N’-(4-chlorobenzoyl)-N,N-di-n-butylthiourea (HL) and its Ni(II) complex have been digitalized by measuring with thermal analyses such as DTA, TG with hardware technical equipment. By entering the raw experimental data taken into the prepared software program, thermodynamic data such as enthalpy and entropy can be calculated as well as kinetic parameters such as activation energy, reaction order, and rate constant. In this study, a software program was developed that can be used in the calculation of the desired thermodynamic parameters by using the experimentally measured ΔT, Δm, and dΔm values. The accuracy of the results was compared with the thermal results of the NaN3 reference material.In gas and liquid phase reactions, the conversion of starting materials to products is directly controlled by the rate of the reaction, whereas in solid state reactions, it is determined by the rate of diffusion. When working with thermal analysis methods, tens of thousands of data to be obtained from measured differential thermal analysis (DTA), thermal gravimetry (TG), and differential thermal gravimetry (DTG) curves must first be digitized, and then these digitized data must be converted to concentrations and thermal values using appropriate software programs. Companies that produce devices using thermal analysis techniques generally adapt the encrypted software programs they use according to the needs of the user companies. Since the devices work like a black box, it is impossible for users to make any changes to the packaged programs. In addition, since the program cannot be seen, its logic cannot be understood most of the time. The program developed in this study will be put at the service of every researcher, and the system of the program can be used on any Windows Software compatible computer. While preparing the program, an easy-to-understand and flexible MATLAB environment was used. The thermal analysis data of synthesized  Events such as sintering, melting, sublimation, or phase change can occur due to the increase in molecular, atomic, or ion movements when a pure solid substance is heated in an inert gas atmosphere , 2. If iIf there is more than one solid in the environment, there may be more interaction. As a result of double decomposition and addition reactions, new compounds can be formed, as well as solid solutions and eutectic mixtures , 6.Thermal analysis methods such as TG and DTG are being used more and more each day to examine the kinetics of pyrolysis reactions . Two basIn this study, the software program prepared based on the Broido Method is presented. By entering the raw experimental data received, besides thermodynamic data such as enthalpy and entropy, kinetic parameters such as activation energy, reaction order, and rate constant can also be calculated. In this study, a software program that can be used in the calculation of the desired thermodynamic parameters was developed by using the experimentally measured ΔT, Δm, and dΔm values.MATLAB 5.2 environment was preferred in the study. MATLAB is a technical program developed for high-performance numerical computations and graphical programming. It offers numerical analysis, matrix operations, signal processing, and graphics drawing as a whole in an easy-to-use environment. MATLAB is derived from the words ‘MATrix LABoratuary’. In MATLAB, the basic element is a ‘matrix’ that requires no sizing. It is possible to solve many numerical problems in a very short time with MATLAB. It is also possible to write algorithms as in the Basic, Fortran, or C programming languages. MATLAB also includes a set of ready-made solution packages and functions written for specific applications, called ‘toolboxes’. Toolboxes consist of M-files needed to solve a problem. The algorithms used in MATLAB are based on the “least squares method” used in numerical integral and derivative calculations with the help of computers. It is possible to use MATLAB on PC, Macintosh, SUN, UNIX, VAX, Apollo, HP, DECStation, SGI, RS/6000, and Convex computer systems.The prepared program was applied for calculating kinetic parameters of thermal decomposition of newly synthesized thiourea derivative and its complex. N’-(4-chlorobenzoyl)-N,N-di-n-butylthiourea and the Ni(II) complex of these ligands were synthesized and characterized. Decomposition knetic studies were carried out on the program prepared using thermal analysis data .2 complex are shown in 2 complex shows an initial mass loss in the temperature range of 501–612 K, corresponding to the decomposition of the complex to Ni(SCN)2. The mass loss at this stage is attributed to the evolved moieties di-n-butylbenzamide . These agree with the literature data  gives the heating rate.If If the integration of both sides of the obtained has the expression. Pyrolysis reactions are generally first order. The integration of the right-hand side of the equation was explored by Vallet and published as a monograph in 1961 [25]. According to this, the following :Equation dT is written from If the right-hand side of  is combiSolution of the integral part of If this expression is substituted at ,is given by the expression. Integrate both sides of :Substituting the value of z in (2.34)Herethe expressions can be represented by m and J. When 3/E3)] is constant. Accordingly, the equation can be written as (In itten as :Plotting 1/T against lnln(1/y) results in a linear graph. (The linear plot of ln1/T versus lnln(1/y) gives good results in the range of 0.999 > y > 0.001.)The PC program was written and compiled using C++ programming language in the MATLAB 5.2 environment. Preparation of the program was in easy and flexible MATLAB concept. By using the TG data of the synthesized ligands and chelates, the program can perform the kinetic analysis of the pyrolysis reactions with the help of the Broido kinetic method .In the program, only the percent amount of mass change and the relevant temperature from the TG analysis results are used as data. By using the prepared software program, raw experimental data and thermodynamic data such as entropy can be calculated as well as kinetic parameters such as activation energy, reaction order, and rate constant. The program accepts the reaction degree as ‘1’ for the Broido kinetic method and the flow diagram is given in 3 was examined as a reference and the program written in the ‘MATLAB 5.2’ environment was tested. TG/DTG/DTA analyses were performed in a differential thermal analyzer with analytical grade NaNO3 and α-Al2O3 reference material obtained from Merck.In this study, the kinetics of the pyrolysis reaction of NaNO−1 N2 gas flow rate and dynamic nitrogen atmosphere using a Pt reaction vessel. The TG/DTG/DTA diagrams of the NaNO3 compound are given in 3 (51.2 kJ/mol) was consistent with that in the literature (45.3 kJ/mol).Seventeen milligrams of sample was used for the measurement made at 10 K minTG/DTA/DTG and GC/MS analyses were interpreted and it was determined that the ligand was decomposed in accordance with the reaction , 3.1b.The decomposition kinetic data of the HL ligand calculated in the software program using the Broido method are given in TG/DTA/DTG and GC/MS analyses were interpreted and it was determined that the ligand was degraded in accordance with the reaction .2] complex, calculated in the software program using the Broido method, are given in The decomposition kinetic data of the  complex were investigated using TG/DTG/DTA and GC-MS combined systems.In this study, the decomposition mechanism and kinetics of the previously synthesized and characterized 3 and α-Al2O3 were used as reference materials. Seventeen milligrams of sample was used and the measurements were made under 10 mL/min N2 gas flow rate using a Pt reaction pan. It is seen that the calculated energy value (51.2 kJ/mol) for NaNO3 is compatible with that in the literature (45.3 kJ/mol). It was determined that there is an absolute error of 1.1% between the values in the literature and the calculated values of the NaNO3 compound. This error is thought to be caused by the calibration of the device and the purity level of the substance. Thus, the accuracy of the kinetic parameters calculated in the program written in MATLAB 5.2 environment according to the Broido method of the decomposition reactions with the NaNO3 compound was tested.TG/DTG/DTA analyses were performed in a differential thermal analyzer. Analytical grade NaNO*, S, A) of the HL ligand and its Ni(II) complex obtained with the prepared PC software are given in Calculated kinetic parameters (E


Liposomes have received tremendous attention as a class of versatile pharmaceutical vehicles of great potential over the past several decades. However, the application of liposomes encounters major challenges due to the knowledge gaps in their in vivo delivery process. Immunoglobulin M (IgM) displays both pervasiveness and complexity in regulating the biological functions as well as eliciting adverse effects of liposomes. Understanding, mitigating, and exploiting the duality of IgM are prerequisites for achieving various biomedical applications of liposomes. In this review, the intricate relationship between liposomes and their biological environments has been summarized, with an emphasis on the regulatory effects of IgM on in vivo performance of liposomes. Corresponding solutions have also been discussed to evade IgM‐mediated opsonization for safe and efficient drug delivery. In this review, the intricate relationships between the regulatory effects of immunoglobulin M (IgM) and liposome performance in vivo are organized and contemplated. Corresponding strategies are proposed to evade IgM‐mediated opsonization for safe and efficient drug delivery. With this mechanistic knowledge as guidance, it is beneficial to design liposomes for in vivo delivery in a controlled manner. Meanwhile, some liposomal products, with the example of Doxil, DaunoXome, Myocet for the treatment of cancers and Ambisome for antifungal therapy, have reached the market. Despite these successes, substantial challenges remain for clinical translation and application of liposomes. For example, the delivery efficiency of liposomes was only 0.5% as reported, representing nonspecific interaction rather than specific targeting. Increasing adverse effects represented by hypersensitivity reactions make the clinical application of liposomal therapeutics now in a plight. In addition, the major improvement of Doxil appears to be the reduction of cardiotoxicity compared to free doxorubicin, rather than a significant amelioration of the survival time of patients. Significant improvements are still required to promote the safety and efficiency of delivering therapeutics to target cells.The use of liposomes as potential drug delivery systems was envisioned shortly after their discovery by Bangham in 1964.1 Afte Validating the key plasma proteins that dominate in vivo performance of liposomes and revealing the regulatory mechanisms are vital for guiding rational medication in clinical practice. Among the numerous plasma proteins, immunoglobulin M (IgM) has received increasing attention due to its ubiquity and complexity in regulating the biological functions of liposomes in vivo.This gap between laboratory findings and clinical application largely results from the limited knowledge of the in vivo delivery process of liposomes. The mechanistic study on reciprocal interaction between liposomes and the immune system is a key scientific issue in the field of nano‐biological effects and for rational design of liposome‐based nanomedicines. In complex pathophysiological environments, liposomes interact with numerous plasma proteins to form the “protein corona” that regulates circulation time, biodistribution, drug release, immunotoxicity, and cellular interactions of liposomes.11 ValIn this review, we first describe ubiquitous IgM adsorption on liposomal surfaces. Then, modulatory effects of IgM on the in vivo performance of liposomes  have been highlighted, such as altered pharmacokinetics, reduced pharmacodynamics, and induction of adverse reactions. We also summarize the corresponding strategies to improve in vivo performance of liposomes by manipulating the reciprocal interaction between IgM and liposomes.22.1Figure The protein coronas usually consist of two layers, the inner layer (hard corona) formed by proteins tightly bound to the liposome and the outer layer (soft corona) composed of loosely bound proteins. The boundary between the two layers is blurred and the corona is always in a thermodynamic equilibrium during blood circulation.The knowledge gap in the interplay between liposomes and the physiological environment, for example, the blood and interstitial fluids, is probably one of the major reasons responsible for the limited clinical use of liposome‐based therapeutics. Most of the commercially available liposomal products are administered systemically. Once administered, liposomes are immediately surrounded by various plasma proteins , forming the so‐called “protein coronas” as shown in There are dozens of plasma proteins with varying identities and quantities, and the composition of protein coronas can be greatly influenced by the complexity of the physiological environment. As a result, the impact of protein adsorption on liposomes can be multifold. It might mask the pre‐set chemical or biological functions of liposomes and govern the in vivo fate, including circulation lifetime, biodistribution, cellular uptake, and targeting ability. Even though the liposomes are surface‐modified for certain chemical or biological functions, they can be easily masked by the corona. Moreover, the hydration layer around the liposomes is displaced upon protein binding, and the resulting protein aggregation and conformational changes may trigger immune responses in hosts.142.2 Therefore, validating the key plasma protein(s) dominating the in vivo performance of liposomes and revealing the regulatory mechanisms play crucial roles to guide the precise medication of liposome‐based therapeutics. Situations can be even more complicated because the composition and content of plasma proteins undergo dramatic changes in patients during disease progression, which severely exacerbates the unpredictability of the characteristics of protein coronas. Kostarelos et al. described the formation of human‐derived protein coronas on Caelyx (doxorubicin‐encapsulated PEGylated liposomes) following the collection of liposomes from the blood circulation of ovarian cancer patients. Immunoglobulins, lipoproteins, and complement proteins were found to be the most abundant proteins, accounting for 28%, 9%, and 4% of the total protein content, respectively. Among immunoglobulins, IgM  was found to be the most abundant in liposomal protein corona, whose content was even higher than that of IgG .Hundreds of plasma proteins have been confirmed to deposit on liposomal surfaces, some of which might have major impacts on the in vivo fates of liposomes, while others do not.15 The which would be discussed in a later section.Although surface modification endows liposomes with better in vivo properties such as prolonged circulation time or specific targeting ability, it may also change the corona composition. By preparing liposomes with different targeting ligands  and molar ratios, we identified that IgM was ubiquitously adsorbed on liposomes with different modifications. IgM plays a critical role in complement activation, negatively correlating with the in vivo performance of liposomes,20 whi An important feature of anti‐PEG antibodies is that they can be detected even in “treatment‐naïve” individuals, with a prevalence of 0.2% in 1984, 25% in 2012, 40% in 2016, and a surprising 97.5% in 2019. This phenomenon is commonly known as pre‐existing anti‐PEG antibodies. Antibody isotype analysis showed that pre‐existing anti‐PEG antibodies were 25% IgM, 18% IgG, and 30% both IgG and IgM in the general population. A genome‐wide association study in 177 Han Chinese individuals identified the variable segment of immunoglobulin heavy chain locus is associated with IgM antibody responses to PEG, providing novel genetic markers for predicting the risk of allergic reactions and the effectiveness of PEGylated drugs. In nearly all animal studies, major serum proteins binding to PEGylated liposomes was identified to be IgM isotype. The reduced efficacy of PEGylated therapeutics in clinical trials has been reported to be associated with the presence of pre‐existing anti‐PEG antibodies. They dramatically altered the half‐life and therapeutic effect of PEGylated drugs in patients.In addition, most of the clinically approved liposomal products are PEGylated that are able to elicit anti‐PEG antibodies.21 An 2.3 sIgM is usually pentameric, containing five pairs of antigen‐binding sites and a joining (J) chain. It is occasionally found in hexamer, with six pairs of antigen‐binding sites but no J chain  and secreted IgM (sIgM). mIgM presented on B cellular surface is a main constituent component of B cell receptor (BCR). It controls B cell maturation, activation, and differentiation. sIgM is mainly found in blood, contributing to immune supervision by recognizing pathogens and mediating immune reactions.32 sIg Since it is the first line of defense against pathogens before the formation of germinal centers where adaptive antibodies are formed, nIgM is polyreactive. Adaptive IgM is secreted mainly by B2 cells several days following pathogens exposure. This process is T‐cell dependent and requires interactions between antigens and BCR on B2 cells. Adaptive IgM is antigen‐specific, with only limited cross‐reactivity with other antigens. Anti‐PEG IgM falls into this category.sIgM can be further classified as pre‐immune “natural” IgM (nIgM) and antigen‐induced “adaptive” IgM. nIgM is produced spontaneously by B lymphocytes, mainly B1 cells and marginal zone B (MZB) cells, without specific immunization.34 Sinα/µR, and pIgR) expressed on various cell membranes and soluble molecules such as complement components  into C4b, thereby promoting the classical pathway of complement activation. This unique structure may well explain its 1000‐fold higher C1q binding affinity compared with that of IgG. sIgM has very potent complement‐dependent cytotoxicity (CDC) effects because it is better at fixing complement compared with IgG, which utilized natural killer cells to exert antibody‐dependent cell‐mediated cytotoxicity (ADCC) via Fc gamma receptors. However, complement activation is a “double‐edged sword”. Unwanted complement activations also cause tissue damage and opsonization of therapeutics. Specifically, complement activation gives rise to powerful anaphylatoxins such as C4a, C3a, and C5a, leading to inflammatory responses and even anaphylaxis in some individuals. The subsequently generated membrane attack complex (C5b‐9) is also responsible for the nonlytic stimulation of vascular endothelial cells, endothelial regulation of hemostasis, and inflammatory cell recruitment.sIgM‐antigen complexes mediated biological activities require interactions of IgM Fc segment with effector molecules including Fc receptors  phenomenon, or directly neutralization. Additionally, adverse reactions represented by hypersensitivity reactions (HSRs) caused by PEGylated liposomes have become new issues. In this section, the effect of IgM on the physicochemical properties and in vivo biological performance of liposomes is presented and discussed in depth.3.13.1.1 Later, they demonstrated that the binding of only a few surface‐bound immunoglobulin molecules, specifically IgG, was needed to trigger complement activation. However, the formation of protein coronas is largely affected by the properties of nanomaterials, such as material composition, surface charge, diameter, hydrophobicity, morphology, and stiffness. As for liposomes, Guan et al. verified that nIgM adsorption was more ubiquitous compared with that of IgG on the liposomal surface of DCDX‐modified liposomes and plays important roles in activating complement via the classical pathway. Furthermore, anti‐PEG IgM antibodies induced by PEGylated liposomes also activate the complement system.Substantial experimental evidence has indicated that nanoparticle‐corona complexes activate the complement system either via the classical, lectin, or alternative pathway. For instance, Chen et al. reported that superparamagnetic iron oxide nanoworms incubated in human plasma and serum rapidly activated the complement system via the alternative pathway.42 Latγ receptors on macrophages), IgM mediates microbial opsonization through classical complement activation pathway, followed by C3 deposition on the microbial surface with consequent recognition through complement receptor 3 (CR3). CR3 is an opsonic receptor that recognizes C3 fragments such as C3b and iC3b, which binds to multiple sites on the surface of liposomes, aiding recognition and rapid clearance of liposomes by macrophages resident in the reticuloendothelial system bearing complement receptors . Taking ganglioside‐containing liposomes as an example, naturally occurring low‐affinity anti‐GM ganglioside IgM antibodies (mainly anti‐GM1) are part of the normal antibody repertoire of healthy humans. However, in the plasma of patients with autoimmune neuropathies like Guillain–Barré Syndrome, elevated titers of high‐affinity anti‐GM IgM antibodies are detected. When the GM1‐liposomes are exposed to blood, the complement system is activated through the classical pathway. Subsequently, the activated complement components deposit on the liposomal surface, serving as opsonins to promote the lysis of GM1‐liposomes and the uptake by hepatic Kupffer cells.The primary outcome of liposomes/IgM complex‐mediated complement activation is surface opsonization. Opsonization is an immune process that uses blood components called “opsonins,” such as antibodies and complement proteins, to label non‐self particles for clearance from the bloodstream by phagocytes. Compared to the opsonic effect of IgG , but with interspecies differences. It was reported that the ABC phenomenon plateaued when the interval was 7 days, beyond which it gradually vanished over weeks. Notably, the time window to observe the ABC phenomenon is consistent with the circulation of IgM (3 weeks). Many factors affect the strength of the ABC phenomenon such as the dosage of the injected liposomes. For example, Doxil had no ABC phenomenon at a dose of 20 mg m−2, while it occurred at a low dose of 0.2 mg m−2. It was hypothesized that a high dose of PEGylated liposome could induce immune tolerance or B cell anergy in mice. Besides PEGylated liposomes, other PEGylated nanocarriers can also have ABC phenomenon, such as PEGylated nanoemulsion, PEGylated PLGA nanoparticles, PEGylated solid lipid nanoparticles.In 2000, Dams et al. discovered the ABC phenomenon in rats, that is, after the first injection of PEGylated liposomes, the repeatedly injected PEGylated liposomes at intervals of several days were rapidly cleared and then accumulated in liver and spleen.49 The Splenic B lymphocytes are involved in recognizing PEGylated liposomes and inducing the production of anti‐PEG IgM antibodies. In rats that received splenectomy before the first injection of PEGylated liposomes, serum IgM concentrations and the amounts of IgM that bound to PEGylated liposomes were significantly decreased and the ABC phenomenon was completely abolished. TI immunogenicity (without the involvement of CD4+ T cells) is considered to be the main mechanism of antibody induction by PEGylated liposomes due to the typical repeating structure of PEG. In such a process, MZB cells are specialized for the capture of PEGylated liposomes through crosslinking of BCR, and rapidly produce massive but low‐affinity IgM antibodies without the help of T cells within 5 days. Subsequent complement‐mediated opsonization induced by anti‐PEG IgM antibodies is an important part of the ABC phenomenon. Therefore, the TI molecular mechanism of ABC could be generalized in the following key steps as shown in FigureAnti‐PEG IgM antibodies elicited following the injection of PEGylated liposomes are shown to account for the ABC phenomenon. The anti‐PEG IgM antibodies exhibit potent neutralizing effects on the subsequently administered PEGylated liposomes in vivo. In animals with circulating anti‐PEG IgM antibodies, the circulation time of PEGylated liposomes is greatly reduced.56 Spl Recently, Deng et al. determined that PEGylated nano‐emulsions (PE) could induce innate immune memory in Kupffer cells. This memory could be transmitted by the PE‐stimulated Kupffer cells to the naïve rats through adoptive transfer, leading to increased hepatic accumulation of PE in the recipient rats, independently of other cells or components until 21 days after the first stimulation. The discovery of KC immune memory revisited the relationship between the innate immune system and PEGylated nanomedicine, providing new insights into improving medication in the clinic.Kupffer cells, as the most abundant phagocytic cells in the tissues, are the main contributors to the elimination of PEGylated liposomes and the induction of ABC phenomenon.59 Rec3.1.3 However, compared to plain liposomes such as Doxil, the number of targeted liposomes that entered the clinical stage is fewer, including MM‐302 (targeting HER2 receptor using an anti‐ErbB2‐scFv for treatment of breast cancer), 2B3‐101 (targeting glutathione receptor at the blood‐brain barrier for treatment of glioma and brain metastases), and MBP‐426 . Meanwhile, a growing number of contradictory literature suggests that active targeting strategies do not eventually result in enhanced tumor accumulation. The connection between the “off‐target” effect of nanomedicines and the formation of protein coronas has not been observed until recent years. For instance, Salvati et al. discovered that proteins could hinder the transferrin from binding to both its membrane‐bound receptors and soluble receptors in biological environments. As a result, the targeting molecules on the transferrin‐conjugated nanoparticles were screened and the specificity in targeting was impaired. Similarly, protein coronas shields also decreased the binding affinity of anti‐HER2 antibodies, cyclic RGD peptides, or small molecule ligands toward their targets.Active targeting strategies that conjugate ligands  to the carrier surface have long been investigated in cancer nanomedicine.61 How However, all of the FA‐enabled nanomedicine failed in clinical trials . Furthermore, FA‐enabled targeted therapies also have inconsistent results in preclinical studies. Recently, we re‐examined the regulatory roles of the key plasma protein in FA‐modified liposomes (FA‐sLip). It was demonstrated that FA functionalization caused avid deposition of nIgM on the FA‐sLip surface and the binding affinity was positively correlated with the FA modification. The nIgM deposition subsequently activated the complement cascade‐like opsonization by liver and spleen‐resident macrophages, resulting in ABC phenomenon eventually. Most importantly, the binding affinity of FA with folate receptors was inhibited and tumor targeting capacity was severely hindered in folate receptor‐positive tumor models. It is of note that human nIgM also demonstrated strong interaction with FA‐sLip and efficiently activated complement. The results have at least three enlightenments for us. First, nIgM was validated as a key plasma protein dominating in vivo performance of FA‐sLip. Second, patient screening was required for the clinical medication of FA‐modified targeted nanomedicines considering the variable titers of IgM in cancer patients. Third, the IgM binding capacity needed to be taken into account in the de novo design of targeted drug delivery systems. Liposomes have long been used as adjuvants to enhance immune effects. FA‐sLip becomes immunogenic just as enhanced immunogenicity of small haptens when combined with a larger carrier. The reason why the enhanced immunogenicity of FA‐enabled nanomedicine was underestimated might be the immune suppression caused by cytotoxic payloads. However, with the continuous emergence of non‐cytotoxic drugs such as antibodies and proteins, the immunogenicity of FA‐sLip as a drug delivery vehicle cannot be ignored.Among them, folic acid (FA) is undoubtedly one of the most extensively exploited “star” small molecules to facilitate targeted delivery of nanomedicine by recognition of folate receptors which are overexpressed in many kinds of human cancer cells.71 How3.23.2.1It is generally accepted that the development of neutralizing antibodies can be expected within days after an initial dose of a biotherapeutic. Neutralizing antibodies, typically low‐affinity IgM‐type immunoglobulins, are initially induced during the initial stages of immune responses. Both TD and TI mechanisms of B‐cell activation have been proposed for IgM generation of immune responses. The early‐onset IgM can later mature into high‐affinity IgG subclasses (IgG1 and IgG4). PEG‐uricase, and PEG‐interferon. Concerns have been raised regarding the immunogenicity of PEGylated liposomes. Hsieh et al. generated two anti‐PEG antibody mouse models, that is endo αPEG (naïve mice administered with PEGylated protein for endogenous anti‐PEG IgM and IgG) and αPEG‐PT . Tumor‐bearing naïve, endo αPEG, and αPEG‐PT mice were then i.v. treated with 111In‐labeled PEGylated doxorubicin‐loaded liposomes. Reduced tumor accumulation and therapeutic efficacy were found in endo αPEG and αPEG‐PT mice. Although direct evidence linking neutralizing antibodies to the limited therapeutic effect of Doxil remains unexplored in clinical trials, it should not be overlooked considering the pre‐existing anti‐PEG IgM antibodies in the population, especially in patients requiring repeated injections.In the clinic, neutralizing antibodies produced in patients treated with certain PEGylated biologics are known to be associated with loss of therapeutic efficacy, with examples of PEG‐asparaginase,29 PEG3.2.2 Pre‐existing or elicited anti‐PEG IgM antibodies by PEGylated liposomes can trigger complement activation. Since the phospholipid composition of liposomes is very similar to that of biological membranes, MAC can also be formed on liposomes. Chen et al. showed that anti‐PEG antibodies (IgM and IgG) bind to PEG molecules on PEGylated liposomal doxorubicin, resulting in complement activation and MAC insertion in the liposomal membrane. Consequently, up to 40% of the encapsulated doxorubicin was released from the liposomes. Senti et al. further investigated the outcomes and mechanisms of complement activation following the interaction of anti‐PEG antibodies with different PEGylated lipid‐based nanoparticles, liposomes loaded with different model drugs, and lipid nanoparticles encapsulated with mRNA. They demonstrated that complement activation initiated by anti‐PEG antibodies could potentially compromise the integrity of lipid nanoparticles, leading to unwanted drug release or exposure of mRNA to serum proteins. In this case, the protective effect of liposomes was lost, the loaded drug was quickly cleared, and even serious toxic side effects were caused due to the burst release of drugs.Once triggered by one of three pathways , complement activation can result in multiple relative functions. For example, C5 along with the other terminal complement factors deposits into the membrane of pathogens to form the membrane attack complex . The MAC forms a small channel in the lipid membrane, leading to cellular contents efflux and cell death, which is the main mechanism of complement‐dependent cytotoxicity.79 Pre3.2.3In recent years, the incidence of side effects of PEGylated liposomes represented by Doxil has increased with their extensive clinical application. Doxil‐related mild to severe hypersensitivity reactions like breathing difficulty, facial swelling, chest pain, flushing, rash, and blood pressure aberrance can occasionally be life‐threatening. The immediate, non‐IgE‐mediated acute hypersensitivity reactions are defined as complement activation‐related pseudo‐allergy (CARPA). For example, up to 45% of patients treated with Doxil without steroids and antihistamines medication developed CARPA, and this reaction still occurred in 4–7.1% of patients premedicated with steroids and antihistamines. The phenomenon reflects an unsolved immune dilemma to the clinical application of these nanomedicines, yet the mechanisms are inadequately explored. It is assumed that CARPA of Doxil is caused by PEGylated liposomes as free doxorubicin alone does not induce these hypersensitivity syndromes. In fact, CARPA is closely related to the activation of the complement system. Anaphylatoxins  generated in complement activation specifically bind to their receptors on immune cells , triggering the release of various vasoactive mediators, including histamine, leukotrienes, tryptase, platelet‐activating factor, thromboxane A2, and prostaglandins. Recent evidence highlighted the causal role of anti‐PEG IgM antibodies in the initiation of CARPA by PEGylated liposomes. Single i.v. injection of a low dose of PEGylated liposomes  in pigs induced massive anti‐PEG IgM antibodies formation in blood, with the concentration peaking on days 6–9 and decreasing slowly in 6 weeks. Bolus injections of the same liposomes during seroconversion led to fatal anaphylactic reactions within 2–3 min in immunized pigs. Similar treatments in naïve animals only resulted in minor reactions. The results suggest that the classical complement activation triggered by rapid binding of anti‐PEG IgM antibodies to PEGylated liposomes is closely related to anaphylactoid shock. In addition, pre‐existing anti‐PEG antibodies have also been linked to CARPA after the infusion of PEGylated medicines.CARPA has been confirmed in both experimental and clinically approved liposomal therapeutics , micelles , iron oxides  and LNP‐based COVID‐19 mRNA vaccine.87 For44.1 In patients suffering from chronic liver diseases and autoimmune diseases, the IgM is predominantly monomeric instead of multimeric. To make matters worse, IgM levels and functions undergo dynamic changes during disease progression and medication, especially in cancer patients receiving chemotherapy, severely exacerbating the unpredictability of liposome clinical use. The immune system disorders in patients may be a crucial factor in individual differences in the efficiency and side effects of clinically approved liposome‐based therapies. It is notable that anti‐PEG IgM antibodies titer in general population also exists individual differences with high antibody levels in a small part of samples. Thus, prescreening the suitable population through IgM affinity tests before medication is critical to maximizing the clinical benefits of liposomal therapeutics.The IgM‐protein corona varies among individuals due to not only proteome heterogeneity but also disease‐related alterations in IgM titers and structures. For instance, in patients with hyper IgM syndrome, a rare primary immunodeficiency disorder, the blood titers of IgM can be abnormally elevated while the concentrations of IgG, IgA, and IgE are decreased.91 In  Post‐rituximab IgM hypogammaglobulinemia has caused clinical concerns because of the significant role of IgM in protective immunity, but it provides an ideal time window for the delivery of liposome‐based therapeutics.Meanwhile, IgM in pathological conditions can be leveraged to improve liposome‐based therapies. For example, post‐rituximab IgM hypogammaglobulinemia refers to low IgM levels resulting from the administration of rituximab. Rituximab is a CD20‐positive B cell‐targeting chimeric monoclonal antibody. It is currently used to treat lymphoma  and a variety of autoimmune diseases . However, rituximab also kills normal B cell subsets that contributed to IgM production. Clinical data suggest that the prevalence of IgM hypogammaglobulinemia following several cycles of therapy with rituximab ranges between 10% and 58%.94 Pos4.2 and clodronate liposomes, can significantly prolong liposome circulation. However, poisonous compounds accumulated in Kupffer cells will result in cell apoptosis or cell necrosis. It usually takes at least 2 weeks for the recovery of Kupffer cells, during which bacteremia will be fatal to patients. A less aggressive approach is based on the efficient MPS blockade by injecting large doses of foreign substances such as dextran sulfate, colloidal carbon, liposomes, and fat emulsions. But dose‐related toxicity considerations limit the application of this strategy in clinical practice and even basic research. An alternative, called MPS‐cytoblockade, is a conceptually different approach by injecting allogeneic anti‐erythrocyte antibodies to force the MPS to boost the clearance of its own intact erythrocytes.The rapid blood clearance of liposomes induced by the MPS significantly restricts the therapeutic efficiency of many liposomal formulations. Administration of macrophage‐depleting toxic compounds, such as gadolinium chloride95 and However, excessive depletion or blockade of Kupffer cells instead would exacerbate the ABC phenomenon because more circulating liposomes in blood (within a range of concentrations) would stimulate B cells to produce more anti‐PEG IgM antibodies.The hepatic clearance capability, the serum anti‐PEG IgM concentration, and the complement activation intensity are three important contributors to the ABC phenomenon of liposomes. Ishida et al. found that the strength of complement activation in mice increased linearly with the increase of serum anti‐PEG IgM antibody levels. But hepatic clearance of PEGylated liposomes was first increased and then saturated, suggesting that hepatic uptake is a limiting step in the ABC phenomenon.102 Ho In addition, mice inter‐strain variabilities have been seriously overlooked as far as the phagocytic capacity of MPS is concerned. We proposed that inter‐strain variabilities in liposome pharmacokinetic profiles could be calibrated using the corrected phagocytic rate (KC= K × (c × Ig) / ), especially for serum opsonin‐sensitive liposomes , where c, Ig, alb and apo represent the total content of complement proteins, immunoglobulins, albumin, and apolipoproteins, respectively.Although liver is the primary organ that contributes to blood clearance of liposomes, the specific mechanisms differ among species. For example, hepatic uptake of liposomes in mice does not involve specific serum opsonin. In contrast, the uptake of liposomes by rat liver is intensely dependent on serum opsonin.104 In4.3 For example, in rats administered with cyclophosphamide to deplete splenic MZB cells, the anti‐PEG IgM response was diminished. Therapeutic doses of Doxil also inhibit the production of anti‐PEG IgM antibodies due to cytotoxicity on B cells, which may be the reason why no Doxil related ABC phenomenon has been reported clinically. In addition, similar to the blockade of the MPS, blocking stimulation of PEG‐specific B cells in the spleen represents a strategy for lessening the production of anti‐PEG IgM antibodies. Constant and intensive antigen stimulation can block the BCR signal pathway and desensitize B cells to induce B‐cell anergy. As a result, antibody production ceased. Lai et al. reported that pre‐administration of a relatively low dose (5.5 mg kg−1) of high molecular weight (40 kDa) free PEG led to a threefold reduction of anti‐PEG IgM antibodies compared to PBS treatment 7 days following injecting of empty PEGylated liposomes. Furthermore, the fusion of free PEG effectively saturated anti‐PEG IgM antibodies, restoring the circulation of PEGylated liposomes in mice with high titers of pre‐existing anti‐PEG IgM antibodies.Co‐administration of immunosuppressive agents  or cytotoxic drugs  has previously been demonstrated to diminish antibody responses to both TD and TI antigens.107 Fo4.44.4.1 Ishida et al. reported a polyglycerol‐derived polymer lipid conjugate which was utilized as a biocompatible alternative polymer for liposome modification. They found that the hydroxymethyl pendant group in the polyglycerol repeating subunit could eliminate the production of anti‐IgM antibodies in rodents, thereby preventing the ABC phenomenon and improving the in vivo performance of polyglycerol‐coated liposomes after repeated injections.Although the stealth coating using PEG greatly reduce nonspecific opsonization, prominent protein coronas have been reported to inevitably form around liposomal surfaces in the bloodstream. Antibody responses induced by protein coronas can minimize the stealth effect for repeated injections . Structural optimization of the PEG moieties is one of the successful approaches to evade corona formation and IgM adsorption. For instance, branched PEG‐modified liposomes induced significantly lower anti‐PEG IgM antibody levels and complement activation than linear PEG‐modified liposomes, holding promise in avoiding the ABC phenomenon, reducing CARPA, and improving the antitumor efficacy of encapsulated drugs.111 Is4.4.2 The pre‐adsorption process has also been reported to be a versatile and convenient method for the attachment of targeting antibodies to nanocarriers’ surfaces. Compared to covalently bound antibodies that were covered by protein coronas and no longer functional, pre‐adsorbed antibodies maintained their function and were not masked by the protein coronas.Coating with a hydrophilic polymer does prevent the adsorption of certain proteins, but paradoxically, this can also evoke recognition of liposomes by the immune system, especially with repeated administration. To overcome this issue, precoating liposomal surfaces with functional proteins and antibodies has been reported as an alternative approach to endow liposomes with stealth properties. Giulimondi et al. showed that pre‐coating of liposomes with artificial human plasma proteins corona drastically reduced the cellular recognition and uptake by circulating leukocytes in peripheral blood.113 ThAnother prominent example is the pre‐adsorption of scFv. scFv is a single‐chain variable antibody fragment with high antigen‐binding activity and small molecular weight. Recently, our group prepared a 30‐kDa anti‐PEG scFv with a high PEG binding affinity for the amelioration of PEGylated liposome‐related ABC phenomenon. In naïve rats without anti‐PEG antibodies, pre‐deposition of such anti‐PEG scFv on the liposomal surface effectively abolished the complement activation due to the lack of Fc fragment. The anti‐PEG scFv also had negligible influence on the intrinsic performance of liposomes due to its small molecular weight. However, in liposome pre‐stimulated rats, anti‐PEG scFv pre‐adsorption effectively competed for the binding of anti‐PEG IgM antibodies and subsequently blocked complement activation, significantly ameliorating the ABC phenomenon of liposomes by a 1.5‐folds increase of the AUC values.115 In addition, we reported a facile anti‐PEG scFv‐based separation method combined with HPLC to quantify free doxorubicin and liposomes‐encapsulated doxorubicin in plasma. The anti‐PEG scFv was used to precipitate PEGylated liposomes by simple incubation and low‐speed centrifugation (100–2000 × g for 10 min), demonstrating sufficient accuracy and sensitivity compared to conventional solid‐phase extraction method. We hope the anti‐PEG scFv could provide new effective methods for protein coronas analysis and pharmacokinetic evaluation of PEGylated liposome drugs and other PEGylated nanomedicines.Notably, we further developed an anti‐PEG scFv‐based affinity chromatography to achieve precise and efficient separation of protein coronas on PEGylated liposomes. It demonstrated a 43‐fold higher protein coronas collection efficiency than the classical centrifugation method.116 In4.4.3LCDX, and its retro‐inverso peptide analog DCDX, have previously been designed by our group to promote precise delivery of liposomal agents to the glioma. However, surface modification with such long (16‐amino acid), stable positively charged peptide triggered avid absorption of IgM, causing enhanced immunogenicity and rapid blood clearance. Electrostatic interaction was identified to play a crucial role because the content of IgM absorption positively correlated with the net positive charges of peptides by amino acid mutations. A short stable peptidomimetic ligand D8 with comparative targeting ability toward its receptor nAChRs has been successfully re‐designed by computer‐aided design. Compared to CDX, D8 successfully improved the immunocompatibility of brain‐targeted liposomes by attenuating nIgM absorption. The results provide insights into developing promising targeted liposomes by ligand structural manipulations to reduce potential immunogenicity.Targeting ligands such as aptamers, peptides, and antibodies are anticipated to achieve a high targeting yield of liposomes to diseased tissues by recognizing corresponding receptors. For instance, a brain‐targeted peptide ligand, e glioma.119 Ho4.5Complement‐mediated opsonization is a prime consequence of IgM adsorption. Therefore, modulation of the complement cascade can be a key strategy for improving the in vivo performance of liposomes. By use of proper surface chemistry design such as carboxyl, methoxyl, and amine groups is certainly a good way, but here we would like to highlight the pivotal role geometric and topological features  played in complement activation.2. Due to topological constraints, most of the C3b molecules are rather difficult to deposit on the surface of particles with high curvature. Thus, small particles are deemed to effectively escape C3b opsonization, whereas they are still capable of activating the complement system and releasing anaphylactic peptides due to the assembled fluid phase C3 convertases. In addition, small molecule structure critically determines how immunoglobulins differentiate self from non‐self to prevent infection. Usually, the buried surface of epitope recognition by antibodies is 12–20 nm2. However, for large immunoglobulins such as sIgM whose cross‐sectional diameter in solution is ≈40 nm, it involves a far larger epitope‐presenting surface than a single paratope and epitope. The surface‐bound sIgM adopts a staple‐like conformation to activate complement, in which the five Fab fragments of sIgM are tightly adsorbed to the particle surface, bent about 60° relative to the Fc5 plane to expose a binding site for C1q. Therefore, the conformation of surface‐bound sIgM can also be greatly influenced by topological features, and so does the subsequent C1q docking. Pedersen et al. have explored the relationship between complement activation and the interaction between sIgM and surfaces with different curvatures. They found that in contrast to a larger or smaller size, fragments of peptidoglycan with sizes of 100 nm were more likely to trigger potent complement activation through the classical pathway. Since the as‐prepared liposomes are typically around 100 nm in diameter, their potent complement‐activating capacity can be explained. More recently, we have investigated the effects of lipid carrier morphology on FA functions and found that FA‐functionalized lipodiscs (FA‐Disc) successfully circumvented IgM‐mediated opsonization and ameliorated the in vivo performance. Owing to the distinct morphology features of FA‐Disc, FA moieties were restricted to its highly curved edges, along with the bound IgM. In this case, it eventually circumvented IgM‐mediated complement activation because the bound IgM was in an inactive conformation. The clearance of FA‐Disc as well as the IgM‐mediated ABC phenomenon was alleviated, too. FA‐Disc maintained its folate receptor binding and tumor‐targeting capacities in vivo. Interestingly, we also revealed that plain lipid nanodiscs displayed unique protein corona patterns. Apolipoproteins preferentially bound to the discoid surface endowed nanodiscs with distinct brain targeting capability. This work may provide new insights into morphology‐driven manipulation of the protein coronas for efficient preferential drug delivery.Activation of all complement pathways leads to the deposition of C3 proteolytic fragments, for example, C3b, on particle surfaces. The buried area of each surface‐bound C3b molecule is estimated to be 40 nmvertases.121 In4.6 developed bioinspired liposomes by modifying the liposomal surface with a short peptide that specifically interacts with the lipid‐binding domain of apolipoproteins. The engineered liposomes actively absorb apolipoproteins  in vivo, consequently exposing the receptor‐binding domain of apolipoproteins to achieve brain‐targeted delivery via multiple apolipoprotein receptor‐mediated transcytosis in mice.The exploitation of protein coronas by recruiting functional endogenous biomolecules to the nanocarriers’ surface has emerged as a promising strategy for improving targeted drug delivery. Liposomes are surface engineered to pick up specific plasma proteins to initiate receptor‐mediated cellular recognition, internalization, and translocation across biological barriers. Potential receptors  are abundant depending on the type of plasma proteins. For example, Zhang et al.126 de The enhancement involves complement receptors 1 and 2 (CR1/2) and FcµR that are expressed on B cells. Since both the IgM‐CR and IgM‐FcµR pathways may contribute to humoral immunity, IgM can be utilized as a self‐adjuvant to modulate the interplay between nanovaccines and immune cells. As mentioned before, DCDX‐modified liposomes heavily absorbed IgM via electrostatic interaction. It inspired us to use DCDX‐modified liposomes for enhanced antigen uptake and presentation by splenic MZB cells via both CR and FcµR pathways. DCDX‐modified liposomes induced a stronger and more durable IgG1 titer than plain ovalbumin‐loaded liposomes. Since the biological functions of IgM are diverse, this work suggested that manipulating IgM as a functional self‐adjuvant may provide new impetus for improving in vivo performance of liposome‐based nanovaccines.IgM is the most potent immunoglobulin to activate the complement system and thus may work as an efficient immune response enhancer.32 The5The nano‐bio interfaces are far more complex than that of conventional small molecular drugs. Although increasing clues have been learned about the in vivo performance of liposomes over the past decades and some general principles have emerged, detailed relationships of liposomes with plasma protein coronas formation, immune cells interplay, and pathophysiological response remain elusive. The theoretical system of the in vivo delivery process of liposome formulations is still in a blank state. Without this mechanistic knowledge as a guide, it is difficult to design liposomes for in vivo delivery in a controlled manner.FigureAmong these abundant sources of protein coronas, IgM was identified to be one of the key plasma proteins regulating the in vivo performance of liposomes . As shown in FigureBased on the regulatory effect of IgM on the delivery process in vivo, corresponding strategies can be adopted to improve the performance Figure5.1) Considering the individual variability of IgM, it is necessary to pre‐screen suitable recipients before medication. Adjusting doses according to the IgM levels and activities of recipients might be an alternative to calculating the dose based on body surface area. Extreme cautiousness is urged especially for patients with potentially serious side effects. Premedication with transient MPS blockade or IgM suppression might improve the therapeutic efficiency.2) IgM adsorption and sequent complement cascade are the culprits that negatively regulate the in vivo performance of liposomes. Thus, design strategies to attenuate IgM adsorption onto liposomal surfaces such as structural optimization of the PEG moieties, pre‐adsorption of functional proteins and antibodies to shield IgM deposition, structural re‐design of the targeting ligand to weaken IgM binding affinity, modulation of complement cascade by liposomal topological features, can achieve desired surface properties and optimal in vivo performance.3) IgM can be manipulated as a functional self‐adjuvant to modulate the interplay between liposomes and immune cells, providing new impetus for the development of liposome‐based nanovaccines for humoral immunity. Since IgM has multiple biological functions, there may be other applications to be explored based on the interaction of liposomes with IgM.Plenty of understanding of the fundamental mechanisms governing IgM corona formation on NP surfaces is still missing, particularly in complex physiological settings rather than in simple ex vivo plasma samples. Therefore, predicting the voyage of liposomes in vivo is extremely challenging due to the knowledge gaps as well as the complexity and heterogeneity of human physiology. At least these following basic questions remain for building up the theoretical system of liposomal in vivo delivery with higher clinical translational potentials.First, more details on the structural information of the IgM‐liposome complex are needed, especially for liposomes with specific IgM affinity like FA‐functionalized liposomes.Second, considering different routes of administration, how do liposomes interact with IgM from other sources in addition to serum, such as gastrointestinal fluids? More principles and mechanisms need to be explored. According to a recent report which determined the cryo‐electron microscopy structure of human IgM‐BCR, mIgM and sIgM have similar monomeric structure, so the binding mode of liposomes with mIgM deserves further exploration for more potential biological functions.Third, each B cell contains 100 000–200 000 mIgM‐BCR complexes on its membrane, playing crucial roles in B cell maturation, activation, and differentiation.128 AcLast but not least, we need to condense and summarize general rules from fragmented clinical information. For instance, in cancer patients, varying IgM levels and binding avidity with liposomes might be a causal factor for individual differences in response to the application of liposome‐based therapeutics. The clinical value lies in the potential of IgM as an indicator to predict the in vivo performance of liposomes, which in turn instructs the de novo design and optimization of liposomes and guides clinical individualized precision medication.The authors declare no conflict of interest.H.W., S.L., and X.W. contribute equally to this work. H.W. conducted the literature review and drafted the manuscript. S.L. and X.W. revised the manuscript and draw the figures. K.J. and H.L. provided suggestions for manuscript content. C.Z. critiqued and edited the manuscript and contributed expert advice on the topic. All authors read and approved the final manuscript.


The United States Veterans Health Administration (VHA) Office of Rural Health funds Enterprise-Wide Initiatives (system-wide initiatives) to spread promising practices to rural Veterans. The Office requires that evaluations of Enterprise-Wide Initiatives use the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework. This presents a unique opportunity to understand the experience of using RE-AIM across a series of evaluations. The authors conducted a study to document the benefits and pitfalls of using RE-AIM, capture the variety of ways that the team captured the elements of RE-AIM, and develop recommendations for the future use of RE-AIM in evaluation.The authors first conducted a document review to capture pre-existing information about how RE-AIM was used. They subsequently facilitated two focus groups to gather more detailed information from team members who had used RE-AIM. Finally, they used member-checking throughout the writing process to ensure accurate data representation and interpretation and to gather additional feedback.Four themes emerged from the document review, focus groups, and member checking. RE-AIM: provides parameters and controls the evaluation scope, “buckets” are logical, plays well with other frameworks, and can foster collaboration or silo within a team. Challenges and attributes for each RE-AIM dimension were also described.Overall, participants reported both strengths and challenges to using RE-AIM as an evaluation framework. The overarching theme around the challenges with RE-AIM dimensions was the importance of context. Many of these benefits and challenges of using RE-AIM may not be unique to RE-AIM and would likely occur when using any prescribed framework. The participants reported on the RE-AIM domains in a variety of ways in their evaluation reports and were not always able capture data as originally planned. Recommendations included: start with an evaluation framework (or frameworks) and revisit it throughout the evaluation, consider applying RE-AIM PRISM  to gain a broader perspective, and intentionally integrate quantitative and qualitative team members, regardless of the framework used. Rural Veterans are also significantly more likely to enroll in the US Veterans Health Administration (VHA) for healthcare than Veterans who live in more urban areas .The VHA established the Office of Rural Health (ORH) in 2006 with the goal of conducting, coordinating, promoting, and disseminating research on Veterans that live in rural communities .In 2017, ORH began requiring evaluations of Enterprise-Wide Initiatives to use the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework  to strucRE-AIM is an implementation framework that considers elements of implementation (such as characteristics of sites and those that provide the intervention) and program performance . Additionally, the RE-AIM dimensions consider the steps necessary to successfully monitor and improve program implementation and success . ORH sel1.Document the benefits and pitfalls of using RE-AIM2.Document the variety of ways that the team captured the elements of RE-AIM3.Develop recommendations for the future use of RE-AIM in evaluation.While there are many papers and reports of individual evaluations that used RE-AIM as an evaluation framework –13, and 2.2.1.The authors are Masters  and PhD (AM) educated Health Science Specialists with degrees in Public Health, Social Work, Sociology, Clinical Psychology, and Women, Gender, and Sexuality studies. They have 4–15 years of experience in evaluation and work at the Seattle-Denver Center for Innovation in Veteran-Centered and Value-Driven Care (COIN). The Seattle-Denver COIN has completed six Enterprise-Wide Initiative evaluations, with four more currently underway . Four ofThe authors took an approach reminiscent of collaborative autoethnography to learn from their previous experiences and inform future evaluations of Enterprise-Wide Initiatives. In collaborative ethnography, the personal experiences of the researchers are the primary data . This prThe first step was a document review to identify how each RE-AIM dimension was measured, challenges with using RE-AIM as an evaluation framework, data collection methods, project goals, outcomes of interest, and adaptations to RE-AIM.The document review was followed by focus groups. Completing the document review first grounded the authors in how RE-AIM was used in the evaluations, which allowed them to ground the focus group questions in the existing documentation and use focus groups to explore personal experiences that were not captured in the existing documents. The aim of the focus groups was to gain a deeper understanding of experiences using RE-AIM to conduct an evaluation.Finally, the authors also applied member checking techniques to ensure that the identified themes truly reflected the experiences of additional team members who worked on the evaluations that were surveyed.2.2.2.2.1.The authors started by compiling relevant information and materials from prior and current evaluations. Documents reviewed included project regulatory documents, evaluation plans, logic models, local department presentations, and quarterly and annual reports . Some prFour of the authors  completed a review  of all d2.2.2.All authors collaborated on focus guide development which was informed by document review. The authors chose to conduct focus groups over individual interviews in order to allow for “depth and detail” through group discussion of experiences with using RE-AIM as an evaluation framework . Focus gKey players from each project, including the principal investigator, project manager, qualitative methodologist, and quantitative methodologist, were invited via email to participate in one of two planned virtual focus groups. Each focus group targeted a subset of projects so teams could build on each other's comments. Author AM, who had less direct experience with evaluating Enterprise-Wide Initiatives than the other authors, facilitated two focus groups over Microsoft Teams. This allowed the remaining authors to participate in one focus group as a project team member and observe the other. A total of 15 team members (including four of the authors) were invited and 12 participated. At least one team member from each identified project participated. The focus group guide was sent to all participants in advance of the focus groups. Focus groups lasted approximately 60 min and were recorded and automatically transcribed in Microsoft Teams. Participants were informed that their participation was voluntary and that they could leave at any time.2.3.2.3.1.After the focus groups, the authors reviewed the recordings and transcripts to identify themes. Each author independently listened to the focus group audio and conducted careful line-by-line reading of the transcript of one of the two focus groups to identify key concepts and meaning units . The aut2.3.2.The authors presented the identified themes at a standing meeting comprised of team members who evaluated Enterprise-Wide Initiatives. The purpose of this presentation was to check the validity, trustworthiness, and credibility of the analysis. All focus group invitees were invited to participate in member checking and member checking was opened up to other roles that were not invited to focus groups, such as qualitative analysts, quantitative analysts, and programmers , 39.Finally, the authors shared an early version of this paper with all team members who participated in evaluations of Enterprise-Wide Initiatives. Additional feedback was solicited over email, in a team meeting, and in one additional meeting that was set up to capture the perspectives of team members who were unable to participate in other member checking activities. The themes presented below were synthesized across results of the document review, focus group analysis, and member checking.This work was deemed quality improvement by the Eastern Colorado Research and Development Committee because it was designed and conducted for the purposes of improving internal VA processes in support of activities carried out in accordance with the VA mission and the institutional requirements.3.15 evaluators  were invited to focus groups and 12  attended. Nine focus group participants  and an additional 11 team members who were not invited to focus groups  participated in member checking. At least one person from each evaluation participated in focus groups and member checking .Themes are grouped into overarching comments about RE-AIM and specific comments about measuring each element of RE-AIM.Input from member checking is integrated into the themes and noted where substantial changes were made due to member checking. Overall, member checking strengthened the existing themes and did not identify new themes.3.1.3.1.1.Using RE-AIM in evaluation planning led to early identification of data collection methods and clearly defining each RE-AIM dimension as it related to the subject of the evaluation. Participants found it “really helpful” to do up front work to define RE-AIM dimensions before beginning the evaluation. They also commented that without the RE-AIM framework they could “write a 100-page report.”Early on we knew that we needed to use RE-AIM… so we made a joint display that had the qual and quant methods in the table with every [dimension] of the RE-AIM framework listed… and listed our data sources or data variables that we would be using.” “Even though RE-AIM was seen as useful to control the scope, some participants expressed that it was challenging when there was pertinent information that didn't fit clearly into a RE-AIM dimension. The Office of Rural Health provides a standardized report template and the participants reflected that this led to “stuffing,” “pushing,” or “fitting” findings into RE-AIM. Another challenge was when findings could be reported under multiple RE-AIM dimensions, specifically overlap between adoption and implementation. This made it difficult to decide where to include the information in the report.And [Adoption] often overlaps so much with implementation… you're either double talking into sections or you're just trying to figure out ‘what am I gonna fit here or here’ when often it’s an interplay between implementation and rollout and barriers and facilitators. And then whatever you're calling adoption and how that actually played out and what your metrics are.” (Project Manager)“Other participants commented that RE-AIM was broad by nature and didn't limit the evaluation: “I don't think I would want us to turn away from an outcome that was appropriate for, you know, representing the impact of a program” . Another participant explained: “let [the RE-AIM dimensions] guide data collection, but don't be constrained in terms of expanding the data collection and analysis”  These participants advised that this supplemental information that didn't “fit” elsewhere in RE-AIM could be reported in the implementation section.3.1.2.Participants felt that RE-AIM was logical, and that any good evaluation would have data for each of the RE-AIM dimensions. They discussed “filling the RE-AIM buckets,” or making sure that there was data for each element of RE-AIM, when planning the evaluation. One participant commented that they felt that use of RE-AIM was not overly restrictive because the dimensions were so “high level” and another commented that “if you do a full summative evaluation from soup to nuts, you should have stuff in each of those categories” RE-AIM is just kind of a logical construct like “OK, we need to find the patients and we need to see how the team is adopting it within the VA. All those things are logical things and evaluation is going to work on.” “It tends to be that one or two of the components get the vast preponderance of the energy and effort and the otheroneskind of end up being, I don’t want to go as far as, say, is vestigial. But we're approaching something like that depending upon what the stakeholder interests are.” (Quantitative Methodologist)Even though there is no explicit guidance that every RE-AIM dimension should be given the same amount of attention, participants still felt that it was important to explicitly recognize that there should not be an expectation that each RE-AIM dimension hold the same weight. They shared that the focus will naturally shift for different partners, initiatives/programs, and stages of implementation. Some partners were interested in effectiveness , while others were more interested in adoption . Participants stated that they work closely with partners to ensure that the evaluation focuses on the most important or salient components, rather than trying to give equal weight to each element of RE-AIM: “3.1.3.Wedon’t think RE-AIM is always the best implementation science framework for the EWIs [Enterprise-Wide Initiatives] we've worked on to really show the barriers and facilitators” . Participants reported having success when pairing RE-AIM with other frameworks such as CFIR “During member checking, a third perspective emerged. Some team members shared that they believed that the collaboration or lack thereof was due to the Principal Investigator's style and not the evaluation framework that was used. One of these team members commented that they were not aware of RE-AIM prior to participating in member checking.3.2.All evaluations reported RE-AIM dimensions in ways that were specific to the program that they evaluated. Cross cutting themes regarding measuring RE-AIM were: (1) some outcomes were reported in multiple places, sometimes places that did not seem to fit the definition of the RE-AIM element, (2) some proposed outcomes were dropped because of inability to identify appropriate data sources, and 3) the participants utilized resources from the VA Office of Rural Health  the part when des3.2.1.3.2.1.1.Some participants identified limitations with the application of Reach, especially in early implementation phases. They commented that the number of patients served in early implementation is not representative of the number of patients that will be served when the initiative reaches maintenance. In these cases, participants provided supporting information in their reports to provide context when reporting reach.I find that Reach isn’t that helpful in the [Enterprise-Wide Initiatives] we've worked on because the programs we've been evaluating take a lot of effort to build… the reach metric is just often problematic with us for our efforts and honestly not that useful. Whereas I think other programs that can draw from like big secondary data sources or whatever might have a better, more effective use of reach.” “The document review revealed that nearly all evaluations reported the number and proportion of eligible Veterans served and included information on representativeness. Other items included provider impressions of Veterans who do and do not participate, potential locations for future sites, the number of encounters, program utilization by service line and modality, and fidelity to key program components. One evaluation team proposed but was ultimately unable to capture the number of referrals to the program, number of patients offered the program, and the number of patients who decline the program.3.2.2.3.2.2.1.Some participants noted that cost may not be an appropriate metric for programs that are in early implementation. They reflected that new programs are often working to increase reach, which can lead to increased cost, and do not reflect the cost of the program once it reaches maintenance. Participants reported that one project looked at the prevention of death as an alternative to cost.This notion that we're going to save money and provide more care at the same time is rarely realized in practice.” “Cost isn’t an effective measure, it’s not really that meaningful because there’s just such a huge startup cost to starting these things and then trying to quantify the cost per patient and it just looks like ‘why are we doing this? It’s so expensive.’ but because it hasn’t already reached Adoption. Or Maintenance. That that number is not really representative of what the actual cost would be if this was something that’s stained.” “We were able to show that the program was expensive but–like, because the program prevented death among participants we were able to show like a cost benefit in a sense…. it’s really hard because people, I mean the people creating these programs tend to want to show a positive return on investment because they think that’s what they need in order for the program to be sustained.” “Other measures of effectiveness that were identified in the document review included descriptions of the experience of participating in the program from both VHA and Veteran perspectives, perceived quality of care, satisfaction, acceptability, and feasibility. Some evaluations included measures of cost. Other information reported in effectiveness was specific to each program or initiative and included access to care, the frequency and values from clinically relevant lab draws, medications prescribed, adverse events, training results, and relational coordination . Outcome3.2.3.3.2.3.1.One participant described that adoption was intended to be measured as yes/no and it was challenging to find a metric that “feels adequate” to report adoption. Participants described reporting barriers and adaptations in addition to raw numbers of sites and staff who adopted the initiative to provide context and more authentically represent adoption.I think adoption is also problematic to me because if you if you bring it down to a ‘yes’ or ‘no’ and whether it’s yes or no overall or yes or no, these program elements were adopted or yes or no, you know, these sites were adopted and what does that mean? There’s, you know, it can seem punitive, or like, I can see where partners might be concerned… But there’s reasons [sic], right. And often it’s not just like, no, they didn’t care. It’s often like ‘we did something different because of XY or Z,’ But there’s not a great–it doesn’t feel like adoption to me is the right way to talk, to talk about that narrative, really.” “The document review revealed that while the evaluation reports and other artifacts included the number of settings that initiated the program being evaluated, they did not always include information about representativeness of staff and settings. Other information included in adoption was barriers and facilitators to site and VHA provider/staff participation, adaptations to the program, services and modalities implementing the program, equipment purchased, and staffing. This was another place where one evaluation planned but was unable to collect data on referrals to the program.3.2.4.3.2.4.1.Participants reported that RE-AIM is broad by nature, and that any additional or supplemental data collected would fit “under” implementation in the RE-AIM report and “if it didn't fit RE-AIM, I don't think I would want us to turn away from an outcome that was appropriate for, you know, representing the impact of a program”  One team member explained that even though RE-AIM may seem limiting if it is seen as the only way to design the evaluation, “let [the RE-AIM constructs] guide data collection, but don't be constrained in terms of expanding the data collection and analysis.”There’s a little bit of tension between working toward RE-AIM and the best thing for the evaluation and the best thing for working with your partners. Which may have a lot of overlap or… it may diverge and not clearly fit RE-AIM” “The document review uncovered that teams included information about site context and fidelity to core program components as well as adaptations when reporting implementation. They also included information about the number and types of visits conducted, cost, acute care, acceptability, feasibility, and barriers and facilitators to implementation among other measures.3.2.5.3.2.5.1.Participants reported that many evaluations that began the evaluation process when in pre-implementation or early-implementation did not reach maintenance before the 3-year evaluation ended and that there often was not robust maintenance data to report. Participants reported that they utilized qualitative data about what the partners thought was necessary to maintain the program and referred to this as “perceived maintenance.” The participants noted that “perceived maintenance” may not predict actual maintenance and associated barriers and facilitators. Some participants shared that another strategy they used was to include maintenance data from the first wave of a program to demonstrate what the expected maintenance might look like.A lot of the projects that I've worked on don’t actually reach Maintenance, so it’s more about perceptions of Maintenance and sustainability that we are measuring there because we're not at a place where there is actual Maintenance.” “As most of the programs were evaluated before they reached maintenance, teams included perceptions of sustainability, suggestions for future program development, and plans for program sustainment in the maintenance section of their reports.4.The purpose of this manuscript was to capture the authors’ teams’ experiences using RE-AIM as an evaluation framework across a series of US Veterans Health Administration Enterprise-Wide Initiative evaluations.Benefits and pitfalls to using RE-AIM as an evaluation framework (Aim 1) were identified in this manuscript. Overall, participants saw both strengths and challenges to using RE-AIM. Participants reported that RE-AIM provided parameters and controlled the evaluation scope, was logical, and paired well with other frameworks. While using RE-AIM as a framework sometimes seemed to improve team communication in some cases, this was not always true and may have more to do with the Principal Investigator than the RE-AIM framework. Other challenges included the importance of providing additional context, limitations of the RE-AIM dimensions, and challenges when RE-AIM seemed to miss important outcomes.These challenges can be mitigated by thoughtful identification of ways to capture RE-AIM elements (Aim 2) and developing and implementing recommendations for future use of RE-AIM in evaluation (Aim 3). The document review revealed that some measures were reported in multiple RE-AIM elements, some of which may seem to be the “wrong” place, and that some measures were planned and later dropped. While there are tools available on the RE-AIM website that expand on how to measure the RE-AIM elements, the participants primarily consulted guidance from the funder to guide evaluations. This guidance was not as comprehensive as the tools available on the RE-AIM website. Additional recommendations are shared below.The overarching theme around the challenges with RE-AIM dimensions was the importance of context. Participants reported that without implementation context, often provided by qualitative data, quantitative data about RE-AIM could misconstrue the actual viability of a program, especially in early implementation. While RE-AIM was initially created as a quantitative framework, more recent work points to the importance of including qualitative data when using RE-AIM . The intFinally, many of these benefits and challenges of using RE-AIM may not be unique to RE-AIM and would likely occur when using any prescribed framework. In order to understand benefits and challenges unique to RE-AIM, one would need to conduct a comparative analysis of evaluations that were required to use different frameworks. This work sets up the opportunity to look at the transferability of experience between models.4.1.Several findings from this paper were previously addressed in an article written by the founders of RE-AIM . In thisThe findings in this manuscript echo previous work, such as Kwan et al.  and QuinUnlike this manuscript, Kwan et al. and Quinn et al., reported challenges with data acquisition. While this manuscript identified data acquisition issues in the document review, this was not a salient theme in the focus groups. This may be because this paper examines US Veterans Health Administration programs, and, as the largest integrated health system in the United States, there is a plethora of administrative data available. Additionally, this may not be salient in part due to the limited participation of quantitative team members, further discussed in the limitations.4.2.The biggest limitation to this project was that the authors reviewed their own work. While they took precautions, such as member checking and ensuring that all themes and examples were represented in the data, it is still likely that their own experiences played a disproportionate role in the findings. Review by those not intimately involved in the series of evaluations may identified a different set of themes.A second limitation was that the RE-AIM framework is a requirement for evaluations of Office of Rural Health Enterprise-Wide Initiatives. This colored the experience of the participants. Focus group participants shared extensively about the requirement to use RE-AIM, the standardized reporting format, and the challenges that some faced when switching to RE-AIM after an evaluation was designed with a different framework. This detail was not included in the manuscript because it was specific to evaluations of Enterprise-Wide Initiatives. Evaluators who independently chose RE-AIM as an evaluation framework likely have different experiences using RE-AIM.Finally, the perspectives of participants with qualitative expertise  far outw4.3.-Start with an evaluation framework and continue to revisit the framework throughout the evaluation.-Don't limit oneself to one evaluation framework; even if a specific framework is required, pairing it with additional frameworks may be necessary to ensure that the evaluation is comprehensive and meets the needs of the partners, other interested parties, and the evaluation funder.-Think carefully about applying economic analysis; ensure that team members conducting economic analysis have the appropriate expertise, supplement economic analysis with contextual information, and consider conducting further research on applying economic analysis to programs that have not yet met sustainment.-Consider RE-AIM PRISM  to gain a broader perspective, as the developers of RE-AIM have endorsed this expanded framework.-Be intentional about integrating quantitative and qualitative team members; it is easy to lose sight of the possibilities for mixed methods and collaboration over the course of an evaluation.In closing, while RE-AIM was not without its challenges, participants found RE-AIM to be a logical and practical framework for evaluating Enterprise-Wide Initiatives, especially when providing contextual information to support findings. More work is needed to understand the experience of using dissemination and implementation frameworks in evaluation. This includes further examination of the experience of quantitative team members, exploring the experiences of evaluators who chose the RE-AIM framework on their own accord, and comparing the experience of using RE-AIM with the experience of using other frameworks. This research will both provide guidance for evaluators who utilize the frameworks and shine a light on gaps and misconceptions that framework developers can address with additional guidance and/or resources.Recommendations drawn from this analysis include:


The program positively affected and improved head nurses’ knowledge and perception of workplace cyberbullying; however, it elevated their symptom experience and had no immediate impact on turnover intention. The developed program could be applied as a valuable educational strategy in the nursing field. Head nurses act as intermediaries between individuals and the organization. Therefore, they must respond with in-depth knowledge and perceptions of cyberbullying to fulfill their responsibilities of identifying, mediating, and managing cyberbullying among hospital team members.This study aimed to (1) develop a version of the cognitive rehearsal program that is suitable for cyberbullying and (2) apply the m-learning method to evaluate its effectiveness among head nurses. This study was conducted in July 2021 and comprised 69 South Korean university hospital head nurses. It was evaluated using a nonequivalent control group pretest-posttest and a quasi-experimental design. The program was developed using the Analysis, Design, Development, Implementation, and Evaluation (ADDIE) step process, consisting of 10 scenarios. Variables with proven reliability were used in the program effect measurement. The differences between the experimental and control groups were examined using an independent  Bullying in nursing organizations is more common than in other establishments ,2. WhileBased on a literature review and meta-analysis, a number of theories serve as the theoretical foundation for the causes and effects of workplace cyberbullying ,9,10,11.Cyberbullying, which occurs in cyberspace, is more serious than traditional bullying, as victims can be continuously tormented regardless of their physical location . CyberbuThus far, interventions aimed at preventing workplace bullying include the cognitive rehearsal program by Griffin , in whicRecent studies involving Korean nurses reported that the workplace cyberbullying incidence rate was 8–10% . NeverthNurses experience time and space constraints due to the nature of their work. In nursing education, mobile and internet technology that enables continued learning via text, audio, video, and image data without time and space constraints has been expanding . During H1: Compared to the control group, the experimental group will experience fewer negative symptoms and exhibit a lower turnover intention.H2: Compared to the control group, the experimental group will exhibit higher levels of perception and knowledge.This study was evaluated with a nonequivalent control group pretest-posttest and a quasi-experimental design.t-test effect size of d = 0.66, significance level (α) of 0.05, and statistical power (1 − β) of 0.80 indicated that the minimum sample size was 30. Considering drop-outs, 35 participants were selected for the experimental and control groups.The study setting was a university hospital with 1700 beds located in “I” city (Republic of Korea). The hospital employed 78 female head nurses. The inclusion criteria were: worked as a head nurse for at least six months at the hospital, had no experience in workplace cyberbullying training, had a minimum of 10 workers within the department, and could attend workshops. Those whose job title was changed to general nurse were excluded. The total sample size for the effect evaluation was calculated using G-power 3.1.9.2. Moreover, the effect size from a previous study that examined the influence of a face-to-face cognitive rehearsal program for nurses  was usedThe initial 70 candidates who volunteered to partake were recruited to participate in the pretest-posttest survey and the program. After excluding one control group participant due to incomplete responses, the data from the remaining 69 were included in the final analysis. For group allocation, cards with the letter “A” or “B”  were placed in a non-transparent box, from which each participant personally picked a card .A research assistant informed the participants about the study objectives during a one-hour, one-day head nurse workshop on 29 July 2021. The pretest was administered to the 70 participants to investigate their symptom experience, perception and knowledge of cyberbullying, and turnover intention.n = 35) remained in the workshop venue to observe the workplace cyberbullying cognitive rehearsal m-learning program; when necessary, they were repeatedly directed to a password-protected Vimeo site throughout the day to watch the video using mobile phones or computers. The participants completed the posttest within 24 h after finishing the program and submitted it to the nursing department’s administrative office.The experimental group (n = 35) was prohibited from watching the Vimeo video and did not receive any intervention. The following day, they completed the post-test questionnaire and submitted it to the nursing department’s administrative office. Those who wanted to complete the program were provided with the link to the Vimeo site and its password two days after the questionnaire’s completion. The participants who finished all questionnaires were provided a gift certificate .The control group  step process. ADDIE was proposed as evidence of instructional design by Peterson  and is wThe cognitive rehearsal training program previously developed by Griffin  and StagThe program’s goals were established based on the analysis results and were designed to: (a) present a problem by exposing the participants to two cognitive rehearsal scenarios, (b) conduct the training program, and (c) respond after presenting 10 cognitive rehearsal scenarios again.A professor with three years of research experience in workplace cyberbullying administered the 40-min training program to the nurses via video. An explanation of the program goals and learning methods was displayed on the screen, and a voice speech described the cases. After the nurses learned all the content, the final evaluation was performed.The developed program’s content validity was assessed by a four-member expert panel, after which it was partially revised and supplemented. The content validity coefficient ranged from 0.95 to 1.00. Regarding the revised and supplemented content, a national-level response was added to the response methods. The completed program was uploaded to Vimeo for playback using a mobile phone or a computer. The use of Vimeo software required a set password to access any content that was copyright patented .The nurses’ symptom experience was measured using the brief symptom tool, originally developed as the Brief Symptom Inventory-18 by Derogatis  and subsThe measure for knowledge of workplace cyberbullying was developed by the researcher by referring to the knowledge assessment of bullying among medical students in Quetta . The conEach correct and incorrect or missing answer was assigned one and zero points, respectively. The total score was converted to a 100-point scale; higher scores indicated greater levels of knowledge. The final expert validity was a CVI of 0.95. The pilot and the present study’s reliabilities using the Kuder–Richardson formula 20 were 0.65 and 0.72, respectively.The researcher constructed the tool for the perception of workplace cyberbullying by referring to the scale employed to measure the perception of bullying among medical students in Quetta . It consTurnover intention refers to the purpose of voluntarily leaving the present job for another institution . It was t-tests. The pretest-posttest differences’ normality in the dependent variables of the two groups was examined using the Shapiro–Wilk test. According to the aforementioned normality, an independent t-test (perception) or the Mann–Whitney U test  was utilized to analyze the differences between the two groups. Furthermore, the statistical significance was set at p < 0.05. For the measurement tools’ reliability, the Kuder–Richardson formula 20 and Cronbach’s alpha were used.SPSS/WIN 23.0  was used for data analysis. The homogeneity of the characteristics and pretest-dependent variables between the experimental and control groups were examined by the frequency, mean, chi-square tests, Fisher exact tests, and This study was approved by the Institutional Review Board of “G” University (IRB NO.1044396-202106-HR-129-01) and adhered to the Declaration of Helsinki. The participants were informed that the collected data would be anonymously codified, would only be used for this study’s purposes, and that they could refuse or withdraw their consent at any time. Additionally, written informed consent was obtained from each participant before the study commenced.n = 69) were female and did not work in a three-shift system. The clinical experience’s mean length was 28.24 ± 5.10 and 26.79 ± 4.59 years in the experimental and control groups, respectively. Regarding the experience of face-to-face workplace bullying in the past six months, two cases each in the experimental (5.7%) and control (5.9%) groups were reported. With respect to facing workplace cyberbullying in the past six months, there were three (8.6%) and no (0%) cases in the former and latter, respectively. The pre-intervention pretest results demonstrated no statistically significant differences in the general characteristics and dependent variables between the two groups , knowledge of workplace cyberbullying , and perception of workplace cyberbullying .There was an increase in the experimental group’s scores from the pretest to the posttest for symptom experience (3.10 ± 1.12 to 3.47 ± 1.13 points), knowledge of workplace cyberbullying (84.06 ± 11.59 to 95.19 ± 5.32 points), and perception of workplace cyberbullying (3.98 ± 0.52 to 4.45 ± 0.82 points). Consistent with H2, a statistically significant difference was observed between the experimental and control groups’ symptom experience .The turnover intention in the experimental group decreased from 2.04 ± 0.83 to 1.94 ± 0.80 points at the pretest and posttest, respectively. However, there was no statistically significant difference between both groups (As a result of hypothesis verification, the difference values of the experimental group compared to the control group were confirmed to be significant for symptom experiences, knowledge, and perception. However, there was no statistically significant difference between both groups for turnover intention; H1 is not supported .Workplace cyberbullying in nursing organizations is a new concept whose influences should be evaluated by developing effective intervention programs on the same level as face-to-face bullying. However, such programs have thus far been unavailable for head nurses, who serve as organizational managers . This stPrevious studies that directly applied cognitive rehearsal interventions for workplace bullying for nurses ,14,15 usDifferent from existing studies, the cognitive rehearsal program for cyberbullying applied in this study was proven effective as a new educational technique using mobile technology ,14,15,23This research’s effect evaluation of the two groups demonstrated statistically significant differences in symptom experience, knowledge, and perception of workplace cyberbullying, but not in turnover intention. Similar to a face-to-face workplace bullying intervention study that applied cognitive rehearsal, the experimental group’s knowledge and perception scores in this study were significantly higher than that of the control group, thus indicating the effectiveness of cognitive rehearsal m-learning programs for workplace cyberbullying ,15. In KUnlike previous studies, this study’s program did not affect turnover intention ,23. It iThis study had several limitations. First, the participants only included head nurses; thus, repeating the effect evaluation with general and chief nurses is suggested. In addition, all participants in the study were female, making it difficult to compare the effects of interventions based on sex. To evaluate the effects of sex on such interventions, a future study that includes male subjects is suggested. Second, randomization was not possible because the experimental and control groups were selected out of convenience. Subsequent studies should consider randomized sampling. Third, due to insufficient research on cyberbullying, the program operation and application durations were set to a relatively short time. Therefore, additional tests on the effective operational time and method are needed. Regarding the effect evaluation’s dependent variables, symptom experience requires more replication studies with an expanded research population and comparison with general staff nurses. Moreover, other dependent variables, such as the cyberbullying reporting rate, should be considered. Lastly, turnover intention remained unaffected when it was evaluated immediately after the program’s application; thus, it is essential to assess the effect by extending the evaluation period.This study’s cognitive rehearsal m-learning program for workplace cyberbullying provided indirect experience of cyberbullying cases that are difficult to learn directly in clinical settings. The results showed that the developed program had a negative effect and increased head nurses’ physical symptoms. However, it also had a significantly positive educational impact by enhancing their knowledge and perception of workplace cyberbullying. Thus, the developed program could be an effective educational strategy in the nursing field.The application of a cognitive rehearsal m-learning program can be used to effectively train head nurses on workplace cyberbullying within a short period. It can efficiently enhance their knowledge and perceptions about cyberbullying. Therefore, such a program could enable head nurses to more successfully manage their staff. Additionally, the program could also be expanded to various target populations, nursing education, and medical institutions.