selective response to interviews among participants. For instance, directors and teachers who responded might have been more or less committed and positive toward the program or the program goals. In addition, we interviewed a limited range of program representatives involved in day-to-day program implementation, leaving out the probable different perceptions and needs from those in other roles.The process evaluation results of the study provided an overall picture of the extent to which the different activities of the HPS obesity prevention program were carried out as planned, and showed how characteristics of the intervention including multiple interacting components, the degree insufficient task descriptions, and materials are provided to implementers, and unclear coordination lines influenced the development of activities. Our results also showed how contextual factors represented by the number of stakeholders and target groups involved, time availability and perceived importance, and commitment towards program goals impeded uniform program delivery and reach across the four target schools.The study findings thus highlight the importance of understanding the conditions of each recipient organization (school) as well as the individual expectations and needs of the different target groups, so that planning can vary to fit activities and strategies to the context, which could subsequently ensure better program implementation.


Sustaining health promotion programs (HPP) is critical to maintain their intended health benefits, community capacity, and to optimize resources and investment. However, not all programs are sustained beyond their initial implementation period. This is partly due to uncertainty regarding sustainability: lack of a clear definition; infrequent use of a sustainability framework; and lack of understanding of the factors that influence sustainability. The aim of this systematic review is to identify barriers and facilitators that promote or inhibit the sustainability of HPP, particularly those that can be considered in program planning.priori framework to initially code barriers and facilitators, which were then grouped into factors for HPP sustainability. Factors were then analyzed for frequency, importance, and relevance, and categorized into one of three tiers.Two search strategies were used: 1) electronic database searching; and 2) grey literature searching. Inclusion criteria included papers published since 1998, in English, focusing on the sustainability of HPP that explicitly used a sustainability framework and specifically reported on facilitators and barriers to sustainability. Exclusion criteria included papers that addressed environmental, system or sector sustainability. Quality assessment was conducted on all included papers and a quality assessment tool was developed for grey literature. Data analysis included a thematic analysis, using an a priori framework, and five additional ones based on the results of our analysis. Tier 1 factors were the most important for sustainability with organizational capacity scoring the highest; tier 3, the least important.Sixteen papers were included in this review. Eleven definitions of sustainability and 13 sustainability frameworks were used. A total of 83 barriers and 191 facilitators were identified and categorized into 14 factors: nine from the a This review provides clarity regarding existing definitions of sustainability and sustainability frameworks. It identifies fourteen factors that influence program sustainability, which practitioners can consider when planning, developing and implementing HPP. In addition, it is important for practitioners to clearly articulate program elements that should be sustained, define sustainability as it relates to the context of their program, select a sustainability framework to guide their work, and consider these factors for sustainability. The Ottawa Charter defines health promotion as the process of enabling people to increase control over their health, and its determinants, in order to improve health . Health The issue that a program was established to address remains or recurs.Programs see their funding withdrawn before activities have been fully realized and outcomes reached, despite significant human, fiscal and technical start-up costs.Diminished community support and lack of trust in communities with a history of programs that were abruptly/inappropriately terminated .While sustainability is often thought of within the lens of funding; for instance, finding replacement funding when initial program funding comes to an end , 9, sustSeveral key challenges affect HPP sustainability. First, there is a lack of consistency regarding what ‘sustainability’ means and how it is defined , 14–17. In order to promote the sustainability of HPP, the factors that promote long term sustainability need to be understood , 14, 21.This systematic review aims to identify the barriers and facilitators that influence the sustainability of HPP. Particular attention is given to those factors that can be considered during the program planning processes. Our research question is “what factors facilitate or influence the sustainability of health promotion programs?” In conducting this review, we assessed the body of literature on this topic to determine how sustainability is being defined, which frameworks are used to plan for sustainability and the overall implications for the health promotion and public health fields.We conducted a systematic review that focused on identifying barriers and facilitators for HPP sustainability. For this study, we used Grant and Booth’s 2009 definition of a systematic review: an evidence synthesis that adheres to guidelines on conduct of a review/method for integrating or comparing the findings from qualitative studies, looking for ‘themes’ or ‘constructs’ that lie in or across qualitative studies .The authors (AB and SH) jointly developed a protocol for completing this review in advance, which included the following steps outlined in PRISMA guidance: 1) identifying a search strategy; 2) determining inclusion and exclusion criteria; 3) screening papers; 4) assessing methodological quality; 5) extracting data; and, 6) synthesizing results .Two search approaches were used: 1) electronic database searching; and 2) grey literature searching. A three concept database systematic search strategy was developed by a librarian from Public Health Ontario’s (PHO) Library Services team, which included controlled vocabulary and natural language keywords related to the concepts of health promotion , programs  and sustainability . Searches were limited to articles published in English after 1998. The initial search strategy was developed using the MEDLINE database. The MEDLINE search strategy was peer-reviewed by other members of PHO’s Library Services team. The search syntax and controlled vocabulary terms in the MEDLINE search strategy were then translated for execution in secondary databases. The final search strategies were run in the following five databases on June 18, 2018: 1) Ovid MEDLINE; 2) Ovid PsycINFO; 3) EBSCOhost CINAHL plus with Full Text; 4) EBSCOhost SocINDEX with Full Text; and 5) Scopus.A grey literature search was conducted on July 7, 2018. It included grey literature repositories, custom web search engines, and a general web search ; explicitly used or referenced a sustainability framework or model to ground their HPP or guide their work; specifically reported on facilitators and/or barriers  for the sustainability of a program; offered synthesis level research or primary level studies that included assessments of program sustainability and outcome data related to facilitators and barriers of program sustainability. The 21 year time frame was based on preliminary searches conducted to inform the strategy, and a decision made to coincide with the publication of Shediac-Rizkallah and Bone’s (1998) study, which continues to be cited today. For our review, we used Shediac-Rizkallah and Bone’s definition of sustainability, which defines it in three ways: 1) maintenance of health benefits from a program; 2) continuation of program activities within an organization; and 3) capacity building in the recipient community . We focuExclusion criteria included non-English language papers published before 1998, and those from countries outside of the Organisation for Economic Co-operation and Development (OECD). Papers were excluded if they were about sustainable development, sustainable food systems, or environmental sustainability; focused on sustainable organizations or sustaining systems or sectors ; the program took place in a clinical or hospital setting; focused on sustainability of a partnership or coalition with no focus on program delivery; focused on sustainability of program impacts  and continued capacity of a community to develop and deliver HPP; no outcome data were provided; and commentaries and editorials.The two authors (AB and SH) developed the inclusion and exclusion criteria at the outset of the project.Titles and abstracts of the identified papers from the search were screened by two authors (AB and SH), who first independently screened 20% of search results for inclusion and had an agreement of 86.6%. The remaining 80% was split in half with each half screened independently by one of the two authors (AB and SH). For full text screening, two authors (AB and SH) first independently screened 20% of the articles and had agreement of 90%. The remaining 80% was split by 60% (AB) and 40% (SH) and screened independently. Any discrepancies were resolved by discussion until consensus was reached. For two articles, a third reviewer was consulted to apply the criteria and discuss ratings with both authors until consensus was reached.Titles and abstracts of all grey literature search results were screened for inclusion independently by the two authors (AB and SH). All included papers were full-text screened by independently by both authors (AB and SH) and consensus was reached on all discrepancies through discussion.Methodological quality was assessed independently by a research coordinator (TO) who reviewed all included papers, and by two authors (AB and SH) who each reviewed half of the papers as a second reviewer, using appropriate criteria in regard to the design of the included papers. The Health Evidence Quality Assessment Tool for systematic reviews and meta-analyses was used for systematic reviews . Papers Is the methodology identified? If not, could the methodology be found within two internet clicks to additional resources?Are the authors credible? Is the rationale for the resource clearly identified? Is the methodology clear and transparent?Did the authors use a sustainability framework? If they created their own, is it clear, and is it clear how the various components interact?Are the results transferable? Is it clear how the content could be used by practitioners?To assess the quality of the grey literature, a quality assessment tool was developed by the authors (AB and SH) using combined criteria from Caldwell  and BergQuestions were answered with a scoring of ‘yes’ or ‘no’. If ‘no’ was answered for either of the first two questions, the paper was excluded. Remaining papers were screened with the remainder of the questions for a final score out of 6. Papers scored below 1–2 were considered weak, 3–4 moderate, and 5–6 strong. Moderate and strong papers were included and weak ones were excluded. Methodological quality was independently assessed by two authors (AB and SH) who each reviewed all papers. Any discrepancies were discussed until consensus was reached. Quality appraisal results for included grey literature are found in Additional file A data extraction table was drafted to meet the research focus of this project, and refined by discussion between the authors. Two authors (AB and SH) tested and discussed the table, which created a guide for data extraction and instructions for completing each item to ensure consistency. Two authors (AB and SH) independently conducted data extraction for all included papers to ensure that all appropriate data were extracted from the included papers. Information extracted from each paper included: author and year of publication; study design; methods; purpose; objective or research aim; definition of sustainability; sustainability framework; barriers to sustainability; facilitators to sustainability; implications for the program planning process; conclusion; implications for practice; and future directions or research. Study limitations were also extracted and recorded as part of the conclusion column. Extraction tables from both authors were reviewed line by line and a harmonized data table was created  by re-reading all included articles and searching for instances where the authors of the included papers directly reported that a barrier or facilitator or multiple ones, were particularly important to the sustainability of the HPP. For example, authors stated that the barrier or facilitator was “the most important facilitator” , “integrAll data were compiled into one table , seven primary studies, two workbooks, and one sustainability framework. The targeted outcomes of HPP studied were varied, and included diabetes , obesityAll 16 included papers provided a definition of sustainability. Eleven papers developed their own definitions , 33–36. Individual level: maintaining health benefits for individuals after initial program funding ends, particularly continuing to achieve beneficial outcomes for new clients.Organizational level: continuing program activities within an organizational structure and ensuring that program goals, objectives, and approaches adapt to changing needs over time.Community level: building the capacity of the community to develop and deliver program activities, particularly when the program found success through a community coalition or community capacity-developing process.While each paper included in this review cited a definition of sustainability to guide their own work, many referred to inconsistencies in how HPP define sustainability or that in many cases, HPP did not define sustainability at all –18, 21..All of the 16 included papers cited a sustainability framework. Nine papers used existing frameworks , 36, 41 Community falls prevention program sustainability guidelines and workbook [There were two types of frameworks cited in the papers: most depicted sustainability in terms of barriers and facilitators for sustainability –21, 32 wworkbook , provideOne of the included papers, Schell et al. , presentbarrier inhibits or prevents the sustainability of a HPP while a facilitator promotes sustainability or is associated with sustainability. In total, 83 barriers and 191 facilitators related to sustainability of HPP were extracted from the included papers. Since the Schell et al. framework was used to code barriers and facilitators, the barriers and facilitators from their framework were not included in the analysis results. Two papers from the Office of Adolescent Health [For the purpose of this paper, a t Health , 35 citet Health  cited seTwenty-three barriers and 54 facilitators could not be coded into the domains of the Schell framework. As such, five additional factors were created by the authors to summarise and describe them. This results in a total of 14 factors for sustainability being identified: nine from the Schell framework; and five new ones based on the results of our analysis. Five barriers and five facilitators could not be coded into either the Schell domains or the five new factors, and were considered outliers.Factors were grouped into one of three tiers based on relative prominence in the past 21 years of health promotion sustainability literature. Tier 1 had codes with a frequency of ≥15 up to 54 and were heavily coded as important and/or relevant. These factors were considered to be the most influential for program sustainability , or capacity of the community to continue the program. In addition, practitioners should use a sustainability framework to guide their sustainability planning efforts; several were identified in this review. Given the importance of HPP in improving population health, reducing health inequities, and reducing health care costs, health promoters and public health practitioners should strive to prioritize the sustainability of their programs by actively planning for sustainability throughout program implementation and the program lifecycle. The factors identified in this systematic review could be incorporated into the design of HPP to increase their likelihood of sustainment.Additional file 1: Appendix A and B. Search strategy examples. Full MEDLINE search strategy, and repository and general web search for grey literature.Additional file 2: Appendix C. Results of quality assessment of the included 16 papers with final ratings.Additional file 3. Characteristics of included papers.Additional file 4. Weighting of factors for sustainability by frequency, importance and relevance.


Different polysaccharides—namely dextran, carboxymethyl dextran, alginate, and hyaluronic acid—were compared for the synthesis of nanoporous microsponges particles (NMPs) obtained from a one-pot self-precipitation/cross-linking process. The morphologies and sizes of the NMPs were evaluated comparatively with respect to polymer-to-polymer and cross-linker solvents (water-based vs. DMSO). We found that the radial distribution of the polymer in the near-spherical NMPs was found to peak either at the core or in the corona of the particle, depending both on the specific polymer or the solvent used for the formation of NMPs. The NMP porosity and the swelling capability were evaluated via scanning electron microscopy (SEM). The degradation study indicated that after 10 h incubation with a reducing agent, approximately 80% of the NMPs were disassembled into soluble polysaccharide chains. The adsorption and release capacity of each type of NMP were evaluated using fluorescently labeled bovine serum albumin and lysozyme as model proteins, highlighting a release time typically much longer than the corresponding adsorption time. The dependence of the adsorption-release performance on pH was demonstrated as well. Confocal microscopy images allowed us to probe the different distribution of labeled proteins inside the NMP. The safety and non-cytotoxicity of NMPs were evaluated after incubation with fibroblast 3T3 cells and showed that all types of NMPs did not adversely affect the cell viability for concentrations up to 2.25 μg/mL and an exposure time up to 120 h. Confocal microscopy imaging revealed also the effective interaction between NMPs and fibroblast 3T3 cells. Overall, this study describes a rapid, versatile, and facile approach for preparing a universal non-toxic, nanoporous carrier for protein delivery under physiological conditions. Organic–inorganic nanoporous multifaceted structures, such as micro-nanoflowers , microspThe further development of this method for engineering multifunctional nanoporous microparticles under mild conditions is therefore desirable. Instead of using metal salts, a new co-precipitation method was recently developed by our group to use an organic molecule, i.e., cross-linking agent, to obtain polysaccharides nanoporous microsponge particles (NMPs) without resorting to metal ions . During To that end, in this work, we seek to demonstrate the potential use of the developed cross-linked NMP formulation as a universal template to synthesize nanoporous particles from different polysaccharide building blocks while proving safety and protein loading capability. Specifically, we demonstrate the general use of our technique to prepare porous polysaccharide NMPs from dextran, carboxymethyl (CM) dextran, alginate, and low molecular weight hyaluronic acid. Notably, our simple methodology greatly expands the selection of diverse polysaccharides to be used as templates of the nanoporous microparticles found in the literature. The potential exploitation of the NMP as a platform for protein delivery is extremely high and—as a proof of concept—we report the successful loading of NMPs with two proteins of scientific and pharmacological relevance, i.e., bovine serum albumin (BSA) and lysozyme. Because these proteins have different molecular weights and isoelectric points, they represent significant case studies to assess the loading and release capacity of the polysaccharides NMPs.NMPs provide a platform for delivery a variety of biomacromolecules, such as proteins, lipids, and nucleic acids which can hardly pass through the various biological barriers without the assistance of a carrier. Polysaccharides are widely regarded and studied as potential carrier materials for site specific drug delivery because of their non-toxic, biocompatible and biodegradable properties ,16,17,18Cystamine dihydrochloride (CYS), 1.1 Carbonyldiimidazole (CDI), sodium alginate , dextran sulfate sodium salt , carboxymethyl-dextran sodium salt (CM-Dextran-MW 70 kDa), hyaluronic acid , Rhodamine B isothiocyanate (RITC), DL-Dithiothreitol 99% (DTT), bovine serum albumin, 96% (BSA), dimethyl sulfoxide (DMSO), and lysozyme from chicken egg white, 90% were purchased from Sigma-Aldrich .The cross-linker (CL) solution was obtained from CDI and CYS dissolved in DMSO in a 3:2 weight ratio. The reaction proceeded overnight without stirring. Alternatively, another cross-linker solution was obtained by dissolving CDI and CYS in MilliQ water to obtain a precipitate. Subsequently the precipitate was dissolved in MiliQ at pH 4 by adding hydrochloric acid 2M (as needed). NMR spectra were recorded in deuterated DMSO to characterize the cross-linker .w/v solution of dextran, CM-Dextran, hyaluronic acid, and alginate were individually added to cross-linker solutions without stirring. The resulting NMP particles were centrifuged and washed with MilliQ water three times. The yields of the reactions were calculated as follows: the NMP were formed using fluorescently labeled polysaccharide and the UV-vis absorbance “ab”  of the polysaccharide solution was measured before NMP microparticles formation. Next the suspension was centrifuged and the supernatant analyzed by measuring the UV-vis absorbance “aa”. The yield was estimated by comparing the two absorbances as “(ab − aa)/ab 100%”.One-milliliter samples of 1% Particles were characterized with a Zeiss fluorescence microscope (Axio Scope.A1) equipped with a mercury-vapor short-arc-lamp HB = 50 W/AC at 560 nm emission wavelength and 63 × objective, a confocal microscope , and a SEM . Prior to SEM observation, the samples were vacuum-dried. The above procedure applied identically for polymer derivatives labeled with RITC ref. . SEM andFor NMPs used in fluorescence assays, the standard preparation of polysaccharide in The degradation study was carried out over 24 h when the degradation appeared complete. An aliquot of 1 mL was used. At variable time intervals, the suspension was centrifuged, and the supernatant was analyzed using UV-vis spectroscopy. The supernatant contains the microparticles components after disassembly including the fluorescently labeled polysaccharides. The debris was not analyzed using SEM.Lysozyme and BSA were labeled as follows: Rhodamine B isothiocyanate (RITC) was dissolved into DMSO and 10 uL were added to 1 mg/mL protein solution. The resulting solution was stirred for a few hours at room temperature. To remove residual non-conjugated dyes, the samples were dialyzed using a dialysis membrane 12–14,000 Daltons ; the degree of functionalization was calculated as described in The loading of the proteins was performed as follows: 1.75 mg of RITC-lysozyme and 1.75 mg of RITC-BSA were dissolved in 1 mL PBS at pH 7 (precisely pH 7.4). Next, a suspension of NMP containing 1 mg of polysaccharides was added to the protein solutions. At variable intervals, the resulting suspensions were centrifuged, and the supernatants were analyzed using UV-vis spectroscopy (peaks at 560 nm for RITC). The plateau in the adsorption curve was obtained after 24 h incubation up to 250 h and the loading capacity was calculated using the maximum absorption values in the adsorption curves (@24 h plateau).To study dependence of protein loading and unloading in NMPs as a function of pH solution, an additional 1.75 mg of RITC-lysozyme  and 1.75 mg of RITC-BSA  were solved in 1 mL of PBS at pH 5 and 11 to obtain 4 more solutions. Each solution was monitored during 24 h at room temperature to test the stability of proteins at the different pH levels. d-NMPs and w-NMPs (polysaccharides concentration in both: 1.5 g/L) obtained from CL dissolved in DMSO and in H2O respectively were added to the NMPs, yielding 48 different samples for each combination of {protein (2), polysaccharide (4), CL protocol (2), pH (3)}. At variable intervals, the resulting suspensions were centrifuged, and the supernatants were analyzed with UV-vis spectroscopy (peaks at 560 nm for RITC). The plateau of adsorption curve—that is, the maximum of adsorption—was measured after 24 h. The lysozyme and BSA adsorbed particles were centrifuged and suspended in 1 mL of PBS at pH 5, 7, and 11. The resulting suspensions were centrifuged at variable intervals and the supernatant was analyzed with UV-vis spectroscopy. Every 24 h, fresh PBS was added. The plateau of release curve was obtained at 150 h for the sake of calculation (but the study was carried out for 300 h).2 atmosphere, in DMEM and L-Glutamine supplemented with 10% bovine calf serum . Cells were seeded at a density of 5 × 103 cells/cm2 (on 24-well plates or cover slips) and were allowed to attach for 24 h prior to the experiments until the population confluence reached approximately 80%. The NMPs were premixed with culture medium and incubated with 3T3 fibroblasts for different times and concentrations.Fibroblasts 3T3 were chosen as the model cell line and cultured at 37 °C in a 5% COThe trypan blue was used to determine the number of viable cells incubated with particles. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes such as trypan blue, whereas dead cells do not . The celTo investigate at the confocal microscope the possible internalization of NMP microsponges by 3T3 fibroblasts, NMPs were labeled with fluorescein-5-isothiocyanate (FITC). After culturing the cells for 24 h, they were incubated in plates with FITC-labelled NMP particles (0.25 g/mL) in DMEM 10% serum for 4 h, 24 h, or 48 h. At the end of each time period, the cells were washed with PBS to remove the excess/unbound NMPs and dead cells. The dead and living cells were counted using a Bürker-Türk counting chamber and the percentage of living cells was determined as an estimate of adherent cells after 4 h, 24 h, or 48 h incubation with NMPs. After incubation, cells were fixed and stained with a red fluorescent marker wheat germ agglutinin (WGA) conjugated to Texas Red  for 15 min at 37 °C to label the cell, which selectively recognizes plasma and Golgi/ER membrane structures. Afterwards, the cells were fixed with cold methanol and stained with Hoescht 33258  to visualize the nuclei. Such samples were observed by means of a confocal laser scanning microscopy . Confocal X-Y, X-Z, and Y-Z sections in max intensity projection mode  were taken to display three-dimensional images of MPs cell interaction.2O route). The purpose was to investigate the effects on structure and morphology of the resulting NMPs for a given polymer–solvent pair. The results are summarized in Eight batches (4 × 2) of nanoporous polysaccharide microsponges particles (NMP) were synthetized according to our standard one-pot self-precipitation/cross-linking method ,14 usingRemarkably, the w-NMPs consistently exhibit a core shell structure with a less fluorescent core and a highly fluorescent corona. This indicates that the RITC-polysaccharides are predominately located on the periphery of the NMP. Conversely, the d-NMPs synthetized via the DMSO route shows an even distribution of the fluorescent polysaccharides throughout the NMP for dextran and HA and a fluorescent core in the case of alginate and CM-dextran. These different structures can be ascribed to the different kinetic of crystallization of the CL in water and DMSO. The faster precipitation of the CL in water produces a crystalline core excluding the polysaccharides chains; subsequently, the polysaccharides chains form a composite polysaccharides-CL porous outer corona. During the slow precipitation of CL in the case of DMSO water solvent, the polysaccharide chains remain embedded during the entire process of growth of NMP. A peculiar onion-like structure was observed for alginate-based d-NMP with an inner layer composed primarily of CL. The schematics in From a synthesis standpoint, the reaction yields for each polysaccharide are consistently quite high, which is industrially relevant when considering that such values were observed also while scaling up the reaction.Finally, the degradation of NMP under redox treatment was evaluated as it is an important parameter for applications entailing the slow release of the polysaccharide. As the CL bears a disulfide bridge, we verified the ability of NMP to be deconstructed under redox treatment, similarly to what was done in our prior work . RITC laTo demonstrate the marked capability of NMPs to sequestrate proteins from a medium at physiological conditions (pH 7.4), we chose BSA and Lysozyme as two models of positively and negatively charged proteins respectively. In addition, while BSA is a relatively large protein (MW 65 kDa) and an isoelectric point (pI) of 5.3, Lysozyme is a small enzyme (MW 14 kDa) with a pI of 11.4. Non-labeled NMPs were incubated in aqueous solutions of RITC labeled proteins for 24 h; the adsorption capacity was quantified after the centrifugation of NMP and analysis of the supernatant . The adsThe release of proteins from loaded NMPs was evaluated next by replacing PBS solution and monitoring release via UV-vis spectroscopy at different incubation times up to 180 h. The release curves of all NMPs are shown in The comparison between loading and release efficiency for Lysozyme and BSA in Proteins uptake of d-NMP and w-NMP varies between 0.8–1.2 and 0.4–0.8 mg protein/mg polysaccharide, respectively. Lysozyme is preferentially taken up by d-NMP, whereas serum albumin is equally adsorbed by both d-NMP and w-NMP. This indicates that the composite nanostructured template of d-NMP interacts more efficiently with different type of proteins.On the other end, the efficiency of w-NMP (70–80%) in releasing the adsorbed proteins is higher than d-NMP efficiency (40–70%).The adsorption is nearly completed after 4 h while the release typically occurs on a scale of days.Overall these results demonstrate the capability of NMPs towards uploading proteins and highlight a characteristic asymmetry between adsorption and desorption. In fact, the strong capillary interactions that are responsible for the efficient protein-loading of NMPs is also the cause of slow release during discharge, opposing the osmotic gradient pull.The release profile depends on a number of parameters, and one of the most important of which for many biomedical applications  is the pH encountered by loaded NMP. Therefore, in addition to the above results related to physiological condition , we designed an experiment to assess the dependence of both adsorption and release performance of the eight NMP batches at pH 5 and 11. Hence, the six PBS solutions at pH 5, 7, and 11 containing RITC-lysozyme or RITC-BSA (6 samples) were monitored for 24 h at room temperature to ensure the stability of proteins loading and release.The absolute adsorption/release performance depends on the specific NMP polysaccharide  and of release curve (after 150 h) rendered the results in The actual uptake of the protein, apart from the gross value, depends also on how it penetrates and distribute within the NMP, which varies considerably between the 48 cases. Observations from confocal microscopy help appreciating this aspect by rendering the distribution of (RITC-labelled) protein content within each NMP type .Overall, these results indicate that the NMP nanostructures are endowed with a nanoporous morphology and high swelling capacity, which allows them to potentially accommodate bio-therapeutics such as proteins, DNA, and RNA, as well as small solutes dissolved in the up-taken aqueous phase. The protein adsorption inside the NMP is driven by a number of factors, including for example, (i) porosity and permeability of the composite template, (ii) hydrophobic interaction with the CL crystalline domain, and (iii) electrostatic interactions with the negatively charged polysaccharides. These factors are intertwined and dominate the overall distribution of the proteins inside the NMP template. Each NMP system shows a peculiar distribution of the proteins that is highly correlated to the specific and unique nanostructure of each NMP instance. Yet, we may try to better rationalize underlying (multi-factors) trends by using advanced statistical tools in future work. For instance, alginate-based d-NMP adsorb both proteins on the outer layer precluding their access in the core. Conversely alginate-based w-NMP more efficiently accommodate proteins in the hydrophobic core with pH dependence, indicating that the sequestration of proteins by alginate NMP can be controlled in different physiological environments such as the stomach (acidic) or the gut (basic).n = 3, p < 0.05). No value was found significant compared to the relative controls, indicating lack of cytotoxicity of both CL and NMPs. This confirms that NMPs are indeed safe and non-toxic, as observed in our prior study [The toxicity of NMPs was assessed on T3T fibroblasts as survival cells in trypan blue test under several experimental conditions. Cells were grown in complete medium (Ctrl) or in complete medium supplemented with different concentrations ranging from 0.25 mg/mL to 2.25 mg/mL of all eight batches of NMPs, for 24 and 120 h. As the CL exerts a vital role in the fabrication of NMPs, an additional control (CRLK) was performed by adding the CL in complete medium without the polysaccharide. Results are reported in or study . These rTo analyze the interaction between NMPs to 3T3 cells, FITC-labelled NMPs were added to fibroblasts cultured in 24 multiwells. The number of NMPs in each sample was quantified after 4, 24, and 48 h incubation by counting via a Bürker– Türk counting chamber with the unbound NMPs removed from suspension. The results showed that a large fraction of NMPs readily associated with fibroblast within the first 4 h of incubation . HoweverIn order to understand the nature of the uptake and determine whether NMPs localized inside or outside the cells, confocal microscopy analyses were performed. Fibroblasts were incubated with FITC-labeled NMPs (0.25 mg/mL) for 4, 24, or 48 h. After incubation cells were stained with a red fluorescent marker WGA for highlighting plasma and Golgi/ER membrane structures. Results are shown in Typically, microparticles with a diameter longer than 0.5 µm have been known to enter cells via phagocytosis pathways . GeneralIn this work, we demonstrated the general use of one-pot self-precipitation/cross-linking technique to prepare highly porous polysaccharide particles based on polysaccharides, namely dextran, carboxymethyl dextran, alginate, and (low molecular weight) hyaluronic acid. Our methodology involves the cross-linking agent precipitation to form crystalline domains embedding the polysaccharides chains which act as a “glue” between the crystalline domains. The study proves the robustness and versatility of this fabrication method.The efficient loading of BSA and lysozyme into the NMP was shown along with the sustained release of the two proteins. The dependence of the adsorption-release performance on pH was demonstrated as well, which bears implications in medical applications and represents a possible way of tweaking the release profile. Finally, cell interaction was investigated to prove that NMPs are not cytotoxic but markedly biocompatible. Overall, this work highlights that this methodology and nanostructured microsystems could offer an innovative platform for biomedical and clinical applications.